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Apr 2020

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Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
细菌转录起始位点的差异RNA序列全基因组鉴定   

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Abstract

Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence.

Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5′-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis.

Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

Keywords: dRNA-seq (dRNA-seq), Transcriptional start site (转录起始位点), TSS (TSS), RNA purification (RNA纯化), Hot phenol RNA extraction (热酚RNA提取), rRNA depletion (rRNA去除), 5′-monophosphate-dependent exonuclease (TEX) (5′-单磷酸依赖核酸外切酶(TEX)), Phenol chloroform:isoamyl alcohol RNA extraction (苯酚氯仿:异戊醇RNA提取), Bacterial gene regulation (细菌基因调控), RNA precipitation (RNA沉淀), RNA phosphorylation (RNA磷酸化)

Background

Transcription in bacteria is initiated by the RNA polymerase holoenzyme, which recognizes specific sequence elements on the DNA within the promotor region, to which sigma factors are bound (Feklistov et al., 2014). This RNA polymerase holoenzyme binding site defines the Transcriptional Start Site and the direction of transcription. For example, the most common house-keeping sigma factor, named 𝝈70 in Escherichia coli, recognizes two elements centered approximately 10 and 35 bp upstream of the TSS (Feklistov et al., 2014). The RNA polymerase holoenzyme melts the double stranded DNA between 11 nt upstream (position -11) to 3 nt downstream (+3) of the TSS (+1), and the single-stranded DNA can then be used as template for the addition of tri-phosphorylated ribonucleotides. The initiation starts mainly at a specific position, but sometimes “wobbles” of one or more bases up- or downstream are encountered (Murakami and Darst, 2003; Robb et al., 2013; Vvedenskaya et al., 2015). The DNA sequence around TSS have long been recognized as crucial for gene regulation in bacteria (Jacob and Monod, 1961). Depending on the position within the gene structure, which begins with a start codon (usually ATG) and finishes with one of the three stop codons, we can identify two types of transcriptional start sites: primary and secondary. Primary transcriptional start sites (pTSS) are located some nucleotides upstream of the translational start site, while the secondary transcriptional start sites (sTSS) are located within the gene encoding sequence (Figure 1).


Figure 1. Schematic representation of the Primary and Secondary Transcriptional Start Site definition

Until the advent of next-generation sequencing, in order to locate the TSS of a specific RNA, it was necessary to examine each transcript individually, using either the S1 protection assay, primer extension or a 5’ RACE method (Sharma and Vogel, 2014). Owing to the increasing popularity and a decrease in costs of high-through put sequencing, in 2010 differential RNA-seq (dRNA-seq) was developed to simultaneously map all TSS of a genome using Helicobacter pylori as first model organism (Sharma et al., 2010). Since then, this method has been widely employed to determine the TSS of several bacterial species (Berghoff et al., 2009; Jager et al., 2009; Albrecht et al., 2010 and 2011; Bohn et al., 2010; Irnov et al., 2010; Schluter et al., 2010; Sharma et al., 2010; Beckmann et al., 2011; Deltcheva et al., 2011; Filiatrault et al., 2011; Mitschke et al., 2011a and 2011b; Kroger et al., 2012 and 2013; Madhugiri et al., 2012; Ramachandran et al., 2012 and 2014; Sahr et al., 2012; Schmidtke et al., 2012; Wilms et al., 2012; Cortes et al., 2013; Dugar et al., 2013; Mentz et al., 2013; Nickel et al., 2013; Pfeifer-Sancar et al., 2013; Porcelli et al., 2013; Schluter et al., 2013; Voss et al., 2013; Wiegand et al., 2013; Zhang et al., 2013; Voigt et al., 2014; Cervantes-Rivera et al., 2020).

Primary transcripts of prokaryotes carry a triphosphate at their 5’-ends. In contrast, processed or degraded RNAs only carry a monophosphate at their 5’-ends. This is also the case of ribosomal RNA (rRNA) (Schoenberg, 2007). The dRNA-seq approach used here exploits the properties of a 5’-monophosphate-dependent exonuclease (TEX) to selectively degrade processed transcripts, thereby enriching for unprocessed RNA species carrying a native 5’-triphosphate (Schoenberg, 2007). TSS can then be identified by comparing TEX-treated and untreated RNA-seq libraries, where TSS appear as localized maxima in coverage enriched upon TEX-treatment (Sharma et al., 2010).

Until 2013 TSS annotation was performed manually, but this method is arduous and time-consuming. Nowadays many computational tools are available for automatic TSS annotation using dRNA-seq data. These include TSSPredator (Dugar et al., 2013), TSSAR (Amman et al., 2014), TruHMM (Li et al., 2013), TSSer (Jorjani and Zavolan, 2014) and ReadXplorer2 (Hilker et al., 2016).

Here, we present a step by step protocol for TSS determination through comparison of TEX-treated and untreated RNA libraries in Shigella flexneri serotype 5a strain M90T as originally performed in (Cervantes-Rivera et al., 2020). The overall workflow is illustrated in Figure 2.


