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Jan 2022

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An Improved EMSA-based Method to Prioritize Candidate cis-REs for Further Functional Validation
一种改进的基于EMSA的方法来优先考虑候选的顺式RE,以进一步验证其功能   

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Abstract

Cells are the complex product of gene expression programs that involve the coordinated transcription of thousands of genes controlled by cis-regulatory elements (cis-REs). Therefore, identification of cis-REs is the key to decipher the mechanisms underlying the regulation of gene expression. Here, we describe a simple and time-effective protocol of fine-mapping cis-REs by using an electrophoresis mobility shift assay (EMSA)-based, in vitro, high-throughput (HTP) technique called regulatory element-sequencing (Reel-seq). Reel-seq can be applied to identify cis-REs at a high resolution and sensitivity over large genome regions, in a systematic and continuous manner. It can be used to prioritize candidate cis-REs as a complement to the existing approaches, such as massive parallel reporter assay (MPRA), chromatin immunoprecipitation DNA-sequencing (ChIP-seq), and the assay for transposase-accessible chromatin sequencing (ATAC-seq).


Graphical abstract:



Generation of the Reel-seq Library 1 and 2 (A) and identification of cis-REs by an electrophoresis mobility shift assay (EMSA)-based Reel-seq screen (B). NE: nuclear extract; NGS: next generation sequencing.


Keywords: Gene transcription (基因转录), cis-regulating elements (cis-RE) (顺式调节元件 (cis-RE)), High-throughput (HTP) technique (高通量 (HTP) 技术), EMSA (EMSA), PCR (PCR)

Background

Genes make proteins through two steps: transcription and translation. However, specific expression of genes in particular cells is largely regulated by transcription, which is, in general, controlled by millions of promoters and enhancers. Both promoters and enhancers contain short DNA sequences that serve as cis-regulatory elements (cis-Res), which regulate gene transcription by recruiting regulatory proteins (Mikhaylichenko et al., 2018; Schoenfelder and Fraser, 2019). In addition, gene transcription can also be regulated on the epigenetic level, by opening the chromatin structure to alter DNA accessibility, thereby making the underlying cis-REs accessible to regulatory proteins (Crawford et al., 2006; Angeloni and Bogdanovic, 2019; Zhang et al., 2021). Therefore, identification of cis-REs is the key for understanding of biology.


To date, a number of high-throughput (HTP) approaches have been developed to identify cis-REs. Some identify cis-REs directly, by determining the functional impact of each DNA fragment using HTP reporter assays, such as massive parallel reporter assay (MPRA) (Ulirsch et al., 2016; Tewhey et al., 2016), multiplexed editing regulatory assay (MERA) (Rajagopal et al., 2016), clustered regularly interspaced short palindromic repeats interference, flow cytometry and RNA fluorescence in situ hybridization (CRISPRi-FlowFISH) (Fulco et al., 2019), and cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq) (Diao et al., 2017). Some use antibodies specifically against transcriptional factors or epigenetic markers, to identify cis-REs based on protein-DNA interactions, such as chromatin immunoprecipitation DNA-sequencing (ChIP-seq) (Park et al., 2009), and chromatin immunocleavage sequencing (ChIC-seq) (Schmid et al., 2004). Others use the open chromatin regions that are indicative of active regulatory sites, to identify cis-Res, such as DNase-seq (Song and Crawford, 2010), and the assay for transposase-accessible chromatin sequencing (ATAC-seq) (Buenrostro et al., 2015). While all these approaches have their advantages and specific applications in identifying cis-REs, the technical complexity of these methods limits their routine application. In addition, some of these approaches do not have the high resolution to define each single cis-RE, whereas others do not have enough sensitivity and fidelity to reveal cis-REs. Recently, we developed a simple and time-effective technique that not only complements the pre-existing approaches, but can also be used to prioritize candidate cis-REs (Wu et al., 2022). We refer to this technique as regulatory element-sequencing (Reel-seq). Reel-seq is an in vitro HTP screen technique, designed to identify cis-REs based on protein-DNA interaction that is detected by an electrophoresis mobility shift assay (EMSA). For screening, a Reel-seq construct will be engineered by placing a 35-bp DNA fragment between two primers for PCR amplification, as well as for next-generation sequencing (see Graphic abstract A). A Reel-seq library (e.g., Library 1) will be generated by hundreds of thousands of these constructs synthesized by massive parallel oligonucleotide synthesis, so that an entire DNA region will be reconstructed. To cover the break points between all 35-bp fragments, another Reel-seq library (e.g., Library 2) will be generated in the same fashion, except that each 35-bp fragment in this library will bridge the break point of the two contiguous 35-bp fragments in Library 1. For screening, each Reel-seq library will be first mixed with and without nuclear extract (NE, source of regulatory proteins) isolated from relevant types of cells or tissues, and then analyzed on a TBE native gel. After electrophoresis, unshifted bands from both the buffer-treated control and the NE-treated samples will be isolated, and amplified by PCR. Amplified DNA will then be used for another round of gel shifting. In total, ten rounds of EMSA will be performed so that, if a DNA fragment in the library is a cis-RE, it will be shifted in each round of the gel shift assay. After ten screening rounds, a cis-RE can be identified by its decreased percentage in the unshifted DNA library (see Graphic abstract B). Since Reel-seq is an EMSA-based assay, it is highly specific and sensitive in identifying cis-REs. Moreover, since Reel-seq identifies cis-REs at a high resolution, within a defined 35-bp fragment, each identified cis-RE can be used as a “bait” to pull down the regulatory proteins, by using flanking restriction enhanced DNA pulldown-mass spectrometry (FREP-MS) (Li et al., 2018; Wu et al., 2022), another technique recently developed in our lab. Here, we present the detailed protocol of Reel-seq, for the convenient application of this method to identify cis-REs.

Materials and Reagents

  1. 150 × 21 mm Dish, NunclonTM Delta (Thermo Fisher, catalog number: 168381)

  2. AccuPrimeTM Taq DNA Polymerase System (Thermo Fisher, catalog number: 12339016. Upon receipt, store at -20°C)

  3. Gel Loading Dye, Blue (6×) [New England Biolabs, catalog number: B7021S. Upon receipt, store at room temperature (RT)]

  4. 5% Mini-PROTEAN® TBE Gel, 10 well (Bio-Rad, catalog number: 4565014. Upon receipt, store at 4°C)

  5. PierceTM 10× TBE Buffer (Thermo Fisher, catalog number: 28355. Upon receipt, store at RT)

  6. TE buffer (Thermo Fisher, catalog number: 12090015. Upon receipt, store at RT)

  7. 10,000× GelStarTM Nucleic Acid Gel Stain (Lonza, catalog number: 50535. Upon receipt, store at -20°C)

  8. Low molecular weight DNA ladder (New England Biolabs, catalog number: N3233S. Upon receipt, store at -20°C)

  9. NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, catalog number: 78833. Upon receipt, store at 4°C)

  10. Roche cOmplete, Mini Protease Inhibitor Cocktail (25 tablets) (Emerald Scientific, catalog number: 11836153001. Upon receipt, store at 4°C)

  11. LightShiftTM Chemiluminescent EMSA Kit (Thermo Fisher, catalog number: 20184. Upon receipt, store kit components at -20°C, except for the Chemiluminescent reagents, which are stored at 4°C as described by the manufacturer)

  12. Herculase II Fusion DNA Polymerases (Agilent, catalog number: 600677. Upon receipt, store at -20°C)

  13. UltraPureTM Agarose (Thermo Fisher, catalog number: 16500100. Upon receipt, store at RT)

  14. Safe-Red (APPLIED BIOLOGICAL MATERIALS INC, catalog number: G108-R. Upon receipt, store at -20°C)

  15. All primers are purchased from Integrated DNA Technologies (IDT) as a standard formula

  16. GibcoTM DPBS, no calcium, no magnesium (Gibco, catalog number: 14190235. Upon receipt, store at RT)

  17. Primary human arterial ECs (Lonza, catalog number: CC-2535)

  18. EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM (Lonza, catalog number: CC-3162)

  19. 0. 5× TBE buffer (see Recipes)

  20. 1× GelStar (see Recipes)

