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Apr 2020

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Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq
DRIP-seq法鉴定黑腹果蝇胚胎和组织培养细胞R-loop形成序列   

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Abstract

R-loops are non-canonical nucleic structures composed of an RNA–DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of proteins and to be associated with different histone modifications, suggesting a regulatory function. In addition, R-loops have been demonstrated to form differentially during the development of different tissues in plants and to be associated with diseases in mammals. Here, we provide a single-strand DRIP-seq protocol to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells. This protocol differs from earlier DRIP protocols in the fragmentation step. Sonication, unlike restriction enzymes, generates a homogeneous and highly reproducible nucleic acid fragment pool. In addition, it allows the use of this protocol in any organism with minimal optimization. This protocol integrates several steps from published protocols to identify R-loop-forming sequences with high stringency, suitable for de novo characterization.


Graphic abstract:



Figure 1. Overview of the strand-specific DRIP-seq protocol


Keywords: DRIP (DRIP), R-loop (R-环), S9.6 antibody (S9.6抗体), Drosophila melanogaster (果蝇), RNA-DNA hybrid (核糖核酸-脱氧核糖核酸杂交体), Tissue culture cells (组织培养细胞)

Background

Overview

R-loops are triple-stranded nucleic acid structures that form when an RNA hybridizes with a complementary ssDNA, leading to displacement of the second DNA strand. R-loops were first described as a by-product of transcription and a source of genomic instability that needed to be resolved. However, research over the last decade has shown that R-loops can be associated with specific histone modifications and transcriptional status, induce the targeting of proteins, or act as promoters (Chedin and Benham, 2020; Niehrs and Luke, 2020). To study R-loop function, protocols have been developed to map these structures in multiple organisms. Differences have been observed between species: R-loop-forming sequences in mammals have a GC skew; those in Saccharomyces cerevisiae have an AT skew; and in plants, both GC and AT skews have been reported (Sanz et al., 2016; Wahba et al., 2016; Xu et al., 2017; Hartono et al., 2018). Differences in the properties and localization of R-loop-forming sequences between species and the association of R-loops with different diseases and gene misexpression highlight the importance of developing standard R-loop mapping protocols to perform rigorous comparisons.


R-loop maps in plants have shown that some R-loops can form differentially during development (Fang et al., 2019; Xu et al., 2020) and may potentially be involved in changes in transcriptional status. In mammals, active enhancers have been associated with R-loop formation, and these R-loops can act as promoters (Tan-Wong et al., 2019). The study of R-loops in the context of development in intact organisms with available genetic tools to evaluate their formation, resolution, and function, will allow a better understanding of R-loop biology.


Here, we provide a detailed strand-specific DRIP-seq protocol to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells (Figure 1). Similar to other DRIP protocols, our protocol relies on the S9.6 antibody to identify R-loop-forming sequences but also considers that the weak affinity of the S9.6 antibody for dsRNA can generate false positive signals (Phillips et al., 2013; Hartono et al., 2018; König et al., 2017). This single-strand DRIP-seq protocol in association with the genetic tools available for Drosophila melanogaster should be a powerful system to study R-loop function in the whole organism, during development, and in different tissues (Figure 2, Table 1). DRIP-seq can be combined with genetic manipulation to overexpress or knockdown genes with a view to identifying their effect on R-loop formation, or with cell synchronization to follow R-loop formation during the cell cycle. Finally, the use of sonication rather than restriction enzymes to fragment the genomic DNA means that this protocol could potentially be adapted to other organisms with minimal optimization.


Limitations

The protocol relies on the S9.6 antibody to immunoprecipitate RNA–DNA hybrid-containing fragments. This antibody is not a perfect tool: it shows differential binding affinity based on the sequence of the R-loop with no correlation with the GC content and a preference for longer RNA–DNA hybrids. It can bind dsRNA, albeit with an affinity 5-times lower than that for RNA–DNA hybrids (Phillips et al., 2013; König et al., 2017). The latter two limitations can be overcome during the preparation of nucleic acids. After nucleic acid extraction, ssRNA and dsRNA are removed by incubating the nucleic acids with RNase A in the presence of 0.5 M NaCl to avoid the degradation of RNA in the RNA–DNA hybrids. This first digestion step is followed by an incubation with RNase III to degrade any dsRNA that were not degraded by RNase A. By using two different RNases before immunoprecipitation, we removed all ssRNA and dsRNA but also potentially some R-loops, which can be sensitive to these RNases. Thus, these steps make the experiment more stringent and limit false positives but could be modified to balance stringency with sensitivity, since some R-loops may be sensitive to these treatments. Removal of RNA that can interact with S9.6 is most relevant when the RNA component of the R-loop is to be sequenced; although, we have observed that the RNA–DNA hybrid pulldown efficiency is increased when stringent RNase treatment is used. It is also essential to have an RNase H-treated negative control to evaluate the specificity of the pulldown (Figure 3). We found that commercially available E. coli RNase H does not consistently completely digest RNA–DNA hybrids in nucleic acid preparations. This can be solved by producing and using highly active human RNase H1 and RNase H2. This negative control is especially important during optimization of the DRIP protocol; it ensures that the technique is specific. It is also used to call peaks when DRIP-seq is performed (Figures 2, 4, and 5).


To avoid bias toward longer fragments during the immunoprecipitation step, we fragment the nucleic acids by sonication (Figure 4A). Sonication allows us to obtain a homogenous population of fragments with an average size of 300 bp. Sonication is performed after RNase A treatment to avoid the possibility of generating new RNA–DNA hybrids during the DRIP procedure. Sonication is reported to reduce the recovery of some RNA–DNA hybrids as compared with restriction digestion (Crossley et al., 2020).


Advantages

Advantages compared with other DRIP protocols

This protocol uses a gentle lysis step to extract the nucleic acids. Cell lysis can be performed on tissue culture cells, whole embryos, or dissected tissues from larval, pupal, or adult Drosophila melanogaster. Although the ~200 mg Drosophila embryos needed to perform one DRIP-seq experiment is relatively high, the possibility to freeze tissues and pool them for a single nucleic acid extraction makes it feasible. By dissecting Drosophila melanogaster and performing DRIP on discs, organs, or sorted cell populations, it should be possible to identify cell- or tissue-specific R-loops.


This DRIP protocol, contrary to several protocols developed for mammalian cells (e.g., Sanz et al., 2016), does not use a restriction enzyme cocktail to fragment the nucleic acids. Instead, sonication is used, which leads to the fragmentation of nucleic acids at an average size of 300 bp (Figure 4A). These fragments have a homogenous size and the sonication is highly reproducible. An increase in resolution by using sonication, as compared with restriction digestion, in mammalian cells has recently been demonstrated (Crossley et al., 2020). By using sonication, the protocol can easily be adapted to other organisms with little optimization; the only step that may require optimization is lysis. Sonication has another advantage: it leads to disruption of the displaced single-strand DNA of the R-loop, which makes it possible to prepare strand-specific sequencing libraries using either the DNA or the RNA moiety of the RNA–DNA hybrid (Wahba et al., 2016).


Advantages compared with other methods

Another method to identify R-loop-forming sequences relies on a catalytically inactive form of the RNase H1 enzyme (dRNase H1) (Ginno et al., 2012; Chen et al., 2017). The use of dRNase H1 in cells presents several potential problems, which may explain the differences in the identification of R-loop-forming sequences with this method versus the S9.6 antibody. Firstly, RNase H1 is not the only regulator of R-loops in cells; it has been suggested that topoisomerases are the main enzymes responsible for the resolution of R-loops that form co-transcriptionally in human cells (Manzo et al., 2018; Zhang et al., 2019), while RNase H1 and H2 target R-loops once they are formed. Secondly, a recent article by Lockhart et al., 2019 demonstrated that RNase H1 is activated upon stress, while RNase H2 displays the main activity under physiological conditions in S. cerevisiae. Thirdly, with dRNase H1, R-loop identification may be limited to R-loops that are accessible, protein-free, and normally degraded by RNase H enzymes. Fourthly, expression of dRNase H1 may stabilize R-loops. While this may allow detection of transient R-loops, it could also skew interpretation of where stable R-loops exist. Finally, RNase H1 has two RNA–DNA hybrid binding domains, both of which need to bind to induce degradation of the RNA moiety of the hybrid (Nowotny et al., 2008). This may prevent or limit the detection of smaller RNA–DNA hybrids. Thus, while dRNase H1 may be a useful tool in some contexts, the interpretation of DRIP results may be more straightforward. Comparison of results from both methods could also yield complementary information.


Similar to DRIP, dRNase H1 has been used to isolate RNA–DNA hybrids after extraction of nucleic acids from tissue culture cells; however, bias of the enzyme toward longer hybrids and its weaker affinity as compared with the S9.6 antibody make it less efficient (Ginno et al., 2012).


Finally, native bisulfite sequencing is an alternative high-resolution method for identifying R-loops. Bisulfite converts cytosine to uracil in single-stranded DNA (Yu et al., 2003); thus, this method does not detect RNA–DNA hybrids but the ssDNA strand that is displaced when they form. However, the presence of methyl-cytosine in the genome blocks modification of the ssDNA, and ssDNA can be displaced by the formation of other non-canonical DNA structures such as G-quadruplexes and I-motifs, which could lead to false negative or false positive results, respectively.


The single-strand DRIP-seq protocol presented here has been optimized to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells, and can potentially be used in other organisms with minimal optimization. This protocol could be a standardized means to evaluate R-loops across developmental stages or in different tissues or organisms.



Figure 2. Examples of shared and distinct R-loops among S2 cells and early and later stage embryos. A-B. R-loops form over the CG1513, CG3008, and CG6040 genes in S2 cells and 2-6 H and 10-14 H embryos (red arrow). No R-loop is formed over the CG12923 gene. C. At the alt gene, three R-loops are detected in 2-6 H but not in 10-14 H embryos. Two of these R-loops (red arrows) are also present in S2 cells, while one of them is replaced by an R-loop on the opposite strand (purple arrow). D. R-loop formed over Spn1 is only present in 2-6 H embryos (purple arrows). E. R-loops form over scrt on the + strand in 2-6 H embryos and the - strand in 10-14 H embryos and in S2 cells. “Unstr” corresponds to unstranded DRIP-seq data, “+” and “-” indicate the strand-specific track, and RNase H-treated sample acts as a negative control. The red arrowhead indicates the orientation of the transcript. Note that the “+” and “-” strands refer to the DNA strand such that “+” strand R-loops should arise from transcription in the leftward direction. Thus, in A, the R-loop in the CG3008 gene is in the expected orientation to have arisen from gene transcription, while that in the gene CG1513 would be derived from antisense transcription.


Table 1. Data availability

Materials and Reagents

  1. RNase H1 and H2 expression and purification

    1. Amicon® ultra centrifugal filter, 0.5 ml 10K (EMD Millipore, catalog number: UFC5010BK)

    2. Amicon® ultra centrifugal filter, 0.5 ml 3K (EMD Millipore, catalog number: UFC5003BK)

    3. Econo-column (2.5*20 cm) (Bio-Rad, catalog number: 7374252)

    4. E. coli RosettaTM 2(DE3)pLysS SinglesTM Competent Cells – Novagen (Sigma, catalog number: 71401)

    5. Ni-NTA agarose (Qiagen, catalog number: 1018244)

    6. Glutathione-superflow resin (Takara, catalog number: 635607)

    7. PreScission protease plus (Homemade; commercial enzyme could also be used)

    8. Ampicillin (Bioshop, catalog number: AMP201.100)

    9. LB Broth Miller (Bioshop, catalog number: LBL407.500)

    10. Albumin, bovine serum (Bioshop, catalog number: ALB001.100)

    11. Lysozyme (Bioshop, catalog number: LYS702.5)

    12. Imidazole (OmniPur) (Millipore, catalog number: 5710-OP)

    13. Glycerol (Bioshop, catalog number: GLY001.4)

    14. IPTG (Bioshop, catalog number: IPT002.5)

    15. RNase H2 extraction buffer (see Recipes)

    16. RNase H1 lysis buffer (see Recipes)

    17. RNase H1 wash buffer (see Recipes)

    18. RNase H1 elution buffer (see Recipes)

    19. RNase H size column buffer (see Recipes)

    20. RNase H storage buffer (see Recipes)

    21. Protease inhibitors (see Recipes)


  2. Protease inhibitors and additives

    1. TLCK (Sigma, catalog number: T7254)

    2. Benzamidine (Bioshop, catalog number: BEN601.25)

    3. Pepstatin A (Bioshop, catalog number: PEP605.25)

    4. 1,10-Phenanthroline (Sigma, catalog number: 131377-5G)

    5. PMSF (Fisher Scientific, catalog number: 19538125)

    6. Aprotinin (Bioshop, catalog number: APR200)

    7. Leupeptin (Bioshop, catalog number: LEU011.50)

    8. NP40 (Nonidet P40 substitute, Fluka catalog number: 74385) (can substitute Sigma, catalog number: 74385)

    9. DTT (Bioshop, catalog number: DTT002.100)


  3. Activity testing of RNase H1 and H2

    1. rNTP (NEB, catalog number: N0450S)

    2. UTP α-P32 (Perkin Elmer, catalog number: BLU007H250UC)

    3. T7 RNA polymerase (NEB, catalog number: M0251L)

    4. SSC 20× (see Recipes)

    5. Tri-sodium citrate (Bioshop, catalog number: CIT001.205)


  4. Drosophila melanogaster embryo collection

    1. Fly bottles

    2. Oregon R flies (Dr Éric Lécuyer lab; available through Bloomington Drosophila stock center)

    3. Agar A (Bioshop, catalog number: FB0010)

    4. Sugar (RedPath)

    5. Apple juice

    6. TEGOSEPT, 1 KG (Nipagin) (Diamed.ca, catalog number: GEN20-258)

    7. Homemade sieve made with NITEX (can be obtained from https://flystuff.com/)

    8. Funnel

    9. Fly cages (fly cages, food, and bottles can be obtained from https://flystuff.com/)

    10. Fly food (prepared in-house)

    11. Methanol (Bioshop, catalog number: MET302.4)

    12. Embryo lysis buffer (see Recipes)

    13. Apple juice plate (see Recipes)

    14. 1× PBS (see Recipes)