Figure 2. Workflow of dRNA-seq for whole-genome Transcriptional Start Sites identification

Materials and Reagents

  1. Sterile syringe filter with pore size 0.22 μm (Merck, catalog number: SLGV033RK )
  2. Nitrile gloves
  3. Culture tube of 13 ml (TPP, catalog number: 91016 )
  4. Microfuge tubes 1.5 ml (Eppendorf, Safe-Lock tubes, catalog number: 00 30120086 )
    Note: In principle all prepackaged tubes on the market are RNase free and can be used.
  5. Centrifuge tubes of 50 ml (Sarstedt, catalog number: 62.547.254 )
  6. Pipette tips (VWR, catalog numbers: 89041-404 , 89041-412 , 89041-400 )
    Note: In principle all prepackaged tips on the market are RNase free and can be used.
  7. Semi-micro cuvette (Sarstedt, catalog number: 67.742 )
  8. Shigella flexneri 5a M90T (Sansonetti et al., 1982), can also be purchased from ATCC and available on request from the authors
  9. Tryptone soy broth (TSB) ready to use powder (Merck, catalog number: 105459 )
  10. Tryptone soy agar (TSA) ready to use powder (Merck, catalog number: 105458 )
  11. Congo red (Sigma, catalog number: C6277 )
  12. Diethyl pyrocarbonate (DEPC) (Sigma, catalog number: 40718 )
  13. Sodium dodecyl sulfate (SDS) (Sigma, catalog number: L3771 )
  14. Bromophenol blue sodium salt (Sigma, catalog number: B8026 )
  15. Xylene cyanol FF (Sigma, catalog number: X4126 )
  16. Glycerol (Sigma, catalog number: G5516 )
  17. Formamide (Sigma, catalog number: 47671 )
  18. Ethanol absolute (VWR Chemicals, catalog number: 20821.558 )
  19. Sodium acetate 3 M, pH 5.5 (Ambion, catalog number: AM9740 ).
  20. EDTA, disodium salt, dihydrate (Sigma, catalog number: 324503 )
  21. Sodium hydroxide (Sigma, catalog number: S8045 )
  22. Trizma base (Sigma, catalog number: T1503 )
  23. Acetic acid (Sigma, catalog number: 695092 )
  24. Agarose (VWR Life Science, catalog number: 35-1020 )
  25. DNase I 10 U/ml (Roche, catalog number: 0 4716728001 )
  26. RNaseOUT 40 U/ml (Invitrogen, catalog number: 10777019 )
  27. Chloroform (Sigma, Catalog number: C2432 )
  28. Phenol solution pH 4.3 ± 0.2 (Sigma, catalog number: P4682-400ML )
  29. Isoamyl alcohol (Sigma, catalog number: W205702 )
  30. Sodium acetate pH 5.5 (Ambion, catalog number: AM9740 )
  31. Glycogen 20 mg/ml (Invitrogen, catalog number: R0551 )
  32. Oligos SF-Hfq-F 5′-ACGATGAAATGGTTTATCGAG-3′ and SF-Hfq-R 5′-ACTGCTTTACCTTCACCTACA-3′ (Sigma, custom order)
  33. GeneRuler 1 kb DNA ladder (Thermo Scientific, catalog number: SM0211 )
  34. Linker-RNA-adaptor 5′-GACCUUGGCUGUCACUCA-3′ (Sigma, custom order)
  35. Ribo-Zero rRNA Removal Kit for bacteria (Illumina, catalog number: MRZB12424 )
    Note: This product is not available anymore on the market, but can be replaced with Pan-Prokaryote riboPOOL kit (siTOOLS BIOTECH, Pan-Prokaryote v2), RiboCop rRNA Depletion Kit for Bacteria (Lexogen, catalog number: 125-127 ) or any other rRNA depletion method based on the use of hybridization probes. Replacement products relying on enzymatic-based rRNA depletion such as the Ribo-Zero Plus rRNA Depletion Kit (Illumina, catalog number: 20037125 ) are not suitable.
  36. Terminator-5′-Phosphate-Dependent Exonuclease (TEX) (Lucigene, catalog number: TER51020 )
  37. 5’-Pyrophosphohydrolase 5,000 units/ml (RppH) (New England BioLabs, catalog number: M0356S )
  38. T4 RNA ligase 10,000 U/ml (New England BioLabs, catalog number: M0204L )
  39. T4 polynucleotide kinase (PNK) 10,000 U/ml (New England BioLabs, catalog number: M0201L )
  40. RNaseZap (Ambion, catalog number: AM9780 )
  41. Dream Taq DNA polymerase (Thermo Scientific, catalog number: EP0701 )
  42. dNTPs mix, 2 mM each (Thermo Scientific, catalog number: R0241 )
  43. GelRed nucleic acid gel stain (Biotium, catalog number: 41003 )
  44. ATP solution (100 mM) (Thermo Scientific, catalog number: R0441 )
  45. Tryptone soy broth (TSB) medium (see Recipes)
  46. Tryptone soy agar (TSA) plates (see Recipes)
  47. Congo red solution (see Recipes)
  48. Nuclease-free water (see Recipes)
  49. Sodium dodecyl sulfate (SDS) 10% solution (see Recipes)
  50. EDTA 0.5 M, pH 8 (see Recipes)
  51. 6x DNA Loading buffer (see Recipes)
  52. 2x RNA Loading buffer (see Recipes)
  53. Stop Solution (see Recipes)
  54. Ethanol 80% (see Recipes)
  55. Lysis solution (see Recipes)
  56. 10x TAE (see Recipes)
  57. 1% Agarose (see Recipes)
  58. Phe-CHISAM solution (see Recipes)
  59. CHISAM (see Recipes)