  21. 1× Low Molecular Weight DNA Ladder (see Recipes)

  22. 10% TE buffer (see Recipes)

  23. 1.5% agarose gel (see Recipes)

Equipment

  1. T100 Thermal Cycler (Bio-Rad, catalog number: 1861096)

  2. PowerPacTM Basic Power Supply (Bio-Rad, catalog number: 1645050)

  3. Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (Bio-Rad, catalog number: 1658003FC)

  4. Eppendorf ThermoMixerTM C (Fisher Scientific, catalog number: 13527550)

  5. Thermo Scientific Savant DNA 120 SpeedVac Concentrator (Thermo Scientific, catalog number: BZ10133848)

  6. BLooKTM LED Transilluminator (GeneDireX, catalog number: BK001)

  7. Implen C40 NanoPhotometer UV/Vis Spectrophotometer Mobile System, 110/220 V (Cole-Parmer, catalog number: UX-83070-62)

  8. Cell scraper (VWR, catalog number: 15621-005)

  9. VWR Ergonomic High-Performance Pipettor set (VWR, catalog number: 89079-970)

  10. Thermo Forma Steri Cycle 370 CO2 Incubator (Marshall Scientific, catalog number: TH-370)

  11. Allegra® X-5 Clinical Benchtop IVD Centrifuge, Beckman Coulter® (VWR, catalog number: 89429-566)

Procedure

A simplified flow chart of Reel-seq is present in Figure 1.



Figure 1. A simplified flow chart of Reel-seq

  1. Construction and generation of DNA library 1 and 2

    1. Download the genome DNA sequence of interest from the NCBI website (https://www.ncbi.nlm.nih.gov).

    2. Scroll down from All Databases to Gene.

    3. Input the name of the gene you are interested in, for example, CDKN2A.

    4. Select the gene in Search results.

    5. Select FASTA under the Genomic regions, transcripts, and products.

    6. Define the region of your sequence, by inputting the nucleotide number on the chromosome your gene is located under Change region shown on the right site of the screen. For example, from 2230356 to 22172015, for the 58 kb CDKN2A region on chromosome 9.

    7. Click Update View, and copy the sequence into word file.

    8. Obtain 35-bp fragments continuously starting from the first base of the genomic sequence for Library 1, as shown in the graphic abstract.

    9. Obtain 35-bp fragments continuously starting from base 18 for Library 2, so that these fragments will cover the break points between each of two 35-bp fragments in Library 1. For a 58 kb CDKN2A/B region, you will have 1668 35-bp fragments for both Libraries 1 and 2.

    10. List the 1668 sequences in an Excel file for each library.

    11. Construct Libraries 1 and 2, by adding the common flanking sequences to each 35-bp fragment in the Excel file, as shown in Table 1.


      Table 1. Construction of Reel-seq library

      PE_SP Library 1 sequence G3
      ACACGCACGATCCGACGGTAGTGT 1–35 bp genomic DNA sequence GGATGACGACGATAAGCTCG
      new_SP Library 2 sequence 926RR
      GGTGTGATGCTCGGATCCAGGAAC 18–52 bp genomic DNA sequence GGTAGGACAGTAGTCTCGTG


    12. Order Libraries 1 and 2 from commercial vendors.

      Note: You can order these two libraries together as two sub-libraries, by adding the common sequence AGGACCGGATCAACT on the 5’ end and CATTGCGTGAACCGA at the 3’ end. These common sequences will be served as adaptors for massive parallel oligonucleotide synthesis.


  2. Separation of library DNA for libraries 1 and 2

    1. Upon receipt of the library DNA, resuspend the synthetic DNA Library using DNase-free water to a final concentration of 100 ng/mL, and keep it as a stock at -20°C.

    2. Dilute the synthetic DNA library with 1 μL stock using DNase-free water, to a final concentration of 5, 10, 20, 50, 100, and 200 pg/μL.

    3. PCR amplify the library DNA, by using primers PE_SP (L1_FP): 5’ACACGCACGATCCGACGGTAGTGT3’ and G3 (L1_RP): 5’CGAGCTTATCGTCGTCATCC3’ for Library 1, and new_SP(L2_FP): 5’GGTGTGATGCTCGGATCCAGGAAC3’ and 926RR (L2_RP): 5’CACGAGACTACTGTCCTACC3’ for Library 2.

      Note: These primers were generated in our lab for different purposes and were selected among many primers tested in our pilot Reel-seq screen. These primers are all good for PCR, but some are not good for repeated amplicon amplification, and others are not good for next-generation sequencing, or library preparation. Therefore, please make sure you don’t change these primers for Reel-seq screen.

    4. Perform the PCR reaction with the T100 Thermal Cycler, as formulated in Table 2, with the PCR program listed in Table 3.


      Table 2. PCR amplification of library DNA

      Component Volume (μL)
      Library DNA in different concentrations 5
      Forward primer (10 μM) 1
      Reverse primer (10 μM) 1
      10× AccuPrime PCR buffer 2.5
      AccuPrime Taq DNA polymerase 0.25
      H2O 15.25
      Total 25


      Table 3. PCR amplification program

      Step Temperature Time
      Initial denaturation 94°C 2 min
      30 cycles 94°C 30 s
      60°C 30 s
      68°C 40 s
      Final extension 68°C 5 min
      Hold 10°C


    5. Assemble a 5% Mini-PROTEAN® TBE Gel in a Mini-PROTEAN® Tetra Vertical Electrophoresis Cell.

    6. Resolve all 25 μL of PCR products by adding 5 μL 6× gel Loading Dye on the 5% Mini-PROTEAN® TBE Gel.

    7. Use 10 μL of 1× low molecular weight DNA ladder as a marker.

    8. Run the gel electrophoresis using 0.5× TBE buffer at 100 V for ~60 min.

    9. Disassemble the gel cassette in such a way that the gel is still attached to one side of the glass plate.

    10. Stain the gel, by covering the surface of the gel with 5 mL of 1× GelStar at RT for 20 min.

    11. Visualize the PCR products with BLooKTM LED Transilluminator, and cut the band containing the 79-bp library DNA from the best amplified band (without upper band) (Figure 2).



      Figure 2. Amplification of Libraries 1 (A) and 2 (B).

      A 5% Acrylamide gel showing a 79-bp band containing library DNA was amplified by PCR with different concentrations of library DNA, as indicated.


    12. Place each sliced gel band (~3 × 5 mm in size) into 150 μL of 10% TE buffer in a microtube, and shake at RT overnight.

    13. Collect ~120 μL of liquid containing the library DNA. At this point, you should have one tube for each library with purified library DNA.

    14. Put the tube with the cap open in the Thermo Scientific Savant DNA 120 SpeedVac Concentrator, which is a vacuum centrifuge.

    15. Concentrate the library DNA, by turning on the centrifuge first, and then the vacuum.

    16. Normalize the DNA samples to 10 ng/μL, as measured with the Implen C40 NanoPhotometer UV/Vis Spectrophotometer Mobile System.


  3. Preparation of library DNA for Reel-seq screen

    1. PCR amplify the library DNA for Libraries 1 and 2 obtained from Procedure B.

    2. Prepare the PCR reaction as formulated in Table 2, by using 5 μL of purified library DNA (~10 ng/μL), and run the PCR reaction according to the program listed in Table 3, except that only 15 cycles are required.

    3. Amplify each library DNA in triplicate, so that you will have enough DNA for the Reel-seq screen.

    4. Run 5 μL of PCR product from each reaction on a 5% Mini-PROTEAN® TBE Gel, as described in Procedure B (steps 6–10), to check the quality of PCR amplification. You should observe one major band around 79-bp, with all the reactions having relatively the same amount of DNA.

    5. The amplified library DNA for Libraries 1 and 2 is now ready for the Reel-seq screen.


  4. Isolation of nuclear extract (NE)

    1. Culture ~1.5 × 108 primary human arterial ECs with ERG-2 medium in 150-mm plates in a cell culture incubator with 5% CO2.