    15. 1× PBT (see Recipes)


  5. Cell culture

    1. S2 cells (Invitrogen, catalog number: R69007)

    2. GibcoTM Schneider's Drosophila Sterile Medium (Thermo Fisher Scientific, catalog number: 21720-024)

    3. FBS (Thermo Fisher Scientific, catalog number: 16140-089)


  6. Nucleic acid extraction and preparation

    1. Proteinase K (Biobasic, catalog number: PB0451)

    2. Phase lock gel, heavy (VWR, catalog number: 10847-802)

    3. DNase I (RNase-free) (NEB, catalog number: M0303L)

    4. AmbionTM RNase III (Thermo Fisher Scientific, catalog number: AM2290)

    5. RNase A (Qiagen, catalog number: 19101)

    6. UltraPureTM DNase/RNase-free distilled water (Thermo Fisher Scientific, catalog number: 10977015)

    7. Covaris microTUBE AFA fiber pre-split snap-cap 6*16 mm (Covaris, catalog number: 520045 )

    8. Phenol-chloroform isoamyalcohol (Bioshop, catalog number: PHE512.400)

    9. Chloroform (Bioshop, catalog number: CCL402.1)

    10. Reagent alcohol (Sigma, catalog number: 277649-1)

    11. Tris (Bioshop, catalog number: TRS001.10)

    12. Acetic acid, glacial (Thermo Fisher Scientific, catalog number: 351271-212)

    13. EDTA (Bioshop, catalog number: EDT002.500)

    14. Sodium acetate (Bioshop, catalog number: SAA304.5)

    15. Sodium chloride (Bioshop, catalog number: SOD002.10)

    16. 1× RNase H buffer (see Recipes)

    17. TE (see Recipes)


  7. DRIP

    1. Anti-DNA–RNA Hybrid [S9.6] antibody (Kerafast, catalog number: ENH002, hybridomas are available through ATCC, HB-8730)

    2. DynabeadsTM Protein G for Immunoprecipitation (Thermo Fisher Scientific, catalog number: 10004D)

    3. NucleoSpin® Gel and PCR (Macherey-Nagel, catalog number: 740609.250)

    4. DNA Clean & ConcentratorTM (Zymo Research, catalog number: D4014)

    5. BSA, molecular biology grade (NEB, catalog number: B9000S)

    6. 10× DRIP binding buffer (see Recipes)

    7. 1× DRIP binding buffer (see Recipes)

    8. DRIP elution buffer (see Recipes)


  8. Library preparation and qPCR

    1. NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB, catalog number: E7735S)

    2. NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina® (NEB, catalog number: E7760S)

    3. RNase H (New England Biolabs (NEB), catalog number: M0297L)

    4. PowerUp SYBR Green PCR master mix (Thermo Fisher Scientific, catalog number: A25741)


  9. Agarose and SDS-PAGE gel preparation and staining

    1. SYBRTM Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, catalog number: S11494)

    2. SYPRO Ruby Solution (Lonza, catalog number: 50562)

    3. Glycine (Bioshop, catalog number: GLN001.10)

    4. 0.5× TBE (see Recipes)

    5. 10× DNA loading buffer (see Recipes)

    6. SDS-PAGE running buffer (see Recipes)

    7. Protein loading buffer (see Recipes)


  10. Plasmids

    1. pHis-MBP-hRNaseH1 (Alecki et al., 2020)

    2. pGEX6P1-hsRNASEH2BCA (Addgene, catalog number: 108693)

    3. vg-pETBlue (Alecki et al., 2020)

Equipment

  1. Typhoon Imager (GE Healthcare) for in-gel fluorescence imaging and phosphorimaging

  2. Viaa7 PCR system (Thermo Fisher Scientific) for real-time PCR

  3. Covaris E220 (Covaris) for nucleic acid sonication

  4. Centrifuge Avanti® J-E (Beckman Coulter)

  5. AKTA FPLC (or equivalent chromatography system)

  6. HiLoad 20/60 Superdex 200 column (GE Healthcare, catalog number: GE28-9893-36) (available from Sigma)

  7. Superdex 200 10/300 GL size exclusion column (GE Healthcare, catalog number: 28990944)

  8. Sonicator (Sonics, Vibra cellsTM) (probe sonicator for cell lysis)

  9. Nutator rotating platform to gently mix tubes

  10. Bacterial shaker

  11. 10 cm Petri dish

  12. Liquid nitrogen for flash freezing

  13. -80°C Freezer

  14. Nanodrop spectrophotometer

  15. Water bath

  16. 24°C Incubator

  17. 27°C Shaking incubator for S2 cells; S2 cells can also be grown on plates at room temperature

Procedure

  1. RNase H1 and H2 expression and purification

    1. RNase H1 expression and purification (Figures 3A-3D)

      1. Transform E. coli Rosetta cells with pHis-MBP-hRNaseH1. The next day, inoculate 2 ml LB-ampicillin (100 ng/µl) with a single colony and allow it to grow O.N. at 37°C in a shaking incubator.

      2. Add 1 ml culture from (a) to 200 ml LB-ampicillin and allow it to grow O.N. at 37°C in a shaking incubator.

      3. Add 100 ml culture from (b) to 1 L LB-ampicillin and induce RNase H1 expression with 0.1 mM IPTG for 4 h at 37°C.

      4. Harvest the cells by centrifuging for 20 min at 4,000 × g, 4°C.

      5. Discard the supernatant, freeze the cell pellet in liquid nitrogen, and store for at least a few hours at -80°C.

      6. Thaw the cell pellet on ice and add 20 ml RNase H1 lysis buffer containing 0.1 mg/ml lysozyme, 100 µg RNase A, 100 µg DNase I, and protease inhibitors.

      7. Incubate for 30 min on ice.

      8. Keep the cells on ice and sonicate 12 times for 15 s ON, 15 s OFF at an amplitude of 60%.

      9. Centrifuge for 20 min at 20,000 × g, 4°C.

      10. Keep the supernatant as the crude extract. The supernatant can be frozen in liquid nitrogen and stored at -80°C for future purification.

      11. Wash 3 ml Ni-NTA beads 3 times with 15 ml RNase H1 lysis buffer in a 50-ml tube by centrifuging for 2 min at 207 × g.

      12. Add the crude extract to the beads and incubate O.N. at 4°C on a nutator. Starting from this step, all the washes and elutions must be performed at 4°C to ensure minimal protein degradation.

      13. Wash an Econo column with RNase H1 lysis buffer and transfer the beads and crude extract into the column.

      14. Save the flowthrough (if all the RNase H1 protein did not bind to the beads, the flowthrough can be incubated with the Ni-NTA resin a second time).

      15. Wash 4 times with 15 ml RNase H1 wash buffer supplemented with protease inhibitors and collect each wash.

      16. Elute the protein 5 times with 3 ml RNase H1 elution buffer containing protease inhibitors; collect 1-ml fractions.

      17. Load 2.5 µl each fraction on a 10% SDS-PAGE gel and stain with SYPRO Ruby following the manufacturer’s instructions (Figure 3B).

      18. Pool the elutions with similar concentrations and dialyze them against 1 L chilled RNase H storage buffer.

      19. Equilibrate a HiLoad 20/60 Superdex 200 size exclusion column on an AKTA FPLC with 2-3 CV 20 mM Tris-HCl pH 7.0 containing 150 mM NaCl and 0.2 mM PMSF at 4°C (equilibration can be performed overnight).

      20. Concentrate the sample to ≤ 5 ml if necessary. Centrifuge at ≥ 10,000 × g for 10 min at 4°C to remove any precipitate and load on the Superdex 200 column.

      21. Run the Superdex 200 column at 4°C. Collect 1.5-ml fractions.

      22. Run 5 µl each fraction on a 10% SDS-PAGE gel and stain with SYPRO Ruby.

      23. Pool the fractions containing RNase H1 protein without degradation products and dialyze them twice O.N. at 4°C against 2 L chilled RNase H storage buffer.

      24. Concentrate the protein 2–3-fold with an Amicon ultra column 10,000 MCWO following the manufacturer’s instructions to obtain a protein concentration of 0.5–1 µg/µl.

      25. Aliquot, freeze in liquid nitrogen, and store at -80°C.

      26. Run 2.5 µl on a 10% SDS-PAGE gel and stain with SYPRO Ruby to confirm the quality of the preparation (Figure 3D).

      27. Determine the protein concentration on a NanoDrop using an extinction coefficient = 81,050 M-1cm-1 and a MW = 32 kDa. For 1 L of culture, ~9 mg purified RNase H1 is expected.



      Figure 3. Preparation of human RNase H1 and H2 proteins. A. Overview of RNase H1 and H2 expression and purification. B-C. SDS-PAGE gels of RNase H1 (B) and RNase H2, and (C) induction and purification on an Ni-NTA or GST column. D. SDS-PAGE gel of RNase H1 and H2 protein after purification and dialysis, stained with SYPRO Ruby. E-F. Agarose gel of transcribed DNA after incubation with RNase H1 (E) or RNase H2 (F), showing that both enzymes are active and degrade RNA–DNA hybrids.


    2. RNase H2 expression and purification (Figures 3A-3D)

      1. Transform E. coli Rosetta cells with pGEX6P1-hsRNASEH2BCA. Inoculate two 5-ml starter cultures of LB-ampicillin (100 ng/µl) with a single colony and grow O.N. at 37°C with shaking.

      2. The next day, inoculate 1 L LB-ampicillin with the 10-ml culture and allow to grow for 4 h at 37°C on a nutator.

      3. Allow the culture to sit for 1 h at RT to cool. Save 500 µl to run on an SDS-PAGE gel as the uninduced fraction.

      4. Induce with 0.1 mM IPTG O.N. at 20°C with shaking.

      5. Harvest the cells by centrifuging for 20 min at 15,000 × g, 4°C.

      6. Resuspend the pellet in 15 ml 1× PBS containing 0.2% Tween-20, 10 mM MgCl2, and protease inhibitors.

      7. Freeze in liquid nitrogen and thaw at 37°C (3 times) to facilitate cell lysis.

      8. Sonicate on ice 30 times for 30 s ON, 30 s OFF at an amplitude of 60%.

      9. Centrifuge for 20 min at 20,000 × g, 4°C to pellet the cell debris.

      10. Save the supernatant as the crude extract and run 10 µl on a 12% SDS-PAGE gel to verify the induction. The crude extract can be frozen in liquid nitrogen and stored at -80°C for future purification.

      11. Wash 1.5 ml glutathione superflow beads 3 times with 1× RNase H2 extraction buffer.

      12. Add the crude extract to the beads and incubate O.N. at 4°C on a nutator. All the remaining steps should be carried out at 4°C.

      13. Wash the beads twice with 10 ml 1× RNase H2 extraction buffer containing protease inhibitors and once with 4 ml 1× RNase H2 extraction buffer without protease inhibitors.

      14. Resuspend the beads in 2 ml 1× extraction buffer supplemented with 25 µg PreScission protease plus (we prepare our own PreScission; commercial enzyme could also be used).

      15. Incubate O.N. at 4°C on a nutator.

      16. Collect the flowthrough containing the RNase H2 released by PreScission cleavage.

      17. Repeat the incubation with 1× RNase H2 extraction buffer and PreScission protease.

      18. Analyze 10 µl input and washes, 1 µl each elution, and 10 µl beads on a 12% SDS-PAGE gel.

      19. Stain the gel with SYPRO Ruby following the manufacturer’s instructions (Figure 3C).

      20. Pool the elutions containing protein and concentrate them 5-fold with an Amicon ultra column 3,000 MCWO following the manufacturer’s instructions. The final concentration should be ~30 mg/ml in a volume of ~750 µl.

      21. Equilibrate a 24-ml Superdex 200 10/300 GL size exclusion column with 2 CV 20 mM Tris-HCl pH 7.0 containing 150 mM NaCl and 0.2 mM PMSF.

      22. Load the RNase H2 on the column and collect 500-µl fractions.

      23. Run 5 µl each fraction on a 12% SDS-PAGE gel and stain with SYPRO Ruby.

      24. Pool all the fractions containing the RNase H2 complex and dialyze them O.N. at 4°C against 1 L chilled RNase H storage buffer.

      25. Aliquot, freeze in liquid nitrogen, and store at -80°C.

      26. Run 0.1, 0.5, and 2.5 µl on a 12% SDS-PAGE gel next to a standard dilution of BSA and stain with SYPRO Ruby to determine the protein concentration (Figure 3D).

      27. For 2 L of culture, ~5 mg purified RNase H2 trimer is expected.

    3. Activity testing of RNase H1 and H2 (Figures 3E-3F)

      A plasmid containing a sequence prone to forming R-loops is transcribed and used as a template to measure the ability of RNase H1 or RNase H2 to degrade R-loops. We use a plasmid with the vg PRE cloned into pETBlue (Alecki et al., 2020).

      1. Assemble on ice (Table 2).


        Table 2. In vitro transcription assay
        Final concentration
        Volume (µl)
        Tris-HCl 1 M pH 8.0
        40 mM
        2
        MgCl2 100 mM
        8 mM
        4
        NaCl 1 M
        25 mM
        1.26
        Spermidine 100 mM
        2 mM
        1
        DTT 1M
        30 mM
        1.5
        ATP 1 mM
        40 nM
        2
        CTP 1 mM
        40 nM
        2
        GTP 1 mM
        40 nM
        2
        UTP 1 mM
        8 nM
        0.4
        UTP α-P32
        2.6 nM
        0.2
        DNA(vgcorePRE-pET) 100 ng/µl

        5
        T7 RNA polymerase (NEB)

        0.125
        H2O DNase/RNase-free

        30.3
        Final volume

        50

      2. Incubate for 30 min at 30°C.

      3. Stop the reaction by heating the sample for 10 min at 65°C.

        Titrate RNase H1 or RNase H2.