Equipment

  1. Microcentrifuge, refrigerated (VWR, model: Micro Star 17R )
  2. Spectrophotometer (Amersham, model: Ultrospec 2100 pro )
  3. Pipettes (Eppendorf Research plus)
  4. Microwave oven (Whirlpool, model: MD101 )
  5. Centrifuge (Eppendorf, model: 5810R )
  6. NanoDrop ND-1000 spectrophotometer (Saveen & Werner AB)
  7. Orbital Shaker (Edmund Bühler, model: Swip SM25 )
  8. Block heater (VWR, model: 460-0353 )
  9. DynaMag-2 Magnet (Thermo Fisher Scientific, catalog number: 12321D )
  10. T100 Thermo cycler (Bio-Rad, catalog number: 186-1096 )
  11. Chemical hood
  12. Horizontal gel electrophoresis system (VWR, model: 700-0015 )
  13. Electrophoresis power supply (VWR, model: E0202 )

Software

Programs
All programs used in this protocol are freely available

  1. bcl2fastq (https://emea.support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html)
  2. FastQC/0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc) (Wingett and Andrews, 2018)
  3. MultiQC/1.8 (https://multiqc.info/) (Ewels et al., 2016)
  4. trimmomatic/0.36 (http://www.usadellab.org/cms/?page=trimmomatic) (Bolger et al., 2014)
  5. samtools/1.9 (http://www.htslib.org/doc/samtools.html) (Li et al., 2009)
  6. bowtie2/2.3.5.1 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) (Langmead and Salzberg, 2012)
  7. IGV/2.3.82 (https://software.broadinstitute.org/software/igv/) (Thorvaldsdottir et al., 2013)
  8. ReadXplorer/2.2.3 (https://www.uni-giessen.de/fbz/fb08/Inst/bioinformatik/software/ReadXplorer) (Hilker et al., 2014; Hilker et al., 2016)

Databases

  1. GenBank (https://www.ncbi.nlm.nih.gov/genome/182?genome_assembly_id=493862)
  2. RefSeq (https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP037923.1, https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP037924.1)
  3. RegulonDB (http://regulondb.ccg.unam.mx/)
  4. dRNA-seq raw data are available on the SRA database under the accession numbers: SRR8921222(dRNA-Seq_TEX_Positive), SRR8921223 (dRNA-Seq_TEX_Negative) (https://dataview.ncbi.nlm.nih.gov/)

Procedure

Before starting the RNA purification be sure that you have cleaned pipets, centrifuges, bench, the chemical hood, equipment for gels and the marker pen with RNaseZap to ensure that everything is RNase free. At every step of the protocol you should wear nitrile/latex gloves to avoid RNase contamination.