    2. Wash the cells three times using DPBS.

    3. Scrape the cells in 5 mL of DPBS with 1× Roche cOmplete mini protease inhibitor cocktail.

    4. Collect the cells by centrifuging at 450 g for 5 min, using the Clinical Benchtop IVD Centrifuge.

    5. Isolate the NE using NE-PERTM Nuclear and Cytoplasmic Extraction Reagents, according to the manufacturer’s instructions, except that only half volume of Nuclear Extraction Reagent (NER) is used to extract NE. This will yield NE with a concentration of ~10 μg/μL.

    6. Use Roche cOmplete mini protease inhibitor cocktail to prevent protein degradation, according to the manufacturer’s instructions.

    7. Divide the NE in ~20 μL aliquots, and store it at -80°C for at least one year.

      Note: Cell culture conditions and media will be dependent on the cell line you use to isolate NE.


  5. Titration of NE for EMSA

    1. Use LightShiftTM Chemiluminescent EMSA Kit to perform the titration, by mixing all the reagents as formulated in Table 4.


      Table 4. Titrate nuclear extract concentration reaction

      Component Volume (μL)
      Library DNA from C 5
      10× Binding Buffer 2
      Poly dI-dC (1 μg/μL) 1
      100 mM MgCl2 1
      1% NP-40 1
      50% Glycerol 1
      Nuclear Extract 0, 0.5, 1, 2, 3
      H2O Add to 20
      Total 20


    2. Use 5 μL of library DNA from Procedure C to titrate the NE, by adding 0, 0.5, 1, 2, and 3 μL of NE isolated in Procedure D.

    3. Incubate the mixture at RT for 2 h or at 4°C overnight.

    4. Perform EMSA using a 5% Mini-PROTEAN® TBE Gel, by adding 5 μL of 5× loading dye provided in the kit to each 20 μL of reaction mixture.

    5. Run all 25 μL of PCR product from each reaction on a 5% Mini-PROTEAN® TBE Gel, as described in Procedure B (steps 6–10), and visualize the PCR products with BLooKTM LED Transilluminator.

    6. Use densitometry to determine the reaction that shows ~50% library DNA shifted in the sample compared to the control (no NE added), and use the amount of NE (for example, 2 μL) for the following Reel-seq screen (Figure 3).

      Note: If a 79-bp fragment is not observed clearly, you should apply more cycles, such as 20 cycles for the PCR in step 2 of Procedure C.



    Figure 3. Titration of NE.

    Acrylamide gel showing that different amounts of NE were used to titrate the amount of NE that results in ~50% library DNA shifting in the sample compared to the control.


  6. Reel-seq screen

    1. Prepare EMSA reaction using LightShiftTM Chemiluminescent EMSA Kit, according to Table 5, in triplicate. At this point, you should have three controls and three samples for each library.


      Table 5. EMSA reaction

      Component Control (μL) Sample (μL)
      Library 1/2 DNA (Procedure C) 5 5
      10× Binding Buffer 2 2
      Poly dI-dC (1 μg/μL) 1 1
      100 mM MgCl2 1 1
      1% NP-40 1 1
      50% Glycerol 1 1
      Nuclear Extract (Procedure D) 2 (H2O) 2
      H2O 7 7
      Total 20 20


    2. Shake the reaction mixtures (300 rpm) at RT for 2 h or at 4°C overnight.

    3. Perform EMSA using a 5% Mini-PROTEAN® TBE Gel, by adding 5 μL of 5× loading dye provided in the kit to each 20 μL of reaction mixture.

    4. Purify the 79-bp unshifted library DNA band from the three controls and the three samples (Figure 4A), as described in Procedure B (steps 6–14).

    5. PCR amplify the library DNA for Libraries 1 and 2 obtained above.

    6. Prepare the PCR amplification reaction as formulated in Table 2, and run the PCR with the program listed in Table 3, except that only 15 cycles are required.

    7. Run 5 μL of PCR product from each reaction on a 5% Mini-PROTEAN® TBE Gel, as described in Procedure B (steps 6–10), to check the quality of PCR amplification with the BLooKTM LED Transilluminator (Figure 4B).

    8. Repeat Procedure F ten times.

      Notes:

      1. You should observe one major band around 79-bp with all the reactions (three controls and three samples for each library), and having relatively the same amount of DNA (Figure 4B). The amplified library DNAs are now ready for the next round of Reel-seq screen.

      2. Also, in general, applying positive controls for Reel-seq screen is a good idea. When we performed this Reel-seq, we did not have any positive controls. This is because no cis-REs had ever been identified in the 58-kb CDKN2A/B region we screened. However, one can always use cis-REs from other regions of the human genome as positive controls, if one can make sure that these cis-REs function in the cells you use for your Reel-seq screen.



    Figure 4. Reel-seq screen.

    Acrylamide gel showing the results of EMSA (A) and PCR (B) in round 1 of Reel-seq screen, with three buffer-treated controls and the three NE-treated samples, for one of the two libraries.


  7. Preparation of a next-generation sequencing library using two PCR reactions

    1. Select the Reel-seq PCR products from rounds 1, 4, 7, and 10, to prepare the next-generation sequencing library. As each round has three controls and three samples, a total of 24 sub-libraries will be generated for each library.

    2. Using 1 μL of the Reel-seq PCR product for each control and sample, perform the first PCR reaction according to Table 6.

      Note: We used Herculas II Fusion DNA Polymerase, as previously described (Larman et al., 2013).

    3. Run the PCR according to the program listed in Table 7.


      Table 6. PCR amplification before high throughput sequencing

      Component Volume/Reaction (μL)
      PCR product from cycle 1, 4, 7, and 10 1
      F (L1-PE/L1-New-seq, 10 μM) 1
      R (R1-G3/R1-926RR, 10 μM) 1
      5× Herculase II Reaction Buffer 5
      Herculase II Fusion DNA Polymerase 0.25
      dNTP 0.25
      H2O Add to 25
      Total 25


      Table 7. PCR amplification program before high throughput sequencing

      Temperature

      Time

       

      95°C

      2 min

       

      95°C

      20 s

      Repeat 29 cycles

      58°C

      30 s

      72°C

      40 s

      10°C

      5 min

       

    4. Use L1/PE_SP and R1/G3 primers for Library 1, and L1/new-SP and R1/926RR primers for Library 2. The sequences of all these primers are listed in Table 8.

      Note: We selected PCR products from rounds 1, 4, 7, and 10 for sequencing, based on the capacity of next-generation sequencing, the number of DNA fragments in the Reel-seq library, a 30–50% mutation rate introduced by PCR in the Reel-seq screen, and the cost of sequencing. The data generated from these four rounds is good enough for statistical analysis.


      Table 8. Primers for next-generation sequencing library preparation

      L1/PE_SP

      (L1_FP)

      5’ACACTCTTTCCCTACACGACACACGCACGATCCGACGGTAGTGT3’

      R1/G3

      (L1_RP)

      5’GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCGAGCTTATCGTCGTCATCC3’ 

      L1/new_SP

      (L2_FP)

      5’ACACTCTTTCCCTACACGACGGTGTGATGCTCGGATCCAGGAAC3’

      R1/926RR

      (L2-RP)

      5’GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCACGAGACTACTGTCCTACC3’
      L2 5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC3’
      R2 (Barcode) 5’CAAGCAGAAGACGGCATACGAGATXXXXXXXGTGACTGGAGTTCAGACGTGT3’


    5. Check the quality of the first PCR reaction, by running 5 μL of PCR product on a 2% agarose gel with 2 μL of Gel Loading Dye. A relatively even band at 132 bp should be observed across all the reactions (Figure 5A and B).



      Figure 5. Sequencing library preparation.

      Agarose gel showing the PCR products from the first PCR (A and B) and second PCR (C and D). C: Control; S: Sample.


    6. Perform the second PCR reaction according to Table 9, using 2 μL of the first PCR product for each control and sample, and run the PCR using the program listed in Table 10.


      Table 9. Second PCR amplification reaction before high-throughput sequencing

      Component Volume (μL)
      First PCR product 2
      L2 1
      R2 (Barcode) 1
      5× Herculase II Reaction Buffer 10
      Herculase II Fusion/ DNA Polymerase 0.5
      dNTP 0.5
      H2O 35
      Total 50


      Table 10. Second PCR amplification program before high throughput sequencing

      emperature Time Note
      95°C 2 min  
      95°C 20 s Repeat 9 cycles
      58°C 30 s
      72°C 40 s
      72°C 5 min  

    7. Use L2 and R2 (Barcode) primers for both libraries, as listed in Table 8. In total, 48 R2 primers with 48 different barcodes are needed for amplifying 48 sub-libraries.