      4. Assemble on ice (Table 3).


        Table 3. Activity testing of purified RNase H1 or RNase H2
        Per reaction (µl)
        H2O DNase/RNase-free 5
        10× RNase H buffer 1
        Transcribed vgcorePRE-pET 3
        RNase H1 or RNase H2 1
        Final volume 10


        Prepare serial dilutions of RNase H1 (10-fold steps) or RNase H2 (5-fold steps) in storage buffer.

      5. Incubate for 1 h at 37°C.

      6. Add 1 µl 10× DNA loading buffer.

      7. Load on a 1% agarose 0.5× TBE gel.

      8. Stain with SYBRGold and image.

      9. Incubate the gel for 15 min in H2O.

      10. Incubate the gel for 20 min in 75 mM NaOH.

      11. Incubate the gel for 15 min in 0.5 M Tris-HCl 1.5 M NaCl.

      12. Incubate the gel for 30 min in 6× SSC.

      13. Transfer the gel O.N. to a HYBOND membrane (downward transfer) with the wick in 20× SSC.

      14. Expose the membrane to a phosphorimager screen.

      15. Scan on a Typhoon or equivalent phosphorimager (Figures 3E-3F).


  2. Embryo collection

    Note: This site (https://openspim.org/Drosophila_embryo_sample_preparation) provides a more detailed explanation of how to collect embryos.

    1. Transfer 5 bottles of flies into a cage containing a 10-cm apple juice plate smeared with yeast paste to allow the flies to lay eggs. To collect enough embryos, 2–4 cages are used.

    2. Allow the flies to lay eggs on the apple juice plates for 1 h before starting a timed collection.

    3. Change the apple juice plates and allow the flies to lay eggs for 4 h.

    4. Change the apple juice plates and allow the embryos to age in the incubator on the plate for 2 h or 10 h in order to collect 2-6 h or 10-14 h Drosophila embryos.

    5. Remove the yeast, add household bleach diluted 1:2 with H2O to the plate, and incubate the embryos for 2 min to dechorionate them.

    6. Transfer the embryos to a homemade sieve (we cut a large hole in the cap of a 50-ml tube and glue nylon mesh over it (a cell strainer (40-100 µm mesh size) can also be used) and wash with water.

    7. Dry excess liquid on paper towel and transfer the embryos to a pre-weighed 1.7-ml tube.

    8. Record the weight of the embryos, freeze in liquid nitrogen, and store at -80°C.


  3. Nucleic acid extraction from Drosophila melanogaster embryos

    Notes:

    1. To have enough material for one DRIP-seq experiment, start with 500 μl or 200 mg embryos

    2. Before they are washed with methanol, embryos stick to plastic. To minimize the loss of material, only glass vials and glass pipettes are used until the methanol wash; 8-ml glass scintillation vials work well for this step. For all steps involved in nucleic acid preparation, low-binding tubes and tips are used.

      1. Transfer 500 μl or 200 mg embryos to a glass vial.

      2. Wash embryos with 4 ml 1× PBS.

      3. Remove the PBS and wash the embryos with 4 ml 1× PBT.

      4. Transfer the embryos to a bottle containing 3 ml 1× PBS and 3 ml N-heptane.

      5. Shake for a few seconds to mix the 2 phases.

      6. Remove the lower phase (PBS) and leave the interphase intact.

      7. Add 3 ml methanol and shake vigorously for 1 min.

      8. Remove the top and interphase.

      9. Wash the embryos with 3 ml methanol.

      10. Transfer the embryos to a 15-ml tube and wash with 4 ml 1× PBS.

      11. Resuspend the embryos in 4 ml embryo lysis buffer and incubate for 2 h at 50°C. Every 15 min, invert the tube to mix.

      12. Centrifuge at 4,000 × g for 15 min. Transfer the supernatant to a 50-ml tube.

      13. Add 4 ml phenol/chloroform/isoamyl alcohol. Incubate for 1 h on a nutator at 4°C.

      14. Centrifuge at 4,000 × g for 15 min. Transfer the upper phase to a 50-ml tube.

      15. Add 4 ml phenol/chloroform/isoamyl alcohol. Incubate for 1 h on a nutator at 4°C.

      16. Centrifuge at 4,000 × g for 10 min. Transfer the upper phase to a clean 50-ml tube.

      17. Add 4 ml chloroform/isoamyl alcohol. Incubate for 1 h on a nutator at 4°C.

      18. Centrifuge at 4,000 × g for 10 min. Transfer the upper phase to a clean 50-ml tube.

      19. To precipitate the nucleic acids, add 200 μl 3 M KOAc pH 5.2 and 2.8 ml isopropanol. Incubate for 30 min on a nutator at 4°C.

      20. Gently transfer the white filaments to a 1.7-ml microfuge tube containing 1 ml 70% ethanol. Use a 1-ml pipette tip or cut the end off a 200-µl pipet tip to transfer the filaments without breaking.

      21. Wash 3 times with 1 ml 70% ethanol without centrifugation.

      22. Remove as much ethanol as possible.

      23. Centrifuge for 1 min at 1,000 × g and remove the ethanol. This step can be repeated to remove the residual ethanol.

      24. Air-dry the nucleic acid pellet for 1–4 h depending on the size of the pellet (until it becomes transparent).

      25. Resuspend in 1 ml TE O.N. at 4°C on a nutator. At this step, the nucleic acid is viscous. Nucleic acid can be stored for a few weeks at -20°C before nuclease digestion and purification for DRIP.


  4. Nucleic acid extraction from Drosophila S2 cells

    1. Drosophila S2 cells are grown at room temperature in Schneider’s media containing 10% FBS.

    2. Transfer 2 ×107 cells to a 50-ml tube.

    3. Pellet the cells at 500 × g for 5 min.

    4. Remove the supernatant and wash the cell pellet with 10 ml 1× PBS.

    5. Pellet the cells at 500 × g for 5 min.

    6. Resuspend the cells in 3 ml TE and transfer to three 1.7-ml tubes.

    7. Add 26 μl 20% SDS and 60 μg proteinase K to each tube. Mix gently by inverting the tube several times.

    8. Incubate O.N. at 37°C.

    9. Transfer to 2-ml phase lock tubes.

    10. Add 1 volume phenol/chloroform/isoamyl alcohol. Shake and centrifuge at 14,000 × g for 5 min.

    11. Transfer the supernatant to a 50-ml tube containing 2.4 volumes of 100% ethanol and a 1/10 volume of 3 M NaOAc pH 5.2.

      Note: It should be possible to substitute the KOAc used for Drosophila embryos for NaOAc.

    12. Invert the tube gently to precipitate the nucleic acids.

    13. Transfer the nucleic acids to a 1.7-ml tube containing 1 ml 70% ethanol.

    14. Wash 5 times with 1 ml 70% ethanol by removing as much ethanol as possible without centrifugation.

    15. Remove all the ethanol.

    16. Centrifuge for 1 min at 1,000 × g and remove all the ethanol. This step can be repeated to remove the residual ethanol.

    17. Air-dry the nucleic acid pellet for a few hours until the pellet becomes transparent.

    18. Resuspend in 1 ml TE O.N. at 4°C on a nutator. At this step, the nucleic acid solution is viscous. Nucleic acid can be stored for a few weeks at -20°C before nuclease digestion and purification for DRIP.


  5. Nuclease digestion and sonication of nucleic acids

    Note: All the measurements on the NanoDrop are carried out using dsDNA parameters. The samples collected to measure the concentration on the NanoDrop and run on a gel are: 1) gDNA prior and 2) after RNase A treatment; 3) after sonication; 4, 5) after RNase III ± RNase H treatment.

    1. Quantitate nucleic acids on a NanoDrop (concentration should be around 2 μg/μl and A260/A280 ≥ 2.0).

    2. Run 1 μl nucleic acids on a 1% agarose 1× TAE gel to verify that the gDNA is intact. Stain the gel with SYBR Gold. gDNA should be above 10 kb and a smear of RNA should be visible between 100 bp and 1.5 kb.

    3. RNAase A digestion: Incubate 250 mg nucleic acids in the presence of 0.5 M NaCl and 0.1 mg/ml RNase A in a final volume of 1.5 ml for 3 h at 37°C.

    4. Transfer to 2-ml phase lock tubes and add 1 volume phenol/chloroform/isoamyl alcohol.

    5. Mix vigorously and centrifuge at 14,000 × g for 5 min.

    6. Transfer 500 μl upper phase to a 1.7-ml tube containing 1 ml 100% ethanol and 50 μl 3 M NaOAc pH 5.2.

    7. Invert gently to precipitate the nucleic acids.

    8. Transfer the nucleic acids to a 1.7-ml tube containing 400 μl 70% ethanol.

    9. Remove all the ethanol.

    10. Dry the nucleic acids for 10–30 min depending on the size of the pellet.

    11. Resuspend in 500 μl TE by pipetting gently up and down and incubate on ice for at least 30 min.

    12. Measure the concentration of nucleic acids on a NanoDrop and adjust the volume to a concentration below 40 ng/μl (A260/A280 should be 1.8–2.0). Save 1 μl to run on an agarose gel to verify complete degradation of free RNA.

    13. Sonication using a Covaris E220: Split the sample into 130-µl aliquots (~5 µg) for sonication in a 130-µl Covaris microtube. Sonicate using the following parameters (Table 4):


      Table 4. Parameters for the sonication using a Covaris E220
      Peak incident power (W) 140
      Duty factor 10%
      Cycles per burst 200
      Treatment (s) 80


    14. Pool the sonicated nucleic acids.

    15. Save 1 μl to load on the gel to verify sonication. From a band above 10 kb before sonication, a smear should be observed with an average size of 300–400 bp after sonication.

    16. RNase H and RNase III digestion: Split the nucleic acids from step 14 into two aliquots. To both aliquots, add 100 µl 10× RNase H buffer and 2 units RNase III. Bring the volume to 1 ml. To one of the aliquots (RNase H-treated), add 10 μg RNase H1 and 10 μg RNase H2.

    17. Incubate both digests O.N. at 37°C.

    18. Transfer to 2-ml phase lock tubes and add 1 volume phenol/chloroform/isoamyl alcohol.

    19. Mix vigorously and centrifuge for 5 min at 14,000 × g.

    20. Transfer 500 μl upper phase to a 1.7-ml tube containing 1 ml 100% ethanol and 50 μl 3 M NaOAc pH 5.2.

    21. Invert gently to precipitate the nucleic acids.

    22. Centrifuge for 15 min at 16,000 × g.

    23. Remove the supernatant and wash the pellet with 400 μl 70% ethanol.

    24. Centrifuge for 5 min at 16,000 × g and remove all the ethanol.

    25. Allow the nucleic acid pellet to dry for approximately 10 min.

    26. Resuspend in 400 μl TE by gently pipetting up and down and incubate on ice for at least 30 min.

    27. Measure the concentration on a NanoDrop and save 1 μl to run on a gel. This measurement is used to calculate the volume needed for the 4.4 µg used for DRIP (DRIP Step F2a).

    28. Analyze the test aliquots from each step of the procedure (Steps E1-E5) on a 1% agarose 1× TAE gel and stain with SYBR Gold (Figure 4A). Nucleic acids can be stored for a few days at -20°C before DRIP.



      Figure 4. DRIP and nucleic acid preparation. A. Nucleic acid preparation from Drosophila melanogaster embryos. Total nucleic acids after extraction from embryos (left). Nucleic acids after RNase A digestion and before sonication (middle). Nucleic acids after sonication and RNase III +/- RNase H digestion (right). B. Input and elution from a DRIP experiment performed on 2-6 h and 10-14 h Drosophila melanogaster embryos. A faint smear is observed in the elution but not in the elution of the RNase H-treated sample.


  6. DRIP

    Notes:

    1. For DRIP, all the steps are carried out using low-retention tubes and pipette tips.

    2. For sequencing, we perform 3 DRIP experiments as described above in parallel and pool the elutions after the final purification.

    1. Bead preparation

      1. Wash 40 μl Protein G Dynabeads twice with 1× PBS containing 5 mg/ml BSA.

      2. Resuspend the beads in 2 volumes 1× PBS containing 5 mg/ml BSA and add 10 μg S9.6 antibody.

      3. Incubate O.N. at 4°C on a nutator.

      4. Wash the beads once with 1× PBS containing 5 mg/ml BSA.

      5. Keep the antibody-bound beads on ice or at 4°C.

    2. DRIP

      1. For each DRIP, dilute 4.4 μg previously purified DNA (Step E27) based on the NanoDrop measurement in 440 μl TE and add 50 μl 10× DRIP binding buffer.

      2. Save 50 μl for input and incubate the remaining sample with the S9.6-Dynabeads O.N. at 4°C with rotation.

      3. Capture the beads for 30 s using a magnetic rack, remove the supernatant, resuspend the beads in 700 μl 1× DRIP binding buffer, and incubate for 10 min at RT with rotation.

      4. Repeat the washes twice, for a total of 3 washes.

      5. Resuspend the beads in 250 μl DRIP elution buffer and 140 μg proteinase K.

      6. Incubate for 45 min at 50°C; invert the tube every 5 min.

      7. Collect the supernatant (IP/elution).

    1. Input and IP purification

      Note: The nucleic acids are purified on two successive columns, the first of which is used to eliminate SDS from the samples.

      1. The input and IP are purified on Macherey-Nagel (MN) PCR cleanup columns using NTB buffer following the kit instructions. The elution is performed with 50 μl MN elution buffer.