  1. Total RNA purification using the hot phenol method and RNA quality control by electrophoresis
    1. Prepare a pre-culture by inoculating 5 ml of TSB medium with a single colony of S. flexneri 5a M90T from a Congo red tryptone soy agar plate and grow overnight (~16 h) at 37 °C with agitation (150 RPM).
      Note: Ensure that the colonies used are secretion-competent (forming red colonies on the plate due to Congo red absorption) (Sharma and Puhar, 2019).
    2. Subculture by diluting 100 μl of the pre-culture in 10 ml of TSB (1:100 dilution).
    3. Grow to OD600 = 0.3 with shaking. This will take approximately 2 h if the medium is pre-warmed.
    4. Mix the culture with 2 ml of cold stop solution.
    5. Incubate the mixed culture with the stop solution on ice for 30 min.
      Note: To stabilize the RNA and prevent degradation, incubate it for at least 30 min, but not longer than 2 h on ice.
    6. Transfer the culture into a 15 ml tube.
    7. Centrifuge for 5 min at 16,200 x g at 4 °C.
    8. Remove the supernatant by decanting. Keep ~2 ml of supernatant to resuspend the pellet in it.
    9. Transfer the resuspended cells into two 1.5 ml tubes.
    10. Centrifuge for 5 min at 16,200 x g at 4 °C.
    11. Discard the supernatant by decanting.
      Note: At this point the samples could be frozen with liquid nitrogen and stored at -80 °C. Samples are stable for months. 
    12. Purify the total RNA using the hot phenol method as follows.
    13. Resuspend cell pellets in 500 μl of lysis solution at room temperature.
    14. Under the chemical hood add 500 μl of phenol pH 4 pre-warmed to 65 °C. Mix by inversion at least 20 times.
      Note: Pre-warm in a heat block the phenol before starting the RNA purification.
    15. Vortex vigorously for 20 s.
    16. Incubate 5 min at 65 °C in the heat block.
    17. Centrifuge 3 min at 13,800 x g at room temperature.
      Note: If the centrifugation step is done at 4 °C the SDS is going to precipitate and the solution turns white, which does not affect the efficacy of the process but makes it difficult to differentiate the two phases. After this centrifugation time you should see two phases, the upper one should be whitish (when the SDS is cooling down it becomes insoluble). RNA is in the aqueous phase (upper phase), while in the clearly white disk interphase there are protein layers and DNA, whereas lipids and some other cellular components are dissolved in the lower transparent phase that is the organic phase.
    18. Transfer the upper phase (~500 μl) to a new RNase-free tube under the chemical hood.
    19. Add 1 ml of cold (4 °C) absolute ethanol.
    20. Mix thoroughly by vortexing for 5 s until you see a homogeneous mixture.
    21. Centrifuge at 16,200 x g for 5 min at 4 °C.
    22. Discard supernatant that contains the ethanol solution by decanting.
      Note: At this step you can see a white/transparent pellet on the bottom.
    23. Add 1 ml of 80% ethanol at room temperature. Shake the tube vigorously for 10 s.
      Note: Do not use the vortex, it can detach the pellet from the bottom.
    24. Centrifuge at 16,200 x g for 2 min at 4 °C.
    25. Discard the supernatant by decanting.
    26. Centrifuge (short spin) the tubes and discard the remaining liquid by pipetting.
    27. Dry samples at room temperature by leaving the tubes uncapped on the bench.
      Note: Once that the sample is completely dry it turns transparent. It can take 10 min to obtain a completely dry sample.
    28. Resuspend the RNA sample in 100 μl of nuclease free water. 
    29. Check the quality of the purified RNA in a 1% agarose gel in TAE 1x as follows.
    30. Prepare the sample to be analyzed in a 1.5 ml tube. Add 1 μl of sample plus 5 μl of RNA loading buffer 2x and 4 μl of nuclease free water. 
    31. Heat the sample for 5 min at 65 °C for secondary and tertiary structures denaturation.
    32. Cool down the tube in ice for 5 min.
    33. Spin down for 2 s and load the gel.
    34. Run the gel in TAE 1x buffer for 40 min at 100 V.
    35. Stain the gel with 3x-concentrated Gel Red for 10 min.
    36. Wash the gel with 20 ml of nuclease free water for 10 min shaking at room temperature.
    37. Visualize the gel under UV light (Figure 3A). If the total RNA purification was successful, the 23S and 16S are visible as clear, heavy bands, while in the lower part of the gel weaker tRNA bands are observed. It is not possible to see mRNAs. In case of RNA degradation, bands appear as smear.
    38. Quantify the RNA in a NanoDrop spectrophotometer (Figure 3B).
      Note: At least 20 μg (10 μg for each of two conditions) of total RNA are necessary to continue with the protocol. Typical concentrations should be around 500-900 ng/ml. Very successful purifications yield up to 1,500 ng/ml RNA.


      Figure 3. Total RNA quality control. A. Separation of total RNA from three replicates in a 1% agarose gel in TAE of Shigella flexneri 5a M90T as total RNA quality control. B. Examples of typical values obtained from total RNA quantification in a NanoDrop. The 260/280 ratio should be around 1.88-2.00.

    39. Freeze the RNA samples at -20 °C. The RNA samples are stable for months.

  2. DNase treatment to remove contaminating genomic DNA from total RNA
    Protocol volumes are for one reaction, but every step is scalable to two or more reactions.
    1. Dilute 10 μg of total RNA to a final volume of 30 μl using nuclease free water.
    2. Denature RNA in nuclease free water for 5 min at 65 °C in the heat block.
    3. Cool down in ice for 5 min.
    4. Prepare a reaction mix containing the components listed in Table 1. The indicated quantities are sufficient for one sample containing 10 μg of RNA.

      Table 1. DNase reaction mix for one reaction
      Note: 1 Unit of DNase I for 1 μg of total RNA.


    5. Add the reaction mix to the RNA sample tube on ice.
    6. Incubate the reaction mix for 60 min at 37 °C in the heat block.
      Note: Be sure that the heat block is at the correct temperature, DNase works efficiently at 37 °C.
    7. Add to the reaction tube 60 μl of nuclease free water to obtain 100 μl final volume.