      Note: L2 and R2 primers contain adaptor sequences p5 and p7 (underlined in Table 8). Adaptors allow the sequencing library to bind and generate clusters on the flow cell for illumina sequencing. The design and the sequence of these primers with barcodes were described in Kozarena and Tuner (2011).

    8. Check the quality of the second PCR reaction, by running 5 μL of PCR product on 2% agarose gel with 2 μL of Gel Loading Dye. A relatively even band at 188 bp should be observed across all the reactions (Figure 5C and D).

    9. Mix the second PCR products according to Table 11. You will have 186 μL of secondary PCR product mixture for each library.


      Table 11. Mix PCR product for sequencing library purification

      Round Control (μL) Sample (μL)
      1 1 1
      4 3 5
      7 5 10
      10 7 30

      Note: We chose the volumes mentioned in the Table 11 based on our experience. PCR can generate mutations, and more mutations are generated by PCR with library DNA treated with NE. Therefore, we used these numbers to obtain relatively even sequencing reads (free of mutation) for each library.


    10. Purify the mixed PCR product, by using a 5% Mini-PROTEAN® TBE Gel for each library, as described in Procedure B (steps 6–14). Each 186 μL of library DNA should be evenly distributed into 9 wells, with one well left for the marker.

    11. Cut the band at 188-bp, place each sliced gel into 150 μL of 10% TE buffer, and isolate the sequencing library by shaking at RT overnight.

    12. Concentrate the DNA Library to ~50 ng/μL with the SpeedVac Concentrator.

    13. Send both sequencing libraries to the core facility for next-generation sequencing. Use the PE_SP primer for Library 1, and the new_SP primer for Library 2.

    14. Sequence both Libraries 1 and 2 together, using NextSeq 500 with 75-bp single read high output, at 2.5 pM with 1% PhiX spiked in.

      Notes:

      1. As a part of sequencing, you should request for data analysis to list the reads for each 35-bp fragment in all 24 sub-libraries, for both Libraries 1 and 2. Next generation sequencing was performed at the Genomics Research Core at University of Pittsburgh (https://www.genetics.pitt.edu/next-generation-sequencing).

      2. The actual platform you use for next generation sequencing depends on the size of your library. A small library can be sequenced by MySeq; therefore, talk to a sequencing expert to determine the actual sequencing platform you want to use.

Data analysis

  1. Use Excel to perform all data analysis.

  2. Calculate the percentage of each 35-bp DNA fragment in each of the 24 sub-libraries.

  3. Use the three percentages from the three buffer-treated controls and the three NE-treated samples to calculate the P-value of the Student’s t-test for each fragment in rounds 1, 4, 7, and 10.

  4. Calculate the average of the percentage for each DNA fragment from the three buffer-treated controls and the three NE-treated samples in rounds 1, 4, 7, and 10.

  5. Calculate the ratio of the percentage average from the three NE-treated samples versus the three buffer-treated controls in rounds 1, 4, 7, and 10.

  6. Apply these four ratios to calculate the slope for each 35-bp DNA fragment across rounds 1, 4, 7, and 10.

  7. Apply slope < 0 as the first filter, and P < 0.05 in rounds 1, 4, 7, and 10 as the second filter.

  8. Identify the fragments with slope < 0 and P < 0.05 in rounds 1, 4, 7, and 10 as the candidate cis-REs.

    Note: We have successfully used Reel-seq to identify cis-REs over a 200 kb region. However, at this point, we don’t yet know the limitations on how big a region can be screened by Reel-seq. Also, we have estimated the fidelity to be >90% for identifying cis-REs using Reel-seq.

Recipes

  1. 0. 5× TBE buffer (1 L)

    Add 50 mL of 10× TBE to 950 mL of distilled water (dH2O).

  2. 1× GelStar (10 mL)

    Add 1 μL of 10,000× GelStarTM Nucleic Acid Gel Stain to 10 mL of dH2O.

  3. 1× Low Molecular Weight DNA Ladder

    Add 100 μL of Low Molecule Weight DNA ladder to 900 μL of dH2O and 200 μL of 6× DNA Gel Loading Dye.

  4. 10% TE buffer

    Add 1 mL of TE buffer to 9 mL of dH2O.

  5. 2% agarose gel

    Dissolve 2 μg agarose in 100 mL of 0.5× TBE.

Acknowledgments

The Reel-seq protocol presented herein is modified from its original publication in Nucleic Acids Research by Wu et al. (2021). We thank NIH NIA and UPMC for their grant support. This work was supported partly by grants from NIH NIA R01AG056279 (GL), R01AG065229 (GL) and UPMC (GL).

Competing interests

The authors declare no competing financial interests.

References

  1. Angeloni, A. and Bogdanovic, O. (2019). Enhancer DNA methylation: implications for gene regulation. Essays Biochem 63(6): 707-715.
  2. Buenrostro, J. D., Wu, B., Chang, H. Y. and Greenleaf, W. J. (2015). ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109: 21 29 21-21 29 29.
  3. Crawford, G. E., Davis, S., Scacheri, P. C., Renaud, G., Halawi, M. J., Erdos, M. R., Green, R., Meltzer, P. S., Wolfsberg, T. G. and Collins, F. S. (2006). DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 3(7): 503-509.
  4. Diao, Y., Fang, R., Li, B., Meng, Z., Yu, J., Qiu, Y., Lin, K. C., Huang, H., Liu, T., Marina, R. J., et al. (2017). A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat Methods 14(6): 629-635.
  5. Fulco, C. P., Nasser, J., Jones, T. R., Munson, G., Bergman, D. T., Subramanian, V., Grossman, S. R., Anyoha, R., Doughty, B. R., Patwardhan, T. A., et al. (2019). Activity-by-contact model of enhancer-promoter regulation from thousands of CRISPR perturbations. Nat Genet 51(12): 1664-1669.
  6. Kozarewa, I. and Turner, D. J. (2011). 96-plex molecular barcoding for the Illumina Genome Analyzer. Methods Mol Biol 733: 279-298.
  7. Larman, H. B., Laserson, U., Querol, L., Verhaeghen, K., Solimini, N. L., Xu, G. J., Klarenbeek, P. L., Church, G. M., Hafler, D. A., Plenge, R. M., et al. (2013). PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis. J Autoimmun 43: 1-9.
  8. Li, G., Martinez-Bonet, M., Wu, D., Yang, Y., Cui, J., Nguyen, H. N., Cunin, P., Levescot, A., Bai, M., Westra, H. J., et al. (2018). High-throughput identification of noncoding functional SNPs via type IIS enzyme restriction. Nat Genet 50(8): 1180-1188.
  9. Mikhaylichenko, O., Bondarenko, V., Harnett, D., Schor, I. E., Males, M., Viales, R. R. and Furlong, E. E. M. (2018). The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev 32(1): 42-57.
  10. Park, P. J. (2009). ChIP-seq: advantages and challenges of a maturing technology. Nat Rev Genet 10(10): 669-680.
  11. Rajagopal, N., Srinivasan, S., Kooshesh, K., Guo, Y., Edwards, M. D., Banerjee, B., Syed, T., Emons, B. J., Gifford, D. K. and Sherwood, R. I. (2016). High-throughput mapping of regulatory DNA. Nat Biotechnol 34(2): 167-174.
  12. Schmid, M., Durussel, T. and Laemmli, U. K. (2004). ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell 16(1): 147-157.
  13. Schoenfelder, S. and Fraser, P. (2019). Long-range enhancer-promoter contacts in gene expression control. Nat Rev Genet 20(8): 437-455.
  14. Song, L. and Crawford, G. E. (2010). DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb Protoc 2010(2): pdb prot5384.
  15. Tewhey, R., Kotliar, D., Park, D. S., Liu, B., Winnicki, S., Reilly, S. K., Andersen, K. G., Mikkelsen, T. S., Lander, E. S., Schaffner, S. F., et al. (2016). Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay. Cell 165(6): 1519-1529.
  16. Ulirsch, J. C., Nandakumar, S. K., Wang, L., Giani, F. C., Zhang, X., Rogov, P., Melnikov, A., McDonel, P., Do, R., Mikkelsen, T. S., et al. (2016). Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits. Cell 165(6): 1530-1545.
  17. Wu, T., Jiang, D., Zou, M., Sun, W., Wu, D., Cui, J., Huntress, I., Peng, X. and Li, G. (2022). Coupling high-throughput mapping with proteomics analysis delineates cis-regulatory elements at high resolution. Nucleic Acids Res 50(1): e5.
  18. Zhang, Y., Sun, Z., Jia, J., Du, T., Zhang, N., Tang, Y., Fang, Y. and Fang, D. (2021). Overview of Histone Modification. Adv Exp Med Biol 1283: 1-16.