      2. The input and elution are purified on Zymoresearch DNA purification columns following the kit instructions. Elute the input and IP with 50 μl 10 mM Tris-HCl pH 8.0. For sequencing, elute the IP with 8.5 μl 10 mM Tris-HCl pH 8.0 and pool the 3 IPs together.

      3. Run 2 μl each sample on a 1.5% agarose 1× TAE gel and stain for at least 1 h with SYBR Gold. A smear should be visible in the IP but not in the RNase H-treated IP (Figure 4B). We store the samples overnight at -20°C and prepare the libraries the next day. It should also be possible to store the samples at -80°C for a few weeks until library preparation.


    1. Library preparation and qPCR
      1. Library preparation using an NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina®.

        Notes:

        1. The DNA moiety of the RNA–DNA hybrid is sequenced by starting with the second strand synthesis; the RNA moiety is removed in the second strand synthesis by RNase H digestion.

        2. The number of PCR cycles is determined following the manufacturer’s instructions based on the amount of nucleic acid (Step G1a).

          1. Estimate the amount of material in the IP and input on a bioanalyzer. The amount of nucleic acid should be above 1 ng to make the library (Figure 5A).



            Figure 5. DRIP library preparation . Input and elution from 10-14 h embryos before (A) and after (B) the library preparation. The elution is quantitated by a bioanalyzer. The same amount of input and RNase H elution is used to prepare the libraries. The entire immunoprecipitated sample of the RNase H-treated nucleic acid is used to prepare the library since we are unable to determine the amount of nucleic acid present.


          2. Adjust the amount of input to the IP. For the RNase H-treated IP, the amount of material recovered cannot be quantitated. We use the whole sample and are able to generate a high complexity library, although the number of reads is typically 2–5× lower.

          3. Prepare the library using NEB RNA Ultra II for Illumina, start at the second strand cDNA synthesis step (Table 5).


            Table 5. Modified second strand synthesis reaction
            Second strand synthesis reaction Volume
            Input and IP 20 μl
            NEBNext second strand synthesis reaction buffer with dUTP mix (10×) 8 μl
            NEBNext second strand synthesis enzyme mix 4 μl
            Random primer 1 μl
            RNase H (NEB), 1.6 U 0.3 μl
            Nuclease-free water 46.7 μl

          4. Assemble on ice and incubate for 1 h at 16°C.

          5. For the next steps, follow the kit instructions.

      2. Quality control of the library

        1. Using qPCR, verify enrichment of R-loops at 3 positive sites and their absence at 2 negative sites (Figure 6; primer sequences in Table 6).

          1. Dilute 1 μl each library in 10 μl 0.1× TE.

          2. Set up the qPCR plate with a standard curve using gDNA from S2 cells. This gDNA can be prepared as described in section D, digested with RNase A, and stored at -80°C. We prepare a dilution series in 10-fold steps from 25 to 0.025 ng/μl and use 2 μl each standard for qPCR with each primer set (standard curve is 50-5-0.5-0.05 ng total gDNA).

          3. qPCR reactions (5 μl total volume, run in a 384-well plate) consist of 2.5 μl Power Up SYBR Green master mix, 2 μl diluted library or standard genomic DNA from S2 cells, and 0.25 μl each primer diluted to 1 μM in water.

          4. qPCR is run on a Viaa7 instrument with 40 cycles, Tm = 60°C, and extension time = 1 min.



          Figure 6. qPCR after library preparation. 3 positive and 2 negative sites confirmed the DRIP and library preparation of the input, IP, and RNase H-treated IP by qPCR.


          Table 6. qPCR primers

          Primer name Sequence
          Hop F CTACAAGCAGGCGAAGGTTT R-loop (positive)
          Hop R CTTGATCTCAGGGGTGCGAT
          dek F GCGATGAGCCAGAAGATGAG R-loop (positive)
          dek R CTTGGACTCATCAGTGGCAT
          tRNA lys F GCCAAGCTCATTTTCTACGATCT R-loop (highly transcribed gene, positive)
          tRNA lys R GTCCGACAACGCCGATGATA
          Sdr F ACAGCTGATGTCGCTCACAT No R-loop (negative)
          Sdr R CGCTGAATGATCACCAGGTGA
          F20 F (CG12754) TCAAGCCGAACCCTCTAAAAT No R-loop (negative)
          F20 R AACGCCAACAAACAGAAAATG

        2. Using the bioanalyzer

          1. Run 1 μl each library on a bioanalyzer (Figure 5B).

          2. The average size of the fragments should be 400 bp.


      3. Sequence the library on a HiSeqTM sequencing system with a depth of 50 million reads per sample. Paired end sequencing was used here, with a read length of 50 bases.

    Data analysis

    The work-flow for DRIP-seq analysis as described in Alecki et al. (2020) is similar to standard ChIP-seq analysis. The basic work-flow is trimming of the adaptors, quality control of the FASTQ files, alignment to the genome, removal of PCR duplicates, and calling of the peaks. The protocol diverges from ChIP-seq in the steps of splitting data by strand before peak calling and calling peaks against both the input and the RNase H-treated samples. All the analysis steps can be carried out on a Galaxy (usegalaxy.org) or using simple bash scripts. This workflow identifies a stringent set of reproducible peaks that meet strict criteria for being R-loops (i.e., they are sensitive to RNase H).


    Notes:

    1. Others have described the use of a two-fold reduction in read counts in RNase H-treated samples to filter DRIP-seq peaks (Crossley et al., 2020) rather than comparing peaks called in DRIP versus RNase H-treated DRIP. This approach may be more flexible to accommodate sequencing experiments in which the RNase H digestion is suboptimal and should be considered, particularly if RNase H filtering removes a large number of peaks (>10%).

    2. We call peaks from both strands together or after separating the bam files into forward (F) and reverse (R) strands. The strand-specific analysis is more informative since it makes it possible to infer the orientation of transcription that produced the RNA. A fraction of peaks have an R-loop signal on both strands (~10% of peaks); we do not know whether these are technical artifacts (Alecki et al., 2020). Some, particularly in the case of embryo analysis, may be the result of mixed cell populations. It may be desirable to remove them for downstream analysis (Crossley et al., 2020), which can be done using bedtools to intersect the F and R strands.

    3. Crossley et al. (2020) recently described a synthetic spike-in strategy that can be used to normalize DRIP-seq data, allowing quantitative comparisons across conditions. This could be especially valuable when comparing Drosophila of different genotypes.


    1. Trim the adaptors and remove low-quality reads using Trimmomatic (Bolger et al., 2014) or fastp (v0.20.0) (Chen et al., 2018).

    2. Align the reads to the Drosophila genome using Bowtie2 (v 2.3.1) (Langmead and Salzberg, 2012) (--fr –no-mixed–no-unal).

    3. Use Samtools (v. 1.4.1) (Li et al., 2009) to convert the sam files generated by Bowtie2 to bam files, sort the bam files, and create a bam index for each file.

    4. Use Picard (http://broadinstitute.github.io/picard). Mark duplicates to filter PCR duplicates. Sambamba (v0.7.1) (Tarasov et al., 2015) can also be used for this step.

    5. Generate strand-specific bam files (for all samples: DRIP, RNase H DRIP, and Input) based on samflags using the samtools view as follows:

      Forward strand: samtools view –f 99; samtools view –f 147, followed by samtools merge.

      Reverse strand: samtools view –f 83; samtools view –f 163, followed by samtools merge.

      To carry out this procedure in Galaxy, use the samtools view and select “A filtered/subsampled selection of reads”; then set the correct combination of “Require that these flags are set.”

      For example, for the forward strand, first output the –f99 reads with these flags set:

      –read is paired –read is mapped in a proper pair =mate strand =read is the first in a pair;

      then output –f147 reads with these flags set;

      –read is paired –read is mapped in a proper pair –read strand =read is the second in a pair;

      samtools merge is used as above to merge f99 and f147 (F strand) and f83 and f163 (R strand).

    6. Use MACS2 (v2.1.1) (Zhang et al., 2008) to call peaks of DRIP versus Input using broad peak settings, with DRIP as the treatment and Input as the control. For example, for peak calling with paired-end bam files aligned to the dm6 version of the Drosophila genome:

      (macs2 callpeak –t DRIP.bam –c DRIP_input.bam –f BAMPE –g 1.4e+08 –n DRIP –outdir DRIP_peaks –broad).

    7. Use MACS2 to call peaks of DRIP versus RNase H-treated DRIP with broad peak settings, with DRIP as the treatment and RNase H-treated as the control.

    8. Use the BEDTools (Quinlan and Hall, 2010) intersect to retain peaks present in IP versus input and IP versus RNase H IP (bedtools intersect –a IP_versus_IN_peaks.bed –b IP_versus_RNaseHIP_peaks.bed –u >filtered_peaks.bed).

    9. Use the BEDtools intersect to retain only peaks present in both replicates.

    Notes

    1. This DRIP protocol consists of 4 steps, with the most critical being the preparation of the nucleic acids for immunoprecipitation. We encourage the users to perform DRIP-qPCR several times to ensure reproducibility before performing sequencing.

    2. Time-line (Table 7).


      Table 7. Time-line of DRIP-seq on Drosophila melanogaster embryos and tissue culture cells

      Day 1 Embryo collection Tissue culture cell lysis
      Day 2 Embryo lysis and nucleic acid extraction Nucleic acid extraction
      Day 3 Nucleic acid preparation I + bead preparation
      Day 4 Nucleic acid preparation II + immunoprecipitation
      Day 5 Bead washing + elution + purification
      Day 6 Library preparation and qPCR

    3. Embryo collection

      Note that embryos can be collected and stored at -80°C for a few months. This protocol can be performed in one week but it is also possible to stop at several steps. The nucleic acids can be stored at -20°C for a few days after extraction from tissue culture cells or embryos, after the preparation of the nucleic acids (before immunoprecipitation) and before the qPCR or library preparation.

    4. Nucleic acid extraction and preparation

      For DRIP protocols performed on protein-free nucleic acids after cell lysis, it is essential that the cell lysis and purification be performed gently to avoid breaking R-loops or creating new RNA–DNA hybrid by spurious annealing of RNA and complementary DNA. Thus, from extraction until sonication, do not centrifuge the nucleic acids at high speed. To resuspend the nucleic acids, do not vortex; instead pipette gently up and down.

    5. DRIP

      Nucleic acids from other species can be included before immunoprecipitation as a spike-in control to normalize the results between different samples or experimental conditions (Chen et al., 2016).

    Recipes

    1. Embryo lysis buffer

      50 mM Tris-HCl pH 8.0

      100 mM EDTA

      100 mM NaCl

      0.5% SDS

      5 mg/ml proteinase K

    2. 1× RNase H buffer

      50 mM Tris-HCl

      75 mM KCl

      3 mM MgCl2

      10 mM DTT

      pH 8.3

    3. 10× DRIP binding buffer

      100 mM NaPO4 pH 7.0

      1.4 mM NaCl

      0.5% Triton X-100

    4. 1× DRIP binding buffer

      10× DRIP binding buffer diluted in TE

    5. DRIP elution buffer

      50 mM Tris-HCl pH 8.0

      10 mM EDTA

      0.5% SDS

    6. TE

      10 mM Tris pH 8.0

      1 mM EDTA

    7. Apple juice plate

      3.5% agar

      4% sugar

      40% apple juice

      0.30% nipagin diluted in ethanol (2.5% of the final volume)

    8. RNase H2 extraction buffer

      1× PBS

      0.2% Tween-20

      10 mM MgCl2

    9. RNase H1 lysis buffer

      25 mM Tris-HCl

      300 mM NaCl

      5 mM imidazole

      pH 8.0 adjusted with NaOH

    10. RNase H1 wash buffer

      50 mM Tris-HCl

      300 mM NaCl

      20 mM imidazole

      pH 8.0 adjusted with NaOH

    11. RNase H1 elution buffer

      50 mM Tris-HCl

      300 mM NaCl

      250 mM imidazole

      pH 8.0 adjusted with NaOH

    12. RNase H size column buffer

      20 mM Tris-HCl pH 7.0

      150 mM NaCl

      Add 0.1 mM PMSF immediately before use

    13. RNase H storage buffer

      20 mM Tris-HCl pH 7.5

      50 mM NaCl

      1 mM DTT

      0.1 mM EDTA

      20% glycerol

      Add 0.1 mM PMSF immediately before use

    14. Protease inhibitors and additives (Table 8)

      Note: The buffers used to prepare RNase H1 and H2 proteins contain freshly added protease inhibitors, NP40, and DTT, except for the size column buffers, which have only PMSF and DTT added.


      Table 8. List of protease inhibitors and additives

      Final concentration
      TLCK
      13.5 µM
      Benzamidine
      100 µM
      Pepstatin
      3 µM
      Phenanthroline
      55 µM
      PMSF
      100 µM
      Aprotinin
      1.5 µM
      Leupeptin
      23 µM
      NP40
      0.05%
      DTT
      1 mM


    15. 0.5× TBE

      44.5 mM Tris

      44.5 mM Boric acid

      1 mM EDTA pH 8.0

    16. 10× DNA loading buffer

      20% glycerol

      0.1 M EDTA pH 8.0

      1% SDS

      0.25% Bromophenol Blue

      0.25% xylene cyanol

    17. SSC 20×

      3 M NaCl

      0.3 sodium citrate

      pH 7.0 adjusted with HCl

    18. 1× TAE

      40 mM Tris-HCl

      20 mM Acetate

      1 mM EDTA

    19. 1× PBS

      137 mM NaCl

      2.7 mM KCl

      10 mM Na2HPO4

      1.8 mM KH2HPO4

    20. 1× PBT

      1× PBS

      0.1% Triton X-100

    21. 3 M KOAc pH 5.2

      3 M potassium acetate

      pH 5.2 adjusted with glacial acetic acid

    22. 3 M NaOAc pH 5.2

      3 M sodium acetate

      pH 5.2 adjusted with glacial acetic acid

    23. SDS-PAGE running buffer

      25 mM Tris-HCl

      192 mM Glycine

      0.1% SDS

    24. Protein loading buffer

      6× SDS-sample buffer, added to samples at a final concentration of 1×

      0.35 M Tris, pH 6.8

      30% glycerol

      1% SDS

      0.0001% Bromophenol Blue

    Acknowledgments

    This protocol was adapted from Ginno et al. (2012) and published previously in Alecki et al. (2020). Work in the authors’ lab is funded by the Canadian Institutes for Health Research (CIHR).