  3. RNA clean-up by chloroform:isoamyl alcohol extraction and ethanol precipitation
    1. Add 100 μl of CHISAM to the reaction tube under the chemical hood.
    2. Mix with the vortex vigorously for 15 s.
    3. Centrifuge the tube for 2 min at 13,800 x g, 4 °C.
    4. Under the chemical hood transfer ~100 μl from the aqueous phase (upper) that contains the RNA to a new 1.5 ml tube. Discard the organic phase (lower), proteins and salts from the buffer are dissolved in this phase.
    5. Repeat Steps C1-C4.
    6. Add 300 μl of cold absolute ethanol (3 sample volumes), 10 μl of sodium acetate 3 M pH 5.5 (1/10 of sample volume) and 1 μl of glycogen (20 mg/ml).
    7. Vortex the mix vigorously for 5 s.
    8. Incubate the tube at -20 °C for 30 min.
      Note: This step is optional, but it is good to do it to decrease the loss of RNA in the next steps.
    9. Centrifuge at 16,200 x g (or maximum speed) for 30 min at 4 °C.
      Note: Centrifugation step can be for longer time. The minimum is 30 min, but it could be for 2 to 4 h.
    10. Discard the supernatant carefully by decanting.
      Note: On the bottom of the tube there should be a white pellet. Discard the supernatant without detaching the pellet. 
    11. Add 1 ml of 80% ethanol at room temperature to wash the RNA.
    12. Mix the tube by inverting 20 times.
    13. Centrifuge the tube for 5 min at 13,800 x g, 4 °C.
    14. Discard the supernatant by decanting.
    15. Centrifuge (short spin) and discard the remaining liquid with a 100 μl pipette.
    16. Keep the tubes open on ice to dry the samples.
      Note: The time required to dry the samples depends on how well the liquid phase was removed (usually it takes 10-15 min). The pellet is completely transparent once it is completely dry.
    17. Resuspend the total RNA in 23 μl of nuclease free water.
    18. Check the integrity of the DNase-free RNA by visual inspection of the 23S and 16S band on 1% agarose gel as performed after the total RNA extraction.
    19. Prepare the running sample as in Steps A30-A37.
    20. Visualize the gel under UV light (Figure 4A).
    21. Quantify the RNA in the NanoDrop spectrophotometer (the final RNA concentration should be ~600-1,000 ng/ml) (Figure 4B). It is normal to detect lower concentrations after the process, as the presence of DNA increase the absorbance of the sample.


      Figure 4. Total RNA quality control after DNase treatment. A. Separation of total RNA from three replicates in a 1% agarose gel in TAE of Shigella flexneri 5a M90T as total RNA quality control. B. Examples of typical values obtained from total RNA quantification after DNase treatment in a NanoDrop.

    22. Freeze the RNA samples at -20 °C. The RNA samples are stables for months.

  4. PCR to verify the removal of genomic DNA
    To be sure that there is no trace of DNA in the RNA samples it is extremely important to check the absence of genomic DNA by PCR.
    1. Use 1 μl (~1 μg) of the DNase treated RNA sample as a template to check for the absence of genomic DNA.
    2. As a positive control use ~10 ng of genomic DNA from S. flexneri 5a M90T. Genomic DNA can be extracted using the phenol-chloroform protocol as described in (He, 2011).
    3. As a negative control use a reaction without any template.
    4. Prepare the PCR mix (Table 2). The indicated quantities are sufficient for one sample.

      Table 2. Reaction mix for the PCR control

      Note: The oligos used here amplify a ~909 bp long product of the hfq gene (303 nt) from S. flexneri 5a M90T including 300 bp upstream and 300 bp downstream of the hfq gene.

    5. PCR reaction settings (Table 3).

      Table 3. Settings for control PCR


    6. Check the product in a 1% agarose gel as follows.
    7. Prepare the sample to be analyzed. Add 6 μl of sample plus 2 μl of DNA loading buffer 6x.
    8. Run the 1% agarose gel in TAE 1x buffer for 40 min at 100 V.
    9. Stain the gel with Gel Red 3x for 10 min.
    10. Wash the gel with nuclease free water.
    11. Visualize the gel under UV light (Figure 5).


      Figure 5. Control PCR to verify the absence of genomic DNA with the hfq gene plus 300 pb upstream and downstream of Shigella flexneri 5a M90T as target using genomic DNA as a positive control. Agarose gel 1% in TAE 1x. The absence of a PCR product seen as a 909 bp band in lanes 2-4 indicates that the purified RNA is devoid of DNA contaminations.

  5. rRNA depletion
    Ribosomal RNA is the most abundant RNA species. If rRNA is not removed, most reads from sequencing will originate from rRNA. As a consequence, samples which are compared need to all either have or be devoid of rRNA, or else the sequencing depth will be very different and statistical analysis cannot be performed. The TEX treatment described in the next section to enrich for primary RNA removes all RNA that is not tri-phosphorylated including rRNA, making this library rRNA-depleted. Therefore, to allow the comparison of TEX-treated with mock-treated samples, rRNA needs to be specifically depleted first from total RNA before splitting the RNA into two for further treatment.
        rRNA depletion is carried out using a commercial kit, which specifically depletes bacterial rRNA, according to manufacturer’s instructions. Specific depletion of bacterial rRNA generally relies on hybridization with tagged pan-bacterial probes that recognize conserved sequences in rRNA, followed by removal of captured rRNAs, for example by precipitation with magnetic beads that bind the tag of the probes.
    We have used the Ribo-Zero rRNA Removal Kit for Bacteria from Illumina, but unfortunately it is not available anymore on the market. However, it can be replaced with any hybridization-based kit of your choice. We provided some suggestions in the Material section.