简介

[摘要] 细胞是基因表达程序的复杂产物,涉及由 cis 调节元件 ( cis -REs) 控制的数千个基因的协调转录。因此,独联体-REs的鉴定是破译基因表达调控机制的关键。在这里,我们通过使用基于电泳迁移率变动分析 (EMSA) 的精细映射cis -REs描述了一种简单且高效的协议, 体外高通量 (HTP) 技术称为调节元素测序( Reel -seq) 。 Reel-seq 可用于以系统和连续的方式在大基因组区域以高分辨率和灵敏度识别cis -RE。它可用于优先考虑候选cis -REs 作为对现有方法的补充,例如大规模平行报告基因分析 (MPRA), 染色质免疫沉淀 DNA 测序 ( ChIP -seq),以及转座酶可及染色质测序分析 (ATAC-seq)。

图文摘要:

Reel-seq 库 1 和 2 (A) 的生成以及通过基于电泳迁移率变动分析 (EMSA) 的 Reel-seq 筛选 (B)识别cis -REs。 NE:核提取物; NGS:下一代测序。

[背景] 基因使 蛋白质 通过两个步骤: 转录和翻译。然而,特定细胞中基因的特异性表达主要受转录调控,一般而言,转录受数百万个启动子和增强子控制。启动子和增强子都包含作为顺式调节元件 ( cis -Res) 的短 DNA 序列,通过募集调节蛋白 ( Mikhaylichenko ) 来调节基因转录。 等人,2018 年;舍恩费尔德和弗雷泽,2019 年)。此外,基因转录也可以在表观遗传水平上进行调节,通过打开染色质结构来改变 DNA 的可及性,从而使潜在的顺式-REs 可被调节蛋白访问( Crawford et al ., 2006; Angeloni and Bogdanovic, 2019; Zhang等人,2021 年)。因此,顺式-REs的鉴定是理解生物学的关键。
迄今为止,已经开发了许多高通量(HTP) 方法来识别cis - RE。有些人通过使用 HTP 报告基因测定法(例如大规模平行报告基因测定法 (MPRA) ( Ulirsch ) 确定每个 DNA 片段的功能影响来直接识别cis -REs) 等人,2016;图伊 et al ., 2016 ), 多重编辑调控分析 (MERA) ( Rajagopal et al ., 2016),聚集规则间隔短回文重复干扰、流式细胞术和 RNA 荧光原位杂交 ( CRISPRi-FlowFISH ) ( Fulco et al ., 2019),以及通过平铺删除和测序 (CREST-seq) 进行的顺式调节元件扫描 ( Diao 等人,2017)。一些使用特异性针对转录因子或表观遗传标记的抗体,以基于蛋白质-DNA 相互作用来识别 cis-RE,例如染色质免疫沉淀 DNA 测序 ( ChIP - seq) ( Park et al ., 2009 ) 和染色质免疫切割测序(Park et al., 2009)。 ChIC- seq) ( Schmid et al ., 2004 )。其他人使用指示活性调节位点的开放染色质区域来识别顺式-Res,例如 DNase-seq ( Song and Crawford, 2010 ),以及转座酶可及染色质测序分析 (ATAC-seq) ( Buenrostro 等人,2015 年)。虽然所有这些方法在识别cis -REs方面都有其优势和特定应用,但这些方法的技术复杂性限制了它们的常规应用。此外,这些方法中的一些不具有定义每个单顺-RE的高分辨率,而其他方法没有足够的灵敏度和保真度来揭示顺-RE 。最近,我们开发了一种简单且省时的技术,不仅可以补充现有方法,还可以用于优先考虑候选cis -REs ( Wu et al ., 2022 ) 。我们将这种技术称为监管元素测序( Reel - seq ) 。 Reel-seq 是一种体外HTP 筛选技术,旨在根据通过电泳迁移率变动分析 (E MSA)检测到的蛋白质-DNA 相互作用来识别cis -RE。为了筛选,将通过在两个引物之间放置一个 35 bp DNA 片段来设计 Reel-seq 构建体,用于 PCR 扩增以及下一代测序(参见图形摘要 A) 。 Reel-seq 文库(例如, Library 1 )将由通过大规模平行寡核苷酸合成而合成的数十万个这些构建体生成,从而将重建整个DNA区域。为了覆盖所有 35-bp 片段之间的断点,将以相同的方式生成另一个 Reel-seq 库(例如, Library 2 ),除了该库中的每个 35-bp 片段将桥接两者的断点库 1中的连续 35-bp 片段。对于筛选,每个 Reel-seq 文库将首先与从相关类型的细胞或组织中分离的核提取物(NE,调节蛋白来源)混合,然后在 TBE 天然凝胶上进行分析。电泳后,来自缓冲液处理的对照和 NE 处理的样品的未移位条带将被分离,并通过 PCR 扩增。然后将扩增的 DNA 用于另一轮凝胶转移。总共将进行 10 轮 EMSA,因此,如果文库中的 DNA 片段是顺式-RE,它将在每轮凝胶移位测定中发生移位。在十轮筛选之后,可以通过其在未移位的 DN A 文库中降低的百分比来识别顺式-RE (参见图形摘要 B) 。由于Reel -seq 是一种基于 EMSA 的检测方法,因此它在识别cis - RE 方面具有高度特异性和敏感性。此外,由于 Reel-seq在定义的 35 bp 片段内以高分辨率识别 cis -RE,每个识别的cis -RE都可以用作“诱饵”,通过使用侧翼限制 e来拉下调节蛋白增强 DNA下拉质谱( FREP - MS ) ( Li et al ., 2018; Wu et al ., 2022 ),这是我们实验室最近开发的另一种技术。在这里,我们介绍了 Reel-seq 的详细协议,以方便应用该方法来识别cis - REs。

关键字:基因转录, 顺式调节元件 (cis-RE), 高通量 (HTP) 技术, EMSA, PCR



材料和试剂


1. 150 × 21 mm Dish, Nunclon TM Delta( Thermo Fisher,目录号:168381)
2. AccuPrime TM Taq DNA 聚合酶系统(Thermo Fisher,目录号:12339016 。收到后,在 -20°C 下储存)
3. 凝胶加载染料,蓝色(6 × )[ New England Biolabs,目录号:B7021S。收到后,在室温 (RT) 下储存]
4. 5% Mini-PROTEAN ® TBE Gel,10孔( Bio-Rad ,目录号:4565014 。收到后,在 4°C 下储存)
5. Pierce TM 10 × TBE 缓冲液(Thermo Fisher,目录号:28355 。收到后,储存在 RT )
6. TE 缓冲液(Thermo Fisher,目录号:12090015 。收到后,储存在 RT )
7. 10,000 × 凝胶之星 核酸凝胶染色剂(Lonza,目录号:50535 。收到后,储存在-20°C )
8. 低分子量 DNA 梯(New England Biolabs,目录号:N3233S。收到后,在 -20°C 下储存)
9. NE-PER TM核和细胞质提取试剂(Thermo Fisher,目录号:78833 。收到后,在 4°C 下储存)
10. Roche cOmplete ,Mini Protease Inhibitor Cocktail(25 片)(Emerald Scientific,目录号:11836153001 。收到后,在 4°C 下储存)
11. LightShift TM化学发光 EMSA 试剂盒(Thermo Fisher,目录号:20184 。收到后,将试剂盒组分储存在 -20°C 下,化学发光试剂除外,如制造商所述在 4 °C 下储存)
12. Herculase II Fusion DNA Polymerases(Agilent,目录号:600677 。收到后,储存在-20°C )
13. UltraPure TM琼脂糖( Thermo Fisher,目录号:16500100 。收到后,储存在 RT)
14. Safe-Red( APPLIED BIOLOGICAL MATERIALS INC,目录号:G108-R。收到后,储存在 -20°C)
15. 所有引物均购自 Integrated DNA Technologies (IDT) 作为标准配方
16. Gibco TM DPBS,不含钙,不含镁(Gibco,目录号:14190235 。收到后,储存在 RT)
17. 原代人动脉EC(Lonza,目录号:CC-2535)
18. EGM TM -2 内皮细胞生长培养基-2 BulletKit TM (Lonza,目录号:CC-3162)
19. 0. 5 × TBE 缓冲液(见配方)
20. 1 × 凝胶之星 (见食谱)
21. 1 ×低分子量 DNA 梯(见食谱)
22. 10% TE 缓冲液(见配方)
23. 1.5% 琼脂糖凝胶 (见食谱)