    Competing interests

    The authors declare no competing interests.

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简介

[摘要]R-环是由RNA-DNA杂交和取代的单链DNA组成的非标准核酸结构。最初被认为是基因组不稳定性的一个来源,在过去的十年中,R-环被证明参与了蛋白质的靶向性,并与不同的组蛋白修饰相关,表明其具有调节功能。此外,R-环在植物不同组织发育过程中形成差异,并与哺乳动物疾病有关。在这里,我们提供了一个单链drop-seq方法来鉴定果蝇胚胎和组织培养细胞中的R-环形成序列。此协议在分段步骤中不同于早期的滴滴协议。与限制性内切酶不同的是,超声作用产生了一个均匀的、高度可重复的核酸片段库。此外,它允许在任何有机体中以最小的优化使用该协议。该协议集成了几个步骤,从已发表的协议,以确定R-环形成序列具有高严格性,适合从头表征。



图形摘要:





图1。链特定的drop-seq协议概述


【背景】概述
R环是RNA与互补单链DNA杂交时形成的三链核酸结构,导致第二条DNA链的位移。R环最初被描述为转录的副产物和需要解决的基因组不稳定性的来源。然而,过去十年的研究表明,R-环可与特定组蛋白修饰和转录状态相关,诱导蛋白质靶向,或作为启动子(Chedin和Benham,2020;尼赫斯和卢克,2020)。为了研究R-环功能,已经开发了在多种生物中绘制这些结构的协议。物种间的差异已被观察到:哺乳动物的Rloop形成序列具有GC偏斜;那些在酿酒酵母有一个在倾斜;在植物中,GC和AT偏斜都有报道(Sanz等人,2016;Wahba等人,2016年;徐等,2017;Hartono等人,2018年)。物种间R-loop形成序列的性质和定位的差异以及R-loop与不同疾病和基因错误表达的关联,突出了开发标准R-loop作图协议以进行严格比较的重要性。

植物中的R-环图谱表明,一些R-环在发育过程中可以形成差异

(Fang等人,2019年;Xu等人,2020),可能参与转录状态的改变。在哺乳动物中,活性增强子与R-环的形成有关,这些R-环可以作为启动子(Tan Wong等人,2019)。利用现有的遗传工具,在完整生物体发育的背景下研究R环,以评估其形成、分辨率和功能,将有助于更好地理解R环生物学。

在这里,我们提供了一个详细的链特异性drop-seq方案,用于识别果蝇胚胎和组织培养细胞中的R-环形成序列(图1)。与其他滴灌方案类似,我们的方案依赖S9.6抗体来识别R-环形成序列,但也认为S9.6抗体对dsRNA的弱亲和力会产生假阳性信号(Phillips等人,2013;Hartono等人,2018年;K公司ö尼格等人,2017年)。这种单链drop-seq方案与可用于果蝇的遗传工具相结合,应该是研究整个有机体、发育过程中以及不同组织中R-loop功能的强大系统(图2,表1)。DRIPseq可以与基因操作相结合来过度表达或敲除基因,以确定它们对R-环形成的影响,或者与细胞同步来跟踪细胞周期中R-环的形成。最后,使用超声波而不是限制性内切酶来切割基因组

DNA意味着这一方案可能以最小的优化适应其他生物体。



局限性
该方案依赖于S9.6抗体来免疫沉淀RNA-DNA杂交包含片段。这种抗体并不是一个完美的工具:它显示了基于Rloop序列的差异结合亲和力,与GC含量无关,并且偏好较长的RNA-DNA杂交。它可以结合dsRNA,尽管其亲和力比RNA-DNA杂交的亲和力低5倍(Phillips等人,2013;K公司ö尼格等人,2017年)。后两个限制可以在制备核酸的过程中克服。核酸提取后,通过在0.5 M NaCl存在下将核酸与RNase A孵育来去除ssRNA和dsRNA,以避免RNA-DNA杂交中的RNA降解。第一个消化步骤之后是与RNase III孵育,以降解任何未被RNase A降解的dsRNA。通过在免疫沉淀前使用两种不同的核糖核酸酶,我们去除了所有的ssRNA和dsRNA,还去除了一些可能对这些核糖核酸酶敏感的R-环。因此,这些步骤使实验更加严格,并限制假阳性,但可以进行修改,以平衡严格性和敏感性,因为一些R-环可能对这些治疗敏感。当R-环的RNA组分被测序时,去除能与S9.6相互作用的RNA是最相关的;尽管如此,我们观察到当使用严格的核糖核酸酶处理时,RNA-DNA杂交的下拉效率会增加。同样重要的是要有一个RNase H处理的阴性对照来评估下拉的特异性(图3)。我们发现市面上可以买到的E。大肠杆菌核糖核酸酶H不能完全消化核酸制剂中的RNA-DNA杂交。这可以通过生产和使用高活性的人核糖核酸酶H1和核糖核酸酶H2来解决。这种负控制在滴灌协议的优化过程中尤为重要;它确保了技术的特殊性。它还用于在执行drop seq时调用peaks(图2、4和5)。

为了避免在免疫沉淀步骤中偏向较长的片段,我们通过超声波将核酸片段化(图4A)。超声处理使我们能够获得平均大小为300 bp的同质碎片群。超声波是在核糖核酸酶A处理后进行的,以避免在滴注过程中产生新的RNA-DNA杂交体的可能性。据报道,与限制性消化相比,超声波处理会降低一些RNA-DNA杂交的回收率(Crossley等人,2020)。



优势
与其他滴灌协议相比的优势

该方案使用温和的裂解步骤提取核酸。细胞裂解可以在组织培养细胞、整个胚胎或来自幼虫、蛹或成体果蝇的解剖组织上进行。尽管进行一次滴灌seq实验所需的约200mg果蝇胚胎相对较高,但冷冻组织并将其汇集用于单个核酸提取的可能性使其成为可能。通过解剖黑腹果蝇并对盘、器官或分类细胞群进行滴注,应该有可能识别细胞或组织特异性R环。

与针对哺乳动物细胞开发的几种方案(例如,Sanz等人,2016年)相反,这种滴注方案不使用限制性酶混合物来分割核酸。相反,使用超声波,这导致平均大小为300 bp的核酸碎裂(图4A)。这些碎片具有均匀的大小和超声波是高度可重复性。与限制性消化相比,在哺乳动物细胞中使用超声波可提高分辨率(Crossley et al.,2020)。通过使用超声波,该协议可以很容易地适应其他生物与小优化;唯一可能需要优化的步骤是分解。超声波还有另一个优势:它会导致R环移位的单链DNA断裂,这使得使用RNA-DNA杂交的DNA或RNA部分制备链特异性测序文库成为可能(Wahba et al.,2016)。



与其他方法相比的优点

另一种识别R-环形成序列的方法依赖于RNase H1酶(dRNase H1)的催化非活性形式(Ginno等人,2012;陈等人,2017)。dRNase H1在细胞中的应用提出了几个潜在的问题,这可能解释了该方法与S9.6抗体在识别R-环形成序列方面的差异。首先,RNase H1不是细胞中Rloops的唯一调节因子;有人认为拓扑异构酶是负责在人类细胞中形成共转录R-环的主要酶(Manzo等人,2018;张等。,

而核糖核酸酶H1和H2在R环形成后就以它们为靶点。其次,Lockhart et al.,2019最近的一篇文章证明,RNase H1在应激时被激活,而RNase H2在S。酿酒酵母。第三,利用dRNase H1,R-环的鉴定可能局限于R-环,R-环是可接近的,无蛋白质的,并且通常被RNase H酶降解。第四,dRNase H1的表达可以稳定R环。虽然这可能允许检测瞬态R-环,但也可能会扭曲对稳定R-环存在的位置的解释。最后,

RNase H1有两个RNA–DNA杂交结合域,两者都需要结合以诱导杂交RNA部分的降解(Nowotny et al.,2008)。这可能会阻止或限制检测到较小的

RNA-DNA杂交。因此,虽然dRNase H1在某些情况下可能是一个有用的工具,但对滴灌结果的解释可能更直接。比较两种方法的结果也可以得到互补信息。

与滴灌法相似,从组织培养细胞中提取核酸后,用dRNase H1分离RNA-DNA杂交体;然而,与S9.6抗体相比,这种酶倾向于较长的杂交体,并且其亲和力较弱,使得其效率较低(Ginno等人,2012年)。

最后,天然亚硫酸氢盐测序是识别R-环的另一种高分辨率方法。亚硫酸氢盐将单链DNA中的胞嘧啶转化为尿嘧啶(Yu等人,2003年);因此,这种方法不检测RNA-DNA杂交,而是检测它们形成时移位的单链DNA链。然而,甲基胞嘧啶在基因组中的存在阻止了单链DNA的修饰,并且单链DNA可以被其他非规范DNA结构如G-四链体和I-基序的形成所取代,这可能分别导致假阴性或假阳性结果。

本文提出的单链drop-seq方法已经被优化,可以在果蝇胚胎和组织培养细胞中识别R-环形成序列,并且可以在其他生物中使用最小的优化。该方案可以作为一种标准化的方法来评估不同发育阶段或不同组织或生物体中的Rloops。





图2。S2细胞和早期和晚期胚胎之间共享和不同R环的例子。A-B。R环在S2细胞和2-6小时和10-14小时胚胎的CG1513、CG3008和CG6040基因上形成(红色箭头)。在CG12923基因上没有形成R环。C。在alt基因上,2-6h可检测到3个R环,而10-14h胚胎中未检测到。其中两个R-环(红色箭头)也存在于S2细胞中,而其中一个被相反链上的R-环所取代(紫色箭头)。D。在Spn1上形成的R环只存在于2-6小时的胚胎中(紫色箭头)。E。Rloops在2-6h胚胎的+链上形成,在10-14h胚胎的-链上形成

S2细胞。”Unstr”对应于非标drop-seq数据,“+”和“-”表示链特异性轨道,RNase H处理的样品作为阴性对照。红色箭头表示转录本的方向。请注意,“+”和“-”链指的是DNA链,因此“+”链R-环应该从向左方向的转录中产生。因此,在A中,CG3008基因中的R-环在预期的方向上来自于基因转录,而CG1513基因中的R-环则来自于反义转录。



表1。数据可用性


存放的数据(摘自Alecki等人,2020)

滴水序列2-6H俄勒冈州R胚胎GSE127329

滴水序列10-14H俄勒冈州R胚胎GSE127329

滴水序列S2电池GSE127329

关键字:DRIP, R-环, S9.6抗体, 果蝇, 核糖核酸-脱氧核糖核酸杂交体, 组织培养细胞




 

材料和试剂
 

答。rna酶H1和H2的表达与纯化

1.    阿米康® 超离心过滤器,0.5 ml 10K(EMD密理博,目录号:UFC5010BK)

2.    阿米康® 超离心过滤器,0.5 ml 3K(EMD密理博,目录号:UFC5003BK)

3.    Econo柱(2.5*20 cm)(Bio-Rad,目录号:7374252)

4.    E。大肠杆菌RosettaTM 2(DE3)pLysS SinglesTM感受态细胞–Novagen(Sigma,目录号:71401)

5.    Ni NTA琼脂糖(Qiagen,目录号:1018244)

6.    谷胱甘肽超流树脂(Takara,目录号:635607)

7.    预分解蛋白酶plus(自制;也可使用商业酶)

8.    氨苄西林(Bioshop,目录号:AMP201.100)

9.    LB肉汤磨坊(Bioshop,目录号:LBL407.500)

10.  白蛋白,牛血清(Bioshop,目录号:ALB001.100)

11.  溶菌酶(Bioshop,目录号:LYS702.5)

12.  咪唑(OmniPur)(密理博,目录号:5710-OP)

13.  甘油(Bioshop,目录号:GLY001.4)

14.  IPTG(Bioshop,目录号:IPT002.5)

15.  核糖核酸酶H2提取缓冲液(见配方)

16.  核糖核酸酶H1裂解缓冲液(见配方)

17.  核糖核酸酶H1洗涤缓冲液(见配方)

18.  核糖核酸酶H1洗脱缓冲液(见配方)

19.  RNase H大小列缓冲区(见配方)

20.  核糖核酸酶H储存缓冲液(见配方)

21.  蛋白酶抑制剂(见配方)

 

B。蛋白酶抑制剂和添加剂

1.    TLCK(西格玛,目录号:T7254)

2.    苯甲脒(Bioshop,目录号:BEN601.25)

3.    胃蛋白酶抑制剂A(Bioshop,目录号:PEP605.25)

4.    1,10-菲咯啉(Sigma,目录号:131377-5G)

5.    PMSF(Fisher Scientific,目录号:19538125)

6.    抑肽酶(Bioshop,目录号:APR200)

7.    亮肽(Bioshop,目录号:LEU011.50)

8.    NP40(Nonidet P40替代品,Fluka目录号:74385)(可替代Sigma,目录号:74385)

9.    DTT(Bioshop,目录号:DTT002.100)

 

C。核糖核酸酶H1和H2活性测定

1.    rNTP(NEB,目录号:N0450S)

2.    UTP公司α-P32(Perkin Elmer,目录号:BLU007H250UC)

3.    T7 RNA聚合酶(NEB,目录号:M0251L)

4.    子系统控制器20× ((见食谱)