  6. Terminator exonuclease (TEX) treatment to enrich for primary transcripts
    1. Prepare two (TEX- and TEX+) 1.5 ml reaction tubes with 10 μg (10 μl) each of DNase I treated RNA (from the same sample) in nuclease free water.
    2. Denature RNAs for 2 min at 90 °C.
    3. Cool down on ice for 5 min.
    4. Add to each tube the reaction mix described in Table 4. The indicated quantities are sufficient for one sample containing 10 μg of RNA:

      Table 4. Reaction mix for TEX treatment


    Note: The final reaction volume is going to be 20 μl after adding the enzyme for the TEX+ sample or water for the TEX- sample.
    1. Add 1 μl of nuclease free water to the TEX- sample.
    2. Add 1 μl of TEX (1 U/ml) to the TEX+ sample.
    3. Incubate for 60 min at 30 °C in the thermo cycler.
      Note: This step could be carried in a heat block, but the temperature variation and liquid evaporation are important factors that hamper good results, and these effects are stronger in a heat block.
    4. Add 80 μl of nuclease free water to obtain 100 μl final volume.
    5. Add 100 μl of Phe-CHISAM to the reaction tube under the chemical hood.
    6. Mix with the vortex vigorously for 15 s.
    7. Centrifuge the tube for 2 min at 13,800 x g, 4 °C.
    8. Transfer ~100 μl of the upper phase that contains the RNA to a new tube under the chemical hood.
    9. Repeat Steps F9 to F12.
    10. Add 300 μl of cold absolute ethanol (3 sample volumes), 10 μl of sodium acetate 3M pH 5.5 (1/10 of sample volume) and 2 μl of glycogen (20 mg/ml).
    11. Vortex the sample vigorously.
    12. Incubate the tube at -20 °C overnight (around 16 h), but could be longer, for example over the weekend.
      Note: This step is very important. It helps complete recovery of the RNA.
    13. Centrifuge at 16,200 x g for 1 h at 4 °C.
      Note: The centrifugation step could be for longer time. Minimum 1 h, but could be for 2-4 h.
    14. Discard the aqueous phase carefully by decanting.
      Note: On the bottom of the tube there should be a white pellet. Discard the aqueous phase without detaching the pellet. In the case that you do not see any pellet before discarding the supernatant, just add 10 μl of sodium acetate 3 M pH 5.5 (1/10 of sample volume) and 2 μl of glycogen (20 mg/ml) and repeat Steps F16 to F18).
    15. Add 1 ml of cold 80% ethanol at room temperature to wash the RNA.
    16. Centrifuge for 5 min at 13,800 x g, 4 °C.
    17. Discard the aqueous phase by decanting.
    18. Centrifuge (short spin) and discard the remaining liquid with a 100 μl pipette.
    19. Keep the tubes open on ice to dry the samples.
      Note: The time required to dry the samples depends on how well the liquid phase was removed. It can take around 10-15 min. The pellet will be completely transparent once it is completely dry.
    20. Resuspend the RNA in 13 μl of nuclease free water.
    21. Check the integrity of the TEX- and TEX+ treated RNA by visual inspection on 1% agarose gel.
    22. Prepare the running sample as in Steps A29-A36. 
    23. Visualize the gel under UV light (Figure 6).


      Figure 6. Shigella flexneri 5a M90T RNA treated (TEX+) and untreated (TEX-) with TEX

      Note: It is normal not to see clear bands in this gel for the mRNA, which is a mix of different transcripts of variable length. To see a smear in the gel means that the treatment was successful. If you do not have a high quantity of RNA maybe you will see only tRNAs.

    24. Quantify the RNA in the NanoDrop spectrophotometer (final RNA concentration should be ~20 ng/ml).
    25. Freeze the RNA samples at -20 °C. The RNA samples are stables for months.

  7. RNA 5’ Pyrophosphohydrolase (RppH) treatment of RNA to remove terminal phosphates
    In the following steps, the 5′-ends of the RNA prepared in Procedure F is modified with a linker-RNA-adaptor of known sequence, which allows the identification of TSS with single nucleotide precision. This is necessary because the sequence of adaptors added during library preparation for sequencing can be a trade secret (the full sequence of Illumina adaptors was only revealed recently and other platforms using adaptors of unknown sequence exist). During bioinformatic analysis of sequencing results, the sequence of adaptors is occasionally incompletely removed from reads and the remaining nucleotides could be confused for transcribed bases if they happened to align with the DNA sequence. However, this problem can be avoided by introducing the RNA adaptor of known sequence between the adaptors and the TSS. Moreover, the linker-RNA-adaptor allows to univocally identify 5′ ends present before RNA shearing during library preparation. To allow the ligation of adaptors to the 5’ end of the linker-RNA-adaptor during library preparation, the linker-RNA-adaptor must have a triphosphate in the 5′ end.
    1. Denature remaining 10 μl of TEX+ and TEX- treated RNA for 1 min at 90 °C.
    2. Cool down on ice for 5 min.
    3. Prepare RppH mix as described in the Table 5. The indicated quantities are sufficient for one sample:

      Table 5. Reaction mix for RppH reaction of TEX treated RNA


    4. Incubate at 37 °C for 30 min in the thermocycler.
    5. Add 80 μl of nuclease free water and follow the Steps F8-F23.
    6. Resuspend the RNA sample in 13 μl of nuclease free water.
    7. Check the integrity of the RppH treated RNA by visual inspection on 1% agarose gel.
      Note: This step it not strictly necessary, and with every step you are losing some RNA. The gel should look similar as the one shown in Figure 6.
    8. Quantify the RNA in the NanoDrop spectrophotometer.