设备


1. T100 热循环仪( Bio-Rad ,目录号:1861096)
2. PowerPac TM基本电源( Bio-Rad ,目录号:1645050)
3. Mini-PROTEAN ® Tetra 垂直电泳槽( Bio-Rad ,目录号: 1658003FC )
4. Eppendorf ThermoMixer TM C(Fisher Scientific,目录号: 13527550)
5. Thermo Scientific Savant DNA 120 SpeedVac浓缩器( Thermo Scientific,目录号:BZ10133848)
6. BLook TM LED Transilluminator( GeneDireX ,目录号:BK001)
7. Implen C40 NanoPhotometer UV/Vis 分光光度计移动系统,110/220 V(Cole-Parmer,目录号:UX-83070-62)
8. 细胞刮刀(VWR,目录号:15621-005)
9. VWR 人体工学高性能移液器套件(VWR,目录号:89079-970)
10. Thermo Forma Steri Cycle 370 CO 2培养箱(Marshall Scientific,目录号:TH-370)
11. Allegra ® X-5 临床台式 IVD 离心机,Beckman Coulter ® (VWR,目录号:89429-566)


手续_


Reel-seq 的简化流程图如图 1 所示。


 
图1. Reel-seq 的简化流程图


A. DNA文库1和2的构建和生成
1. 从 NCBI网站( https://www.ncbi.nlm.nih.gov )下载感兴趣的基因组DNA 序列。
2. All Databases向下滚动到Gene。
3. 输入您感兴趣的基因名称,例如,CDKN2A。
4. 搜索结果中选择基因。
5. Genomic region, transcripts, and products下选择FASTA 。
6. 定义序列的区域,通过输入染色体上的核苷酸数,您的基因位于屏幕右侧显示的更改区域下。例如,从 2230356 到 22172015,对于 9 号染色体上的 58 kb CDKN2A 区域。
7. 单击更新视图,并将序列复制到 word 文件中。
8. 从文库 1 基因组序列的第一个碱基开始连续获取 35 bp 片段,如图形摘要所示。
9. 获取 35 - bp 片段,这样这些片段将覆盖库 1 中两个 35-bp 片段之间的断点。对于 58 kb CDKN2A/B 区域,您将有 1668 个 35-库 1 和库 2 的 bp 片段。
10. 在 Excel 文件中列出每个库的 1668 个序列。
11. 通过将公共侧翼序列添加到 Excel 文件中的每个 35-bp 片段来构建库 1 和 2,如表 1所示。


表 1 Reel-seq 库的构建


12. 从商业供应商处订购库 1 和 2。
注意:您可以将这两个库作为两个子库一起订购,方法是在 5' 端添加公共序列 AGGACCGGATCAACT,在 3' 端添加 CATTGCGTGAACCGA。这些常见的序列将用作大规模平行寡核苷酸合成的接头。


B. 文库 1 和 2 的文库 DNA 分离
1. 收到文库 DNA 后,使用无 DNase 水将合成 DNA 文库重悬至终浓度为 100 ng/mL,并在 -20°C 下保存。
2. 将合成 DNA 文库稀释至1 μL库存,最终浓度为5、10、20、50、100和200 pg / μL 。
3. PCR 扩增文库 DNA,使用引物 PE_SP (L1_FP):5' ACACGCACGATCCGACGGTAGTGT3'和 G3 (L1_RP):5'CGAGCTTATCGTCGTCATCC3' 用于文库 1, new_SP (L2_FP):5' GGTGTGATGCTCGGATCCAGGAAC3'和 926RR (L2_RP):5' CACGAGACTACTGTCCTACC3' 用于库 2。
注意:这些引物是在我们的实验室中出于不同目的生成的,并且是在我们的试点 Reel-seq 筛选中测试的许多引物中选择的。这些引物都适用于 PCR,但有些不适合重复扩增子扩增,有些则不适合二代测序或文库制备。因此,请确保不要为 Reel-seq 筛选更改这些引物。
4. 进行 PCR 反应 T100 热循环仪,如表 2所示, PCR程序见表3 。


表 2。 文库的PCR扩增 脱氧核糖核酸


表 3. PCR 扩增程序


5. Mini-PROTEAN ® Tetra 垂直电泳槽中组装5% Mini-PROTEAN ® TBE 凝胶。
6. μL来分离所有 2 5 μL的 PCR 产物 6 × 凝胶上样染料在5% Mini -PROTEAN ® TBE 凝胶上。
7. 使用 10 μL的 1 ×低分子量DNA 梯作为标记。
8. × TBE 缓冲液在 100 V 下运行凝胶电泳约 60 分钟。
9. 拆卸凝胶盒,使凝胶仍附着在玻璃板的一侧。
10. ×覆盖凝胶表面对凝胶进行染色 GelStar在 RT 下 20 分钟。
11. BLoOK TM可视化 PCR 产物 LED Transilluminator,并从最佳扩增条带(无上条带)切割含有 79 bp 文库DNA 的条带(图 2 )。


图 2. 库 1 (A) 和 2 (B) 的扩增。 
如图所示,用不同浓度的文库 DNA 通过 PCR 扩增显示包含文库 DNA 的 79-bp 条带的 5% 丙烯酰胺凝胶。


12. 将每个切片的凝胶带(约 3 × 5 mm 大小)放入 150 μL 在微管中加入10% TE 缓冲液,并在室温下摇晃过夜。
13. 收集 ~120 μL 含有文库 DNA的液体。此时,您应该为每个含有纯化文库 DNA 的文库配备一个试管。
14. 将带盖的试管放入Thermo Scientific Savant DNA 120 SpeedVac浓缩器中,这是一种真空离心机。
15. 浓缩文库 DNA,首先打开离心机,然后打开真空。
16. 使用Implen C40 NanoPhotometer UV/Vis Spectrophotometer Mobile System将 DNA 样本标准化为 10 ng/ μL 。


C. Reel-seq 筛选文库 DNA 的制备
1. PCR 扩增从程序 B获得的文库 1 和 2 的文库 DNA。
2. 使用 5 μL纯化的文库 DNA(~10 ng/ μL )制备表 2中配制的 PCR 反应,并根据表 3中列出的程序运行 PCR 反应,但只需要 15 个循环。
3. 将每个文库 DNA 一式三份扩增,以便您有足够的 DNA 用于 Reel-seq 筛选。
4. 运行 5 μL 如程序 B (步骤 6-10)中所述,在5% Mini-PROTEAN ® TBE 凝胶上从每个反应中提取 PCR 产物,以检查 PCR 扩增的质量。您应该观察到一个 79 bp 左右的主要条带,所有反应都具有相对相同数量的 DNA。
5. 文库 1 和 2 的扩增文库 DNA 现在已准备好用于 Reel-seq 筛选。