5.    柠檬酸三钠(Bioshop,目录号:CIT001.205)

 

D。胚胎采集果蝇

1.    飞瓶

2.    俄勒冈州R苍蝇(DrÉ里克洛écuyer实验室;可通过布鲁明顿果蝇库存中心购买)

3.    琼脂A(Bioshop,目录号:FB0010)

4.    糖(红道)

5.    苹果汁

6.    替戈塞普,1 KG(尼泊金)(Diamed.ca,目录号:GEN20-258)

7.    自制NITEX筛子(可从https://flystuff.com/)

8.    漏斗

9.    苍蝇笼(苍蝇笼、食物和瓶子可从https://flystuff.com/)

10.  苍蝇食品(内部准备)

11.  甲醇(Bioshop,目录号:MET302.4)

12.  胚胎裂解缓冲液(见配方)

13.  苹果汁盘(见食谱)

14.  1× PBS(见配方)

15.  1× PBT(见配方)

 

E。细胞培养

1.    S2细胞(Invitrogen,目录号:R69007)

2.    GibcoTM-Schneider果蝇无菌培养基(Thermo Fisher Scientific,目录号:21720-024)

3.    FBS(赛默飞世尔科技,目录号:16140-089)

 

F。核酸提取与制备

1.    蛋白酶K(Biobasic,目录号:PB0451)

2.    重型锁相凝胶(VWR,目录号:10847-802)

3.    脱氧核糖核酸酶I(无核糖核酸酶)(NEB,目录号:M0303L)

4.    AmbionTM RNase III(赛默飞世尔科学公司,目录号:AM2290)

5.    核糖核酸酶A(Qiagen,目录号:19101)

6.    UltraPureTM DNase/RNase游离蒸馏水(赛默飞世尔科技,目录号:10977015)

7.    Covaris microTUBE AFA光纤预裂扣盖6*16 mm(Covaris,目录号:520045)

8.    苯酚氯仿异戊醇(Bioshop,目录号:PHE512.400)

9.    氯仿(Bioshop,目录号:CCL402.1)

10.  试剂酒精(Sigma,目录号:277649-1)

11.  Tris(Bioshop,目录号:TRS001.10)

12.  冰醋酸(Thermo Fisher Scientific,目录号:351271-212)

13.  EDTA(Bioshop,目录号:EDT002.500)

14.  醋酸钠(Bioshop,目录号:SAA304.5)

15.  氯化钠(Bioshop,目录号:SOD002.10)

16.  1× 核糖核酸酶H缓冲液(见配方)

17.  TE(见配方)

 

G。滴水

1.    抗DNA–RNA杂交[S9.6]抗体(Kerafast,目录号:ENH002,杂交瘤可通过ATCC获得,HB-8730)

2.    免疫沉淀用DynabeadTM蛋白G(赛默飞世尔科学公司,目录号:10004D)

3.    核自旋® 凝胶和PCR(Macherey Nagel,目录号:740609.250)

4.    DNA清洁与浓缩TM(Zymo Research,目录号:D4014)

5.    BSA,分子生物学级(NEB,目录号:B9000S)

6.    10× 滴胶缓冲液(见配方)

7.    1× 滴胶缓冲液(见配方)

8.    滴洗液缓冲液(见配方)

 

H。库准备和qPCR

1.    下一个® Illumina的复合寡糖® (索引引物组1)(NEB,目录号:E7735S)

2.    下一个® 用于Illumina的UltraTM II定向RNA文库准备工具包® (NEB,目录号:E7760S)

3.    RNase H(新英格兰生物实验室(NEB),目录号:M0297L)

4.    通电SYBR绿色PCR主混合物(赛默飞世尔科技,目录号:A25741)

 

一。琼脂糖和SDS-PAGE凝胶的制备和染色

1.    SYBRTM金核酸凝胶染色剂(赛默飞世尔科技,目录号:S11494)

2.    SYPRO Ruby解决方案(Lonza,目录号:50562)

3.    甘氨酸(Bioshop,目录号:GLN001.10)

4.    0.5× TBE(见配方)

5.    10× DNA加载缓冲液(见配方)

6.    SDS-PAGE缓冲液(见配方)

7.    蛋白质装载缓冲液(见配方)

 

J。质粒

1.    pHis-MBP-hRNaseH1(Alecki等人,2020年)

2.    pGEX6P1-hsRNASEH2BCA(Addgene,目录号:108693)3。vg pETBlue(Alecki等人,2020年)

 

设备
 

1.     用于凝胶内荧光成像和磷光成像的Typhone Imager(GE Healthcare)

2.     用于实时PCR的Viaa7 PCR系统(Thermo Fisher Scientific)

3.     Covaris E220(Covaris)用于核酸超声

4.     离心机Avanti® J-E(贝克曼库尔特)

5.     AKTA FPLC(或等效色谱系统)

6.     HiLoad 20/60 Superdex 200列(GE Healthcare,目录号:GE28-9893-36)(可从Sigma获得)

7.     Superdex 200 10/300 GL尺寸排除列(GE Healthcare,目录号:28990944)

8.     超声波仪(Sonics,Vibra cellsTM)(细胞裂解探头超声波仪)

9.     Nutator旋转平台,轻轻混合管

10.  细菌振动器

11.  10cm培养皿

12.  速冻用液氮

13.  -80°冷冻机

14.  纳米滴分光光度计

15.  水浴

16.  24°C培养箱

17.  27°C S2细胞摇瓶培养箱;S2细胞也可以在室温下在平板上生长

 

程序
 

答。rna酶H1和H2的表达与纯化

1.     核糖核酸酶H1的表达和纯化(图3A-3D)

答。转换E。大肠杆菌Rosetta细胞与pHis-MBP-hRNaseH1。第二天,接种2毫升乙氨苄西林(100纳克)/µl) 只有一个菌落,让它在37岁时正常生长°在摇动的培养箱里。

b。将(a)中的1 ml培养物添加到200 ml LB氨苄西林中,并使其在37℃O.N.下生长°在摇动的培养箱里。

c。将(b)中的100 ml培养物添加到1 L LB氨苄西林中,并在37℃下用0.1 mM IPTG诱导RNase H1表达4 h°C。

d。在4000℃下离心20分钟收获细胞× g、 4个°C。

e。丢弃上清液,在液氮中冷冻细胞颗粒,并在-80℃下储存至少几个小时°C。

f。在冰上解冻细胞颗粒,并添加含有0.1 mg/ml溶菌酶的20 ml RNase H1裂解缓冲液,100µ核糖核酸酶A,100µg DNA酶I和蛋白酶抑制剂。

g。在冰上孵育30分钟。

h。将细胞置于冰上,以60%的振幅超声12次,打开15秒,关闭15秒。

一。在20000℃下离心20分钟× g、 4个°C。

j。保留上清液作为粗提物。上清液可在液氮中冷冻并在-80℃下储存°用于将来的净化。

k。在50 ml试管中用15 ml RNase H1裂解缓冲液在207℃下离心2 min,洗涤3 ml Ni-NTA珠3次× g。

l。将粗提物添加到珠子中,并在4℃培养°在一个螺母上。从这个步骤开始,所有的洗涤和洗脱必须在4点钟进行°以确保最低限度的蛋白质降解。

m。用核糖核酸酶H1裂解缓冲液洗涤Econo柱,并将珠子和粗提物转移到柱中。

n。保存流通(如果所有的核糖核酸酶H1蛋白没有结合到珠,流通可以与Ni-NTA树脂第二次孵育)。

o。用补充有蛋白酶抑制剂的15毫升核糖核酸酶H1洗涤缓冲液洗涤4次,收集每次洗涤液。

第。用含蛋白酶抑制剂的3ml核糖核酸酶H1洗脱缓冲液洗脱蛋白5次;收集1毫升的部分。

问。荷载2.5µl在10%SDS-PAGE凝胶上,按照制造商的说明用SYPRO Ruby染色(图3B)。

r。将具有类似浓度的洗脱液汇集并用1L冷冻RNase H储存缓冲液透析。

s。在AKTA FPLC上平衡HiLoad 20/60 Superdex 200尺寸排除柱,23 CV 20 mM Tris-HCl pH 7.0,含150 mM NaCl和0.2 mM PMSF,4°C级

(平衡可在夜间进行)。

t。将样品浓缩至≤ 如有必要,5毫升。离心机≥ 10,000 × g,4分钟时持续10分钟°C去除任何沉淀物并在Superdex 200柱上加载。

美国。在4处运行Superdex 200列°C。收集1.5毫升部分。

五。运行5µl在10%SDS-PAGE凝胶上,用SYPRO Ruby染色。

w。将不含降解产物的含有RNase H1蛋白的部分汇集在一起,并在4℃O.N.透析两次°C对2L冷冻RNase H储存缓冲液。

十。按照制造商的说明,用Amicon ultra柱10000 MCWO浓缩蛋白质2-3倍,以获得0.5-1的蛋白质浓度µ克/µl。

是的。等分,在液氮中冷冻,在-80℃下保存°C。

z。运行2.5µl在10%SDS-PAGE凝胶上,用SYPRO Ruby染色,以确认制剂的质量(图3D)。

aa级。使用消光系数=81050 M-1cm-1和MW=32 kDa测定纳米滴上的蛋白质浓度。对于1L培养物,预计约9mg纯化RNase H1。

 

 

 

图3。人核糖核酸酶H1和H2蛋白的制备。答。核糖核酸酶H1和H2的表达与纯化综述。B-C。核糖核酸酶H1(B)和核糖核酸酶H2的SDS-PAGE凝胶,以及(C)在Ni-NTA或GST柱上的诱导和纯化。D。纯化透析后RNA酶H1和H2蛋白SDS-PAGE凝胶,SYPRO-Ruby染色。E-F。与核糖核酸酶H1(E)或核糖核酸酶H2(F)孵育后转录的DNA的琼脂糖凝胶,表明这两种酶都是活性的并降解RNA-DNA杂交。

 

2.     核糖核酸酶H2的表达和纯化(图3A-3D)

答。转换E。大肠杆菌Rosetta细胞与pGEX6P1-hRNASeh2BCA。接种两个5毫升的LB氨苄青霉素发酵剂(100 ng/µl) 只有一个菌落,37岁时生长正常°他颤抖着。

b。第二天,用10mL培养液接种1L-LB氨苄西林,在37℃下培养4h°在一个螺母上。

c。让培养物在室温下静置1小时以冷却。节省500µ在SDS-PAGE凝胶上作为未导入部分。

d。用0.1 mM IPTG O.N.在20°他颤抖着。

e。在15000℃下离心20分钟收获细胞× g、 4个°C。

f。将小球重新悬浮在15 ml 1× 含有0.2%吐温-20、10 mM MgCl2和蛋白酶抑制剂的PBS。

g。在液氮中冷冻,37℃解冻°C(3次)促进细胞溶解。

h。在冰上超声30次,打开30秒,关闭30秒,振幅为60%。

一。在20000℃下离心20分钟× g、 4个°C将细胞碎片制成颗粒。

j。将上清液作为粗提物保存并运行10µl在12%SDS-PAGE凝胶上验证诱导。粗提物可在液氮中冷冻并在-80℃下储存°用于将来的净化。

k。用1.5毫升谷胱甘肽超流珠洗3次× 核糖核酸酶H2提取缓冲液。

l。将粗提物添加到珠子中,并在4℃培养°在一个螺母上。其余步骤应在4点执行°C。

m。用10毫升1洗两次珠子× 核糖核酸酶H2提取缓冲液,含蛋白酶抑制剂和一次4毫升1× 不含蛋白酶抑制剂的核糖核酸酶H2提取缓冲液。

n。将珠子重新悬浮在2 ml 1× 萃取缓冲液补充25µg预分解蛋白酶plus(我们自己准备预分解;也可使用商业酶)。

o。在4℃培养°在一个螺母上。

第。收集含有分裂前裂解释放的核糖核酸酶H2的流通液。

问。用1重复孵育× 核糖核酸酶H2提取缓冲液和预分解蛋白酶。

r。分析10µl输入和清洗,1µ每次洗脱,10µ12%SDS-PAGE凝胶上的l珠。

s。按照制造商的说明用SYPRO Ruby染色凝胶(图3C)。

t。将含有蛋白质的洗脱液汇集在一起,按照制造商的说明,用Amicon ultra柱3000 MCWO将其浓缩5倍。最终浓度应为~30 mg/ml,体积为~750µl。

美国。用含有150 mM NaCl和0.2 mM PMSF的2 CV 20 mM TrisHCl pH 7.0平衡24 ml Superdex 200 10/300 GL尺寸排除柱。

五。将核糖核酸酶H2加载到柱上并收集500-µl分数。

w。运行5µl在12%SDS-PAGE凝胶上,用SYPRO Ruby染色。

十。将含有核糖核酸酶H2复合物的所有组分汇集在一起,并在4℃O.N.透析°C对1L冷冻RNase H储存缓冲液。

是的。等分,在液氮中冷冻,在-80℃下保存°C。

z。运行0.1、0.5和2.5µl在12%SDS-PAGE凝胶上,靠近标准稀释的BSA,并用SYPRO Ruby染色以测定蛋白质浓度(图3D)。

aa级。对于2L培养物,预计约5mg纯化RNase H2三聚体。

3.     核糖核酸酶H1和H2的活性测试(图3E-3F)

含有易于形成R-环的序列的质粒被转录并用作测量RNase H1或RNase H2降解R-环的能力的模板。我们使用一个质粒将vg预克隆到pETBlue中(Alecki等人,2020)。答。在冰上组装(表2)。

 

表2。体外转录分析
 

最终浓度

体积(µ(左)

Tris HCl 1 M pH 8.0

40毫米

2

氯化镁100毫米

8毫米

4

氯化钠1 M

25毫米

1.26

亚精胺100 mM

2毫米

1

DTT 1米

30毫米

1.5

ATP 1毫米

40牛米

2

CTP 1毫米

40牛米

2

GTP 1毫米

40牛米

2

UTP 1毫米

8牛米

0.4

UTP公司α-第32页

2.6牛米

0.2

DNA(vgcorePRE-pET)100纳克/µ我

 