  8. Linker-RNA-adaptor phosphorylation prior to ligation to TEX+ and TEX- libraries
    1. Denature 100 pmol (10 μl) of the linker-RNA-adaptor per sample dissolved in nuclease free water for 5 min at 90 °C.
    2. Cool down on ice for 5 min.
      Note: You can keep the oligo on ice for longer time while you prepare the reaction.
    3. Prepare the T4 PNK mix according the Table 6.

      Table 6. Reaction mix for T4 PNK phosphorylation of the linker-RNA-adaptor


    4. Incubate at 37 °C for 30 min in the thermo cycler.
    5. Add 80 μl of nuclease free water, follow the Steps F8-F23.
    6. Resuspend the RNA sample in 10 μl of nuclease free water.
      Note: After this step the RNA adaptor can be frozen at -20 °C and used later.

  9. Linker-RNA-adaptor ligation to TEX+ and TEX- samples
    The efficiency of linker-RNA-adaptor ligation is around 80-90%.
    1. Denature the phosphorylated RNA adaptor (10 μl) and RNAs treated with RppH (10 μl) at 65 °C for 5 min.
    2. Cool down on ice for 5 min.
    3. Prepare the T4 RNA ligase mix as described in Table 7 (quantities described on the table are for only one sample, if you are processing more samples just scale them up).

      Table 7. Reaction mix for linker-RNA-adaptor ligation


    4. Incubate at 25 °C for 16 h in the thermo cycler.
    5. Add 60 μl of nuclease free water.
    6. Stop the reaction and precipitate the reaction following Steps F9-F23.
    7. Resuspend the RNA sample in 23 μl of nuclease free water.
      Note: Resuspension volume is defined by the sequencing facilities that you have decided to use. The RNA concentration in our case was 150 ng in 23 μl.
    8. Quantify the RNA adaptor by NanoDrop spectrophotometer.

  10. Library preparation for Illumina sequencing
    Library preparation for sequencing and RNA-sequencing is performed at the service platform or company of your choice. Stranded libraries must be prepared to identify the coding DNA strand. Paired-end sequencing with 150 nt read length should be performed. We used a TrueSeq library on a HiSeq 2000 platform. For microbial genomes, 10-20 M reads per sample will provide appropriate sequencing depth, but 5 M it is enough for an acceptable coverage.

Data analysis

All the programs used in this protocol are based on Linux and freely available on the web. We perform all analyses remotely on a high-performance computer cluster. However, if no access to high-speed remote analysis is available, it is possible to run all the analysis locally after downloading the programs, but this may take a long time if the computing power is low.
    Download all the programs, raw data and the reference genome into the working directory. In this protocol as example we have called the working directory dRNA_seq.

Note: If you use Windows system you should install Ubuntu (https://ubuntu.com/download/desktop) and run all the programs in this environment.


  1. Demultiplexing of Illumina reads
    Note: This step is only necessary in case you perform the sequencing in your own laboratory. If it is the case you will get a file called *.bcl.gz, which first needs demultiplexing for downstream analysis. This step is not necessary if you use a sequencing service, because you will get the already processed *.fastq.gz file.
    bcl2fastq
    1. Go to your working directory where you have your *.bcl.gz file.
    2. Execute bcl2fastq
      $ bcl2fastq [options]

  2. Quality control of raw data
    1. FastQC (Figure 7)
      1. Go to your working directory. In this example the working directory:
        $ cd dRNA_seq
      2. Create a new directory for the output of FastQC analysis.
        $ mkdir FastQC_1
      3. Execute FastQC.
        $ fastqc /dRNA_seq/your_file.fq.gz -o /dRNA_seq/ FastQC_1


        Figure 7. FastQC report of quality control. A. Successful quality control. The quality scores should be in the green area for the entire length of the sequenced fragment. B. Unsuccessful quality control. The quality scores for part of the reads are low (found in the orange or red areas).

    2. MultiQC
      Run MultiQC to merge all the FastQC outputs.
      $ multiqc /FastQC_1

  3. Cleaning raw data from low quality sequences and Illumina adaptors
    Trimmomatic
    Run Trimmomatic in your working directory.
    $ java -jar trimmomatic.jar PE /your_raw_data_1.fq.gz your_raw_data_2.fq.gz your_raw_data_paired_1.fq.gz your_raw_data_unpaired_1.fq.gz your_raw_data_paured_2.fq.gz your_raw_data_unpaired_2.fq.gz ILLUMINACLIP:/TruSeq3-PE-2.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:100

  4. Select the fastq files that contains the RNA adaptor sequence
    The easiest way to perform this step is using the command grep in your terminal. The linker-RNA-adaptor at this step should be in DNA format.
    $ zgrep -B 1 -A 2 GACCTTGGCTGTCACTCA your_raw_data_paired_1.fq.gz > Picked_Up.fastq