D. 核提取物 (NE) 的分离
1. 2的细胞培养箱中,用 ERG-2 培养基在 150 毫米板中培养 ~1.5 × 10 8人原代动脉 EC 。
2. 使用 DPBS 清洗细胞三次。
3. × Roche cOmplete迷你蛋白酶抑制剂鸡尾酒刮取5 mL DPBS 中的细胞。
4. 使用临床台式 IVD 离心机以 450 g 离心 5 分钟收集细胞。
5. 使用NE-PER TM核和细胞质提取试剂分离 NE ,但仅使用一半体积的核提取试剂 (NER) 来提取 NE。这将产生浓度为 ~10 μ g / μL的 NE 。
6. 根据制造商的说明,使用Roche cOmplete迷你蛋白酶抑制剂混合物来防止蛋白质降解。 
7. 将 NE 分成 ~20 μL 等分试样,并在 -80 °C 下储存至少一年。
笔记: 细胞培养条件和培养基将取决于您用于分离 NE 的细胞系。


E. 用于 EMSA 的 NE 滴定
1. 使用LightShift TM化学发光 EMSA 试剂盒进行滴定,方法是混合表 4中配制的所有试剂。


表 4. 滴定核提取物浓缩反应


2. 使用程序 C中的 5 μL文库 DNA滴定 NE,方法是添加 0、0.5、1、2 和 3 μL在程序 D中分离的 NE 。
3. 将混合物在室温孵育 2 小时或在 4°C 过夜。
4. 使用5% Mini-PROTEAN ® TBE 凝胶进行 EMSA ,每 20 μL反应混合物中加入 5 μL试剂盒中提供的 5 ×上样染料。
5. 如程序 B (步骤 6-10)中所述,在5% Mini-PROTEAN ® TBE 凝胶上运行每个反应的所有 25 μL PCR 产物,并使用BLoOK TM可视化 PCR 产物 LED 透照器。
6. 使用光密度测定法确定与对照(未添加 NE)相比,样本中的文库 DNA 发生偏移的反应,并使用 NE 的量(例如,2 μL )进行以下 Reel-seq 筛选(图 3 )。
笔记: 如果一个 79 bp 的片段没有被清楚地观察到,你应该应用更多的循环,例如 20 个循环用于程序 C的步骤 2 中的 PCR 。


ķ
图 3. NE 滴定。 
丙烯酰胺凝胶显示使用不同量的 NE 滴定导致与对照相比,样品中约 50% 的文库 DNA 移动的 NE 量。


F. 卷轴序列屏幕
1. 根据表 5 ,使用LightShift TM化学发光 EMSA 试剂盒制备 EMSA 反应,一式三份。此时,您应该为每个库拥有三个控件和三个样本。


表 5. EMSA 反应


2. 在室温下摇动反应混合物 (300 rpm) 2 小时或在 4°C 下过夜。
3. 使用5% Mini-PROTEAN ® TBE 凝胶进行 EMSA ,每 20 μL反应混合物中加入 5 μL试剂盒中提供的 5 ×上样染料。
4. 如程序 B (步骤 6-14)中所述,从三个对照和三个样本(图 4A )中纯化 79 bp 未移位的库 DNA 条带。
5. PCR 扩增上面获得的文库 1 和 2 的文库 DNA 。
6. 表 2的规定准备 PCR 扩增反应,并按照表 3中列出的程序运行 PCR ,但只需要 15 个循环。
7. 如程序 B (步骤 6-10)所述,在5% Mini-PROTEAN ® TBE 凝胶上运行每个反应的5 μL PCR 产物,以检查BLoOK TM的 PCR 扩增质量 LED 透照器(图 4B ) 。
8. 重复程序 F十次。
笔记:
a. 您应该在所有反应中观察到一个 79 bp 左右的主要条带(每个库三个对照和三个样本),并且 DNA 量相对相同(图 4B )。扩增的文库 DNA 现在已准备好进行下一轮 Reel-seq 筛选。
b. 此外,一般来说,对 Reel-seq 筛选应用阳性对照是一个好主意。当我们执行这个 Reel-seq 时,我们没有任何阳性对照。这是因为在我们筛选的 58-kb CDKN2A/B 区域中从未发现过 cis-RE。但是,如果可以确保这些 cis-RE 在您用于 Reel-seq 筛选的细胞中发挥作用,则始终可以使用来自人类基因组其他区域的 cis-RE 作为阳性对照。




图 4. Reel-seq 屏幕。 
丙烯酰胺凝胶显示 EMSA ( A ) 和 PCR (B ) 在 Reel-seq 筛选的第 1 轮中的结果,三个缓冲液处理的对照和三个 NE 处理的样品,用于两个文库之一。


G. 使用两个 PCR 反应制备下一代测序文库
1. 从第 1、4、7 和 10 轮中选择 Reel-seq PCR 产品,以准备下一代测序库。由于每轮有3个对照和3个样本,每个文库一共会生成24个子文库。
2. 对每个对照和样品使用 1 μL的 Reel-seq PCR 产物,根据表 6 执行第一个 PCR 反应。 
注意:我们使用Herculas II 融合 DNA 聚合酶,如前所述 ( Larman et al., 2013 )。
3. 根据表 7中列出的程序运行 PCR 。






表 6. 高通量测序前的 PCR 扩增


表 7. 高通量测序前的 PCR 扩增程序


4. 库 1 使用 L1/PE_SP 和 R1/G3 引物,库 2 使用 L1/new-SP 和 R1/926RR 引物。所有这些引物的序列列于表 8中。
注:我们根据二代测序的能力、Reel-seq文库中DNA片段的数量、30-50%的突变率,选择了第1、4、7、10轮的PCR产物进行测序。 Reel-seq 筛选中的 PCR 和测序成本。这四轮产生的数据足以进行统计分析。


表 8. 用于下一代测序文库制备的引物


5. 检查第一个 PCR 反应的质量,通过在 2% 琼脂糖凝胶上运行 5 μL PCR 产物和 2 μL凝胶上样染料。在所有反应中都应观察到一个相对均匀的 132 bp 条带(图 5A和B )。




图 5. 测序文库制备。
琼脂糖凝胶显示第一次 PCR( A和B )和第二次 PCR( C和D )的 PCR 产物。 C:控制; S:样品。


6. 根据表 9执行第二个 PCR 反应,对每个对照和样品使用 2 μL的第一个 PCR 产物,并使用表 10中列出的程序运行 PCR 。


表 9. 高通量测序前的第二次 PCR 扩增反应
表 10. 高通量测序前的第二个 PCR 扩增程序


7. 对两个文库使用 L2 和 R2(条形码)引物,如表 8中所列。总共需要 48 个具有 48 个不同条形码的 R2 引物来扩增 48 个子库。
注意:L2 和 R2 引物包含接头序列 p5 和 p7(表 8 中带下划线)。适配器允许测序文库结合并在流动槽上生成簇以进行照明测序。这些带有条形码的引物的设计和序列在Kozarena和 Tuner (2011) 中进行了描述。
8. 在 2% 琼脂糖凝胶上运行 5 μL PCR 产物和 2 μL Gel Loading Dye来检查第二个 PCR 反应的质量。在所有反应中都应观察到一个相对均匀的 188 bp 条带(图 5C和D )。
9. 根据表 11混合第二个 PCR产品。每个文库都有 186 μL的二级 PCR产物混合物。


表 11. 用于测序文库纯化的混合 PCR 产物
注意:我们根据经验选择了表 11中提到的卷。 PCR 可以产生突变,更多的突变是通过用 NE 处理的文库 DNA 进行 PCR 产生的。因此,我们使用了这些数字 为每个文库获得相对均匀的测序读数(无突变)。