5

T7 RNA聚合酶(NEB)

 

0.125

不含H2O-DNase/RNase

 

30.3

最终卷

 

50

 

b。30℃孵育30分钟°C。

c。在65℃加热样品10分钟,停止反应°C。

滴定核糖核酸酶H1或核糖核酸酶H2。

d。在冰上组装(表3)。

 

表3。纯化rna酶H1或rna酶H2的活性检测
 

每反应(µ(左)

不含H2O-DNase/RNase

5

10× 核糖核酸酶H缓冲液

1

转录vgcorePRE-pET

3

核糖核酸酶H1或核糖核酸酶H2

1

最终卷

10

 

在储存缓冲液中制备RNase H1(10倍步骤)或RNase H2(5倍步骤)的连续稀释液。

e。37℃孵育1小时°C。

f。添加1µ长10× DNA装载缓冲液。

g。装入1%琼脂糖0.5× TBE凝胶。

h。用SYBRGold和image染色。

一。将凝胶在H2O中培养15分钟。

j。在75 mM NaOH中培养凝胶20分钟。

k。在0.5 M Tris-HCl 1.5 M NaCl中培养凝胶15分钟。

l。将凝胶在6分钟内孵育30分钟× 子系统控制器。

m。将凝胶O.N.转移到HYBOND膜上(向下转移),芯线为20× 子系统控制器。

n。将膜暴露在磷光成像仪屏幕上。

o。在台风或等效的磷光成像仪上扫描(图3E-3F)。

 

B。胚胎采集

注意:此站点(https://openspim.org/Drosophila_embryo_sample_preparation)提供了一个更详细的解释如何收集胚胎。

1.     把5瓶苍蝇转移到一个笼子里,笼子里有一个10厘米长的苹果汁盘子,上面涂上酵母膏,让苍蝇产卵。为了收集足够的胚胎,使用2-4个笼子。

2.     让苍蝇在苹果汁盘上产卵1小时,然后开始定时收集。

3.     更换苹果汁盘子,让苍蝇产卵4小时。

4.     更换苹果汁平板,让胚胎在平板上的培养箱中老化2小时或10小时,以收集2-6小时或10-14小时的果蝇胚胎。

5.     取出酵母,加入用H2O稀释1:2的家用漂白剂,将胚胎孵育2分钟,使其脱胚。

6.     将胚胎移植到自制的筛子上(我们在50毫升试管的盖子上切一个大洞,在上面粘上尼龙网(细胞过滤器(40-100))µm筛孔大小)也可使用)并用水冲洗。

7.     用纸巾擦干多余的液体,并将胚胎转移到预先称重的1.7毫升试管中。

8.     记录胚胎的重量,在液氮中冷冻,在-80℃下保存°C。

 

C。从果蝇胚胎中提取核酸注释:

答。为了有足够的材料来做一个滴滴顺序实验,从500开始μl或200mg胚胎

b。在用甲醇清洗胚胎之前,胚胎会粘在塑料上。为了尽量减少材料的损失,在甲醇洗涤之前,只使用玻璃瓶和玻璃移液管;8毫升的玻璃闪烁小瓶在这一步效果很好。在核酸制备的所有步骤中,都使用低结合管和尖端。

1.     转移500μ1或200毫克胚胎放入玻璃瓶中。

2.     用4毫升1.5毫升的水冲洗胚胎× 公共广播公司。

3.     取出PBS,用4毫升1× PBT公司。

4.     将胚胎移植到一个装有3毫升的瓶子里× PBS和3毫升正庚烷。

5.     摇动几秒钟以混合这两个阶段。

6.     去除下相(PBS),保持相间完整。

7.     加入3毫升甲醇,剧烈摇晃1分钟。

8.     拆下顶部和中间相。

9.     用3毫升甲醇清洗胚胎。

10.  将胚胎移植到15毫升试管中,用4毫升1× 公共广播公司。

11.  再将胚胎置于4ml胚胎裂解缓冲液中,于50℃孵育2h°C。每隔15分钟,将试管倒置混合。

12.  离心机转速4000× g保持15分钟。将上清液转移到50 ml试管中。

13.  加入4毫升苯酚/氯仿/异戊醇。孵育1小时的章动器在4°C。

14.  离心机转速4000× g保持15分钟。将上相转移到50 ml试管中。

15.  加入4毫升苯酚/氯仿/异戊醇。孵育1小时的章动器在4°C。

16.  离心机转速4000× g保持10分钟。将上相转移到干净的50 ml试管中。

17.  加入4毫升氯仿/异戊醇。孵育1小时的章动器在4°C。

18.  离心机转速4000× g保持10分钟。将上相转移到干净的50 ml试管中。

19.  为了沉淀核酸,加入200μl 3 M KOAc pH 5.2和2.8 ml异丙醇。孵育30分钟的章动器在4°C。

20.  将白色细丝轻轻转移到1.7毫升含有1毫升70%乙醇的微管中。用1毫升的移液管尖或从200毫升的吸液管上切下一端-µl移液管尖端转移细丝而不断裂。

21.  在不离心的情况下,用1 ml 70%乙醇清洗3次。

22.  尽可能去除乙醇。

23.  1000℃离心1分钟× g并除去乙醇。可重复此步骤以去除残余乙醇。

24.  根据颗粒的大小,将核酸颗粒风干1-4小时(直到其变得透明)。

25.  在4分钟时在1ml TE O.N.中重新悬浮°在一个螺母上。在这个步骤中,核酸是粘性的。核酸可以在-20℃保存几周°C在核酸酶消化和纯化前进行滴注。

 

D。果蝇S2细胞核酸的提取

1.     果蝇S2细胞在含10%FBS的Schneider培养基中室温培养。

2.     转移2×107个细胞放入50毫升试管中。

3.     在500磅的温度下把细胞压碎× 5分钟。

4.     取上清液,用10ml 1.5ml洗涤细胞颗粒× 公共广播公司。

5.     在500磅的温度下把细胞压碎× 5分钟。

6.     将细胞重新悬浮于3 ml TE中并转移至3个1.7 ml试管中。

7.     加26μl 20%十二烷基硫酸钠和60%μg每管加入蛋白酶K。将试管倒置几次,轻轻搅拌。

8.     37℃孵育O.N°C。

9.     转移至2毫升锁相管。

10.  加入1体积苯酚/氯仿/异戊醇。摇动并在14000下离心× 5分钟。

11.  将上清液转移到含有2.4体积100%乙醇和1/10体积3 M NaOAc pH 5.2的50 ml管中。

注:可以用果蝇胚胎用KOAc代替NaOAc。

12.  轻轻翻转试管以沉淀核酸。

13.  将核酸转移到含有1毫升70%乙醇的1.7毫升试管中。

14.  在不离心的情况下,用1毫升70%乙醇清洗5次,尽可能多地去除乙醇。

15.  去除所有乙醇。

16.  1000℃离心1分钟× g并除去所有乙醇。可重复此步骤以去除残余乙醇。

17.  将核酸颗粒风干几个小时,直到颗粒变透明。

18.  在4分钟时在1ml TE O.N.中重新悬浮°在一个螺母上。在这个步骤中,核酸溶液是粘性的。核酸可以在-20℃保存几周°C在核酸酶消化和纯化前进行滴注。

 

E。核酸酶消化和超声波处理

注:纳米滴上的所有测量均使用dsDNA参数进行。为测量纳米滴上的浓度并在凝胶上运行而收集的样品为:1)在核糖核酸酶a处理前和2)在核糖核酸酶a处理后;3) 超声处理后;4,5)在RNase III+/-RNase H治疗后。

1.     在纳米滴上定量核酸(浓度应在2%左右μ克/μl和A260/A280

≥ 2.0).

2.     运行1μ1%琼脂糖上的l核酸× 用TAE凝胶验证gDNA是否完整。用SYBR金染凝胶。gDNA应该在10kb以上,RNA涂片应该在100bp到1.5kb之间可见。

3.     核糖核酸酶A消化:在0.5 M NaCl和0.1 mg/ml核糖核酸酶A存在下培养250 mg核酸,最终体积为1.5 ml,在37℃下培养3 h°C。

4.     转移到2毫升的锁相管中,加入1体积的苯酚/氯仿/异戊醇。

5.     用力搅拌并在14000下离心× 5分钟。

6.     转移500μl上相至1.7 ml试管,试管中含有1 ml 100%乙醇和50%μl 3 M NaOAc pH 5.2。

7.     轻轻翻转以沉淀核酸。

8.     将核酸转移到一个1.7毫升的试管中,试管中含有400个核酸μl 70%乙醇。

9.     去除所有乙醇。

10.  根据颗粒的大小,将核酸干燥10-30分钟。

11.  在500分钟后重新使用μ轻轻上下移液,在冰上培养至少30分钟。

12.  在纳米滴上测量核酸的浓度,并将体积调整到40 ng以下的浓度/μl(A260/A280应为1.8–2.0)。保存1μl在琼脂糖凝胶上运行,以验证游离RNA的完全降解。

13.  使用Covaris E220进行超声波处理:将样品分成130份-µl等分(~5µg) 在130里做超声波检查-µ科瓦里斯微管。使用以下参数进行超声波检测(表4):

 

表4。使用Covaris E220的超声波参数



峰值入射功率(W)

140

占空因数

10%

每脉冲周期数

200

治疗

80




 

14.  把超声波处理过的核酸放在一起。

15.  保存1μ我要装上凝胶来验证超声波。从超声前10 kb以上的带,超声后应观察到平均大小为300-400 bp的涂片。

16.  核糖核酸酶H和核糖核酸酶III消化:将步骤14中的核酸分成两份。在两份等分试样上加100µ长10× 核糖核酸酶H缓冲液和2单位的核糖核酸酶III。使体积达到1毫升。向一份等分试样(经核糖核酸酶H处理)中添加10μ核糖核酸酶H1和10μ核糖核酸酶H2。

17.  将两种消化液在37℃下培养°C。

18.  转移到2毫升的锁相管中,加入1体积的苯酚/氯仿/异戊醇。

19.  用力搅拌并在14000下离心5分钟× g。

20.  转移500μl上相至1.7 ml试管,试管中含有1 ml 100%乙醇和50%μl 3 M NaOAc pH 5.2。

21.  轻轻翻转以沉淀核酸。

22.  在16000下离心15分钟× g。

23.  去除上清液并用400%的水冲洗颗粒μl 70%乙醇。

24.  在16000下离心5分钟× g并除去所有乙醇。

25.  使核酸颗粒干燥约10分钟。

26.  400分钟后恢复μ轻轻上下移液,在冰上培养至少30分钟。

27.  在纳米滴上测量浓度并保存1μ我想在凝胶上跑步。此测量值用于计算4.4所需的体积µg用于滴水(滴水步骤F2a)。

28.  在1%琼脂糖1上分析程序每个步骤(步骤E1-E5)的试验小份× TAE凝胶和SYBR金染色(图4A)。核酸可以在20℃保存几天°C滴液前。

 

 

图4。滴注和核酸制备. 答。果蝇胚胎核酸的制备。从胚胎中提取的总核酸(左)。核糖核酸酶A消化后和超声波处理前的核酸(中)。超声波和核糖核酸酶III+/-核糖核酸酶H消化后的核酸(右)。B。在2-6小时和10-14小时果蝇胚胎上进行滴注实验的输入和洗脱。在洗脱液中观察到微弱的涂片,但在经RNase H处理的样品的洗脱液中没有观察到。

 

F。点滴注意事项:

答。对于滴注,所有步骤都使用低保留管和移液管头进行。

b。为了测序,我们平行进行了3次上述滴注实验,并在最终纯化后汇集洗脱液。

1.      胎圈准备

答。洗40μl蛋白G动态珠两次,含1× 含5 mg/ml BSA的PBS。

b。在第1卷的第2卷中重新使用珠子× 含5 mg/ml BSA的PBS,加入10μg S9.6抗体。

c。在4℃培养°在一个螺母上。

d。将珠子用1× 含5 mg/ml BSA的PBS。

e。将抗体结合珠放在冰上或4°C。

2.      滴水

答。每滴稀释4.4μg先前纯化的DNA(步骤E27),基于440中的纳米滴测量μ特加50μ长10× 滴胶缓冲液。

b。节省50μl用于输入,并用S9.6-O.N.在4℃孵育剩余样品°C旋转。

c。使用磁性支架捕获珠子30 s,去除上清液,将珠子重新悬浮在700中μl 1级× 滴注结合缓冲液,在RT下旋转培养10分钟。

d。重复洗两次,共洗3次。

e。将珠子重新悬浮250分钟μl滴洗液缓冲液和140μg蛋白酶K。

f。50℃孵育45分钟°C类;每5分钟翻转一次管子。

g。收集上清液(IP/洗脱)。

3.      输入和IP净化

注:核酸在两个连续的柱上纯化,第一个柱用于从样品中去除SDS。

答。输入和IP在Macherey-Nagel(MN)PCR净化柱上使用NTB缓冲液按照试剂盒说明进行纯化。用50%乙醇进行洗脱μl MN洗脱缓冲液。

b。输入和洗脱在Zymoresearch DNA纯化柱上按照试剂盒说明进行纯化。洗脱输入和IP 50μl 10 mM Tris-HCl pH 8.0。对于测序,用8.5洗脱IPμl 10 mM Tris-HCl pH 8.0,将3个IPs混合在一起。

c。运行2μl每个样品放在1.5%琼脂糖溶液中× TAE凝胶并用SYBR金染色至少1小时。涂片应在IP中可见,但在RNase H处理的IP中不可见(图4B)。我们在零下二十点把样品存放一夜°第二天准备图书馆。也可以将样品储存在-80°C几个星期直到图书馆准备。

 

G。库准备和qPCR

1.      使用NEBNext准备库® 用于Illumina的UltraTM II定向RNA文库准备工具包®.

笔记:

一。RNA-DNA杂交的DNA部分从第二条合成链开始测序;RNA部分在RNA酶H消化的第二链合成中被去除。

二。PCR循环次数根据核酸量按照制造商的说明确定(步骤G1a)。

答。估计IP中的物质量并输入生物分析仪。核酸的量应该在1ng以上才能形成文库(图5A)。

 

 

图5。滴灌库准备. 在(A)和(B)文库制备之前,从10-14小时胚胎中输入和洗脱。洗脱液由生物分析仪定量。同样量的输入和RNase H洗脱用于制备文库。由于我们无法确定存在的核酸量,因此使用经核糖核酸酶H处理的核酸的整个免疫沉淀样品来制备文库。

 

b。调整IP的输入量。对于RNase H处理的IP,回收的物质量无法定量。我们使用整个样本,并且能够生成一个高复杂度的库,尽管读取的次数通常是2-5次× 低一点。

c。使用Illumina的NEB RNA Ultra II制备文库,从第二链cDNA合成步骤开始(表5)。

 

表5。改进的第二链合成反应
第二链合成反应

体积

输入和IP

20μ我

含dUTP混合物的第二链合成反应缓冲液(10×)

8μ我

第二链合成酶混合物

4μ我

随机引物

1μ我

核糖核酸酶H(NEB),1.6 U

0.3μ我

无核酸酶水

46.7μ我

 

d。冰上组装,16℃孵育1小时°C。

e。对于接下来的步骤,请按照套件说明进行操作。

2图书馆的质量控制

答。使用qPCR,验证R-环在3个阳性位点的富集和在2个阴性位点的缺失(图6;引物序列见表6)。一。稀释1μ我每10个图书馆μ长0.1× 特。

二。使用S2细胞的gDNA建立具有标准曲线的qPCR板。这种gDNA可按D节所述制备,用RNase A消化,并储存在-80°C。我们以10倍的步骤制备稀释系列,从25 ng到0.025 ng/μl使用2μl每一组底漆的qPCR标准(标准曲线为50-5-0.5-0.05 ng总gDNA)。三、qPCR反应(5)μl总容积,在384孔板中运行)包括2.5μ启动SYBR绿色主混合,2μl S2细胞稀释文库或标准基因组DNA,0.25μl各底漆稀释至1μ我在水里。

iv.qPCR在Viaa7仪器上运行,具有40个周期,Tm=60°C、 延长时间=1分钟。

 

 

图6。库准备后的qPCR。3个阳性和2个阴性位点通过qPCR证实了输入、IP和RNase-H处理IP的滴注和文库制备。

 

表6。qPCR引物
底漆名称

顺序

 

啤酒花F

CTACAGCGAAGGTTT公司

R回路(正极)

啤酒花

CTTGATCTCAGGGGTGCGAT公司

 

德克F

gcgatgaccagagagagag公司

R回路(正极)

 

德克河

CTTGGACTCATCAGTGGCAT公司

 

tRNA赖氨酸F

GCCAAGCTCATTTCTACGATCT公司

R环(高转录基因,阳性)

tRNA赖氨酸受体

GTCCGACACGCCGATA公司

 

特别提款权

ACAGCTGATGTCGCTCACAT公司

无R回路(负)

特别提款权

CGCTGAATGATCACGAGGTGA公司

 

F20层(CG12754)

tcaagcgaccctaaat公司

无R回路(负)

F20右

AACGCCAACAGAAATG公司

 




 

b。使用生物分析仪

一。运行1μl生物分析仪上的每个库(图5B)。

二。碎片的平均大小应该是400bp。

三。在HiSeqTM测序系统上对文库进行测序,每个样本的深度为5000万次读取。这里使用配对末端测序,读取长度为50碱基。

 

数据分析
 

Alecki等人(2020)中描述的滴水序列分析的工作流程与标准ChIPseq分析类似。基本的工作流程是修剪适配器、质量控制FASTQ文件、与基因组比对、去除PCR重复和调用峰。该协议与ChIP-seq在峰值调用前按串分割数据的步骤不同,并针对输入和RNase-H处理的样本调用峰值。所有分析步骤都可以在Galaxy(useglaxy.org)上执行,也可以使用简单的bash脚本。该工作流程确定了一组严格的可重复峰,这些峰符合R-环的严格标准(即它们对RNase H敏感)。

 

笔记:

答。其他人描述了使用RNase H处理样品中读取计数的两倍减少来过滤滴落序列峰(Crossley et al.,2020),而不是比较滴落与RNase H处理滴落中的峰值。这种方法可能更灵活,以适应核糖核酸酶H消化不理想的测序实验,应予以考虑,特别是如果核糖核酸酶H过滤去除大量峰值(>10%)。

b。我们将两个链的峰值一起调用,或者在将bam文件分为正向(F)和反向(R)链之后调用。链特异性分析的信息量更大,因为它可以推断产生RNA的转录方向。一部分峰在两条链上都有Rloop信号(约10%的峰);我们不知道这些是否是技术工件(Alecki et al.,2020)。有些,特别是在胚胎分析的情况下,可能是混合细胞群的结果。可能需要将其移除以进行下游分析(Crossley et al.,2020),这可以使用bedtools使F和R股相交。

c。Crossley等人(2020年)最近描述了一种合成峰值策略,可用于标准化滴灌序列数据,允许在不同条件下进行定量比较。当比较不同基因型的果蝇时,这可能特别有价值。

 

1.    使用Trimmomatic(Bolger et al.,2014)或fastp(v0.20.0)(Chen et al.,2018)修剪适配器并去除低质量读数。

2.    使用Bowtie2(v2.3.1)将读数与果蝇基因组对齐(Langmead和Salzberg,

2012年)(-fr–无混合–无联合)。

3.    使用Samtools(v。1.4.1)(Li等人,2009)将Bowtie2生成的sam文件转换为bam文件,对bam文件进行排序,并为每个文件创建bam索引。

4.    使用Picard(http://broadinstitute.github.io/picard). 标记重复项以筛选PCR重复项。

Sambamba(v0.7.1)(Tarasov等人,2015)也可用于此步骤。

5.    使用samtools视图基于samflags生成特定于链的bam文件(对于所有示例:DRIP、RNase H DRIP和Input),如下所示:

前股:samtools视图–f 99;samtools视图–f 147,后跟samtools merge。

反向股:samtools视图–f 83;samtools视图–f 163,后跟samtools merge。要在Galaxy中执行此过程,请使用samtools视图并选择“读取的筛选/子采样选择”;然后设置“需要设置这些标志”的正确组合。例如,对于前向链,首先输出设置了这些标志的–f99读取:–read is paired–read is mapped in a proper pair=mate strand=read is the first in a pair;然后输出–f147读取并设置这些标志;

–read is paired–read映射到适当的一对–read strand=read是一对中的第二个;samtools merge如上所述用于合并f99和f147(F股)以及f83和f163(R股)。

6.    使用MACS2(v2.1.1)(Zhang等人,2008)调用滴灌峰值与输入峰值,使用宽峰值设置,滴灌作为治疗,输入作为对照。例如,对于与果蝇基因组的dm6版本对齐的成对末端bam文件的峰值调用:

(macs2 callpeak–t drill.bam–c drill\u input.bam–f BAMPE–g 1.4e+08–n drill–outdir drill\u peaks–宽)。

7.    使用MACS2调用滴灌峰值与RNase H处理的滴灌峰值,以滴灌为处理,RNase H处理为对照。

8.    使用BEDTools(Quinlan和Hall,2010)intersect保留IP与输入的峰值,以及IP与RNase H IP的峰值(BEDTools intersect–a IP\u与\u peaks.bed中的\u–b IP\u与\u RNaseHIP peaks.bed–u>过滤的\u peaks.bed)。

9.    使用BEDtools intersect只保留两个复制品中的峰。

 

笔记
 



1.

该滴注方案包括4个步骤,最关键的是制备用于免疫沉淀的核酸。我们鼓励用户在进行测序之前进行多次滴滴qPCR以确保重复性。

2.

时间线(表7)。

 

表7。drop-seq对果蝇细胞作用的时间线

胚胎和组织培养

第1天

胚胎采集

组织培养细胞裂解

 

第2天

胚胎裂解与核酸提取

核酸提取

 

第3天

核酸制备I+珠制备

 

 

第4天

核酸制备Ⅱ+免疫沉淀

 

 

第5天

洗珠+洗脱+纯化

 

 

第6天

库准备和qPCR

 

 

3.     胚胎采集

注意胚胎可以在-80℃下收集和储存°几个月。这个协议可以在一周内完成,但也可以在几个步骤中停止。核酸可以储存在-20°从组织培养细胞或胚胎中提取后,制备核酸(免疫沉淀前)和qPCR或文库制备前几天。

4.     核酸提取与制备

对于细胞裂解后在无蛋白核酸上进行的滴注方案,必须温和地进行细胞裂解和纯化,以避免通过RNA和互补DNA的假退火破坏R-环或产生新的RNA-DNA杂交。因此,从提取到超声,不要高速离心核酸。再悬浮的核酸,不要漩涡;取而代之的是轻轻地上下移液。

5.     滴水

在免疫沉淀之前,可以将其他物种的核酸作为对照的峰值纳入,以使不同样品或实验条件之间的结果正常化(Chen等人,2016)。

 

食谱
 

1.     胚胎裂解缓冲液

50 mM Tris-HCl pH 8.0

100毫米乙二胺四乙酸

100毫米氯化钠

0.5%十二烷基硫酸钠

5毫克/毫升蛋白酶K

2.     1× 核糖核酸酶H缓冲液50 mM Tris-HCl

75毫米氯化钾

3毫米MgCl2 10毫米DTT pH 8.3

3.     10× 滴胶缓冲器

100 mM NaPO4 pH 7.0

1.4毫米氯化钠

0.5%Triton X-100

4.     1× 滴胶缓冲器

10× 滴注结合缓冲液在TE中稀释

5.     滴洗缓冲液

50 mM Tris-HCl pH 8.0

10毫米乙二胺四乙酸

0.5%十二烷基硫酸钠

6.     TE公司

10毫米Tris pH 8.0

1毫米乙二胺四乙酸

7.     苹果汁盘

3.5%琼脂4%糖

40%苹果汁

0.30%尼泊金在乙醇中稀释(最终体积的2.5%)

8.     核糖核酸酶H2提取缓冲液

1× PBS公司

0.2%吐温-20

10毫米氯化镁

9.     RNase H1裂解缓冲液25 mM Tris-HCl 300 mM NaCl 5 mM咪唑pH 8.0用NaOH调节

10.  核糖核酸酶H1洗涤缓冲液

50毫米Tris-HCl

300 mM NaCl 20 mM咪唑pH 8.0,用NaOH调节

11.  核糖核酸酶H1洗脱缓冲液

50毫米Tris-HCl

300 mM NaCl 250 mM咪唑pH 8.0,用NaOH调节

12.  RNase H大小列缓冲区

20 mM Tris-HCl pH 7.0

150毫米氯化钠

使用前立即添加0.1 mM PMSF

13.  核糖核酸酶H储存缓冲液

20 mM Tris-HCl pH 7.5

50毫米氯化钠

1毫米DTT

0.1毫米乙二胺四乙酸

20%甘油

使用前立即添加0.1 mM PMSF

14.  蛋白酶抑制剂和添加剂(表8)

注:用于制备核糖核酸酶H1和H2蛋白质的缓冲液中含有新添加的蛋白酶抑制剂NP40和DTT,但大小柱缓冲液中只添加了PMSF和DTT。

 

表8。蛋白酶抑制剂和添加剂清单
 

最终浓度

特尔克

13.5µ米

苯甲脒

100µ米

胃蛋白酶抑制剂

3µ米

菲咯啉

55µ米

PMSF公司

100µ米

抑肽酶

1.5µ米

亮肽

23µ米

NP40型

0.05%

DTT公司

1毫米

 

15.  0.5× 待定

44.5毫米Tris

44.5 mM硼酸

1 mM EDTA pH 8.0

16.  10× DNA装载缓冲液

20%甘油

0.1 M EDTA pH 8.0

1%十二烷基硫酸钠

0.25%溴酚蓝

0.25%二甲苯氰醇

17.  子系统控制器20×

3 M氯化钠

0.3柠檬酸钠,pH 7.0,用盐酸调节

18.  1× 泰

40 mM Tris-HCl

20毫米醋酸纤维

1毫米乙二胺四乙酸

19.  1× PBS公司

137毫米氯化钠

2.7毫米KCl

10毫米Na2HPO4

1.8毫米KH2HPO4

20.  1× PBT公司

1× PBS公司

0.1%Triton X-100 21。3 M KOAc pH 5.2 3 M醋酸钾pH 5.2,用冰醋酸调节

22.  3 M NaOAc pH 5.2 3 M乙酸钠pH 5.2,用冰醋酸调节

23.  SDS-PAGE运行缓冲区

25毫米Tris-HCl

192 mM甘氨酸

0.1%十二烷基硫酸钠

24.  蛋白质负载缓冲液

6× SDS样品缓冲液,以1的最终浓度添加到样品中×

0.35 M Tris,pH 6.8

30%甘油

1%十二烷基硫酸钠

0.0001%溴酚蓝

 

 

 

致谢
 

本研究方案改编自Ginno等人,2012年,先前发表于Alecki等人,2020年。

作者实验室的工作由加拿大卫生研究院(CIHR)资助。

相互竞争的利益相互竞争的利益

 

作者声明没有相互竞争的利益。

 

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引用:Alecki, C. and Francis, N. J. (2021). Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq. Bio-protocol 11(9): e4011. DOI: 10.21769/BioProtoc.4011.
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