10. 如程序 B (步骤 6-14)中所述,对每个文库使用5% Mini -PROTEAN ® TBE 凝胶纯化混合 PCR 产物。每个 186 μL 文库 DNA 应均匀分布在 9 个孔中,其中 1 个孔用于标记。 
11. 在 188-bp 处切割带,将每个切片凝胶放入 150 μL的 10% TE 缓冲液中,并通过在 RT 上摇晃过夜来隔离测序库。
12. 使用SpeedVac浓缩器将 DNA 文库浓缩至约 50 ng/ μL 。
13. 将两个测序文库发送到核心设施进行下一代测序。对库 1 使用 PE_SP 引物,对库 2 使用 new_SP 引物。
14. NextSeq 500对库 1 和 2 进行测序,单次读取高输出为 75 bp,下午 2.5点,加入1% PhiX 。
笔记:
a. 作为测序的一部分,您应该请求数据分析以列出所有 24 个子库中每个 35 bp 片段的读数,包括库 1 和库 2。 下一代测序在匹兹堡大学的基因组学研究中心进行 ( https://www.genetics.pitt.edu/next-generation-sequencing )。
b. 您用于下一代测序的实际平台取决于您文库的大小。一个小的库可以通过MySeq进行测序;因此,请咨询测序专家以确定您要使用的实际测序平台。


数据分析


1. 使用 Excel 执行所有数据分析。
2. 计算 24 个子库中每个 35 bp DNA 片段的百分比。
3. 使用三个缓冲处理控件和三个 NE 处理样本中的三个百分比来计算第1、4、7和 10 轮中每个片段的学生t 检验的P值。
4. 计算第 1、4、7 和 10 轮中三个缓冲处理对照和三个 NE 处理样本中每个 DNA 片段的百分比平均值。
5. 计算第 1、4、7 和 10 轮中三个 NE 处理样本与三个缓冲处理控件的百分比平均值之比。
6. 应用这四个比率来计算第1、4、7和 10 轮中每个 35 bp DNA 片段的斜率。
7. 应用斜率< 0 作为第一个过滤器,在第1、4、7 和 10 轮中应用P < 0.05 作为第二个过滤器。
8. 在第 1、4、7 和 10 轮中将斜率< 0 和P < 0.05的片段识别为候选顺式- RE。
注意:我们已成功使用 Reel-seq 识别 200 kb 区域上的 cis-RE。但是,此时,我们还不知道 Reel-seq 可以筛选出多大的区域的限制。此外,我们估计使用 Reel-seq 识别 cis-RE 的保真度 > 90%。


食谱


1. 0. 5 × TBE 缓冲液 (1 L)
将 50 m L 的 10 × TBE 添加到 950 mL 的蒸馏水 (dH 2 O) 中。
2. 1 × 凝胶之星(10 毫升)
添加 1 μL的 10,000 × GelStar TM核酸凝胶染色剂至 10 mL dH 2 O。
3. 1 ×低分子量 DNA 梯
添加 100 μL 低分子量 DNA 梯子的 900 μL dH 2 O和 200 μL 6 × DNA 凝胶上样染料。
4. 10% TE 缓冲液
将 1 mL 的 TE 缓冲液添加到 9 mL 的 dH 2 O。
5. 2% 琼脂糖凝胶
μ g琼脂糖溶解在 100 mL 的 0.5 × TBE 中。


致谢


等人在 Nucleic Acids Research 中的原始出版物修改而来的。 (2021 年)。我们感谢 NIH NIA 和 UPMC 的赠款支持。这项工作部分得到了 NIH NIA R01AG056279 (GL)、 R01AG065229 (GL)和 UPMC (GL) 的资助。 


利益争夺


作者声明没有竞争的经济利益。


参考


1. Angeloni , A. 和 Bogdanovic, O. (2019)。增强 DNA 甲基化:对基因调控的影响。 论文生化63(6):707-715。
2. Buenrostro , JD, Wu, B., Chang, HY 和 Greenleaf, WJ (2015)。 ATAC-seq:一种检测全基因组染色质可及性的方法。 电流 Protoc Mol Biol 109:21 29 21-21 29 29。
3. Crawford, GE, Davis, S., Scacheri , PC, Renaud, G., Halawi , MJ, Erdos , MR, Green, R., Meltzer, PS, Wolfsberg , TG 和 Collins, FS (2006)。 DNase-chip:一种使用平铺微阵列识别 DNase I 过敏位点的高分辨率方法。 Nat 方法3(7):503-509。
4. 刁,Y.,Fang,R.,Li,B.,Meng,Z.,Yu,J., Qiu ,Y.,Lin,KC,Huang,H.,Liu,T.,Marina,RJ,等. (2017)。用于哺乳动物细胞中顺式调节元件鉴定的基于平铺缺失的遗传筛选。 Nat 方法14(6):629-635。
5. Fulco , CP, Nasser, J., Jones, TR, Munson, G., Bergman, DT, Subramanian, V., Grossman, SR, Anyoha , R., Doughty, BR, Patwardhan, TA等。 (2019)。来自数千个 CRISPR 扰动的增强子-启动子调节的接触活动模型。 Nat Genet 51(12):1664-1669。
6. Kozarewa , I. 和 Turner, DJ (2011)。用于 Illumina 基因组分析仪的 96 重分子条形码。 方法 Mol Biol 733:279-298。
7. Larman ,HB, Laserson ,U.,Querol,L., Verhaeghen ,K., Solimini ,NL,Xu,GJ, Klarenbeek ,PL,Church,GM, Hafler ,DA, Plenge ,RM等。 (2013)。 PhIP-Seq 表征多发性硬化症、1 型糖尿病和类风湿性关节炎患者的自身抗体。 J自身免疫43:1-9。
8. Li, G., Martinez-Bonet, M., Wu, D., Yang, Y., Cui, J., Nguyen, HN, Cunin , P., Levescot , A., Bai, M., Westra , HJ,等人。 (2018 年)。通过 IIS 型酶限制高通量鉴定非编码功能性 SNP。 Nat Genet 50(8):1180-1188。
9. Mikhaylichenko , O., Bondarenko , V., Harnett, D., Schor, IE, Males, M., Viales , RR 和 Furlong, EEM (2018)。增强子或启动子的活性程度由 eRNA 转录的水平和方向性反映。 基因开发32(1):42-57。
10. 帕克,PJ(2009 年)。 ChIP-seq:成熟技术的优势和挑战。 Nat Rev Genet 10(10):669-680。
11. Rajagopal, N., Srinivasan, S., Kooshesh , K., Guo, Y., Edwards, MD, Banerjee, B., Syed, T., Emons , BJ, Gifford, DK 和 Sherwood, RI (2016)。调节 DNA 的高通量作图。 Nat Biotechnol 34(2):167-174。
12. Schmid, M.、 Durussel , T. 和Laemmli , UK (2004)。 ChiC和ChEC;染色质蛋白的基因组图谱。 摩尔细胞16(1):147-157。
13. Schoenfelder , S. 和 Fraser, P. (2019)。基因表达控制中的远程增强子-启动子接触。 Nat Rev Genet 20(8):437-455。
14. Song, L. 和 Crawford, GE (2010)。 DNase-seq:一种高分辨率技术,用于绘制哺乳动物细胞基因组中的活性基因调控元件。 冷泉港_ 协议2010(2): pdb prot5384。
15. Tewhey , R., Kotliar , D., Park, DS, Liu, B., Winnicki , S., Reilly, SK, Andersen, KG, Mikkelsen, TS, Lander, ES, Schaffner, SF等。 (2016 年)。使用多重报告基因分析直接鉴定数百种表达调节变体。 单元格165(6):1519-1529。
16. Ulirsch ,JC,Nandakumar,SK,Wang,L., Giani ,FC,Zhang,X., Rogov , P.,Melnikov,A., McDonel ,P.,Do,R.,Mikkelsen,TS,等。 (2016 年)。影响红细胞性状的常见遗传变异的系统功能解剖。 165(6)号电池:1530-1545。
17. Wu, T.、Jiang, D.、Zou, M.、Sun, W.、Wu, D.、Cui, J.、Huntress, I.、Peng, X. 和 Li, G. (2022)。将高通量作图与蛋白质组学分析相结合,以高分辨率描绘了顺式调控元件。 核酸研究50(1):e5。
18. Zhang, Y.、Sun, Z.、Jia, J.、Du, T.、Zhang, N.、Tang, Y.、Fang, Y. 和 Fang, D. (2021)。组蛋白修饰概述。 Adv Exp Med Biol 1283:1-16。

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引用:Wu, T. and Li, G. (2022). An Improved EMSA-based Method to Prioritize Candidate cis-REs for Further Functional Validation. Bio-protocol 12(8): e4397. DOI: 10.21769/BioProtoc.4397.
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