  5. Genome reference index
    1. It is quite likely that your reference genome has more than one replicon. We would recommend to merge all of them into one single file to be used as a refence genome.
      $ cat chromosome.fasta plasmid.fasta > reference_genome.fasta
    2. Create the index with bowtie2.
      $ bowtie2-build reference_genome.fasta index_base_name

  6. Genome reference mapping
    Map the selected reads with the reference genome.
    $ bowtie2 -q -N 0 –no-unal -x index_base_name -U your_raw_data_paured_1.fq.gz -S mapping_1.sam

  7. Sort by position and build an index of BAM file
    The output file from Bowtie2 should be sorted and indexed for use in IGV.
    $ samtools view -Sb -o mapping_1.bam mapping_1.sam
    $ samtools sort -o mapping_Sorted_1.bam mapping_1.bam

  8. Visualize the mapping files in IGV (Figure 8)
    1. Create a genome reference index.
      $ samtools index reference_genome.fasta reference_genome
    2. Create your genome file in IGV.
      IGV>Genomes>Create .genome File
      Load your reference_genome.fasta and reference_genome.gbk files
    3. Load your mapping file.
      IGV>File>Load from File


      Figure 8. Screenshot of the IGV browser for alignment visualization. Alignment of TEX+ and TEX- libraries with the reference genome of S. flexneri 5a M90T. The blue histogram shows the coverage of the TEX- sample, the red histogram shows the TEX+ sample coverage. The red highlight shows the putative transcriptional start site.

  9. Identify the transcriptional start sites
    1. Merge the three replicates in one file.
      $ samtools merge output.sam mapping_Sorted_1.bam mapping_Sorted_2.bam mapping_Sorted_3.bam
    2. Load the merged file in ReadXplorer.
    3. Run the TSS analysis with these parameters (Table 8):

      Table 8. Settings for the TSS analysis in ReadXplorer


    4. The output file of ReadXplorer analysis for TSS is a table in plain text with 39 different columns that you can use for a downstream analysis. As an example you can see an extract of some columns of the output file in Figure 9.


      Figure 9. Example of extract from the ReadXplorer output file

    According to the analysis with ReadXplorer, we identified 6,723 pTSS and 7,328 sTSS in S. flexneri 5a M90T (Cervantes-Rivera et al., 2020), which is around the same number of TSS identified in E. coli MG1655 (Conway et al., 2014; Thomason et al., 2015). These results are also available at RegulonDB at http://regulondb.ccg.unam.mx/central_panel_menu/integrated_views_and_tools_menu.jsp.

Recipes

  1. Tryptone soy broth (TSB)
    30 g TSB medium powder
    MilliQ water to 1 L
    Autoclave the solution
  2. Tryptone soy agar (TSA) plates
    40 g Tryptone soy agar powder
    MilliQ water to 1 L
    Autoclave the solution
    Cool to ~50 °C in the water bath, add 10 ml of 1% Congo red solution and pour plates
  3. Congo red solution 1% (w/v)
    1 g of Congo red dye
    100 ml of MilliQ-water
    Filter sterilize the solution, using a 0.2 μm membrane filter.
    Note: The Congo red solution is stable for months at room temperature.
  4. Nuclease free water
    1 ml of Diethyl Pyrocarbonate (DEPC), (0.1% final concentration)
    1 L of MilliQ water
    Mix well over night using a magnetic stirring bar
    Autoclave
    Let cool down to room temperature prior to use
  5. Sodium dodecyl sulfate (SDS) 10% (w/v)
    10 g of SDS powder
    Nuclease free water 100 ml
    Autoclave the solution
  6. EDTA 0.5 M, pH 8
    18.61 g disodium ethylenediaminetetraacetate dihydrate (EDTA)
    2 g Sodium hydroxide
    Nuclease free water 100 ml
    Mix well and sterilize
    Note: EDTA is solved at pH 8.
  7. DNA loading buffer 6x
    0.125 g bromophenol blue (0.025% final concentration)
    0.125 g xylene cyanol (0.025% final concentration)
    30 ml Glycerol
    Nuclease free water to 100 ml
    Mix well, it is stable at room temperature for years
  8. RNA loading buffer 2x
    0.125 g bromophenol blue (0.025% final concentration)
    0.125 g xylene cyanol (0.025% final concentration)
    100 ml of formamide
    Mix well, it is stable at room temperature for years
  9. Stop Solution
    95 ml of ethanol
    5 ml of phenol pH 4
    Final volume 100 ml
    Mix well, store it at 4 °C
  10. Ethanol 80%
    80 ml of ethanol
    20 ml of nuclease free water
    Mix well and store it at 4 °C
  11. Lysis solution
    5 ml of SDS 10% (0.5% final concentration)
    0.6 ml of sodium acetate pH 5.5 (20 mM final concentration)
    2 ml of EDTA pH 8 (10 mM final concentration)
    Nuclease free water to 100 ml (92.4 ml)
    Mix well, store at room temperature.
    Note: At cold temperature the SDS precipitates. If this happens warm up for 15 s at maximum power in the microwave oven.
  12. 10x TAE
    11.42 ml of acetic acid
    48.4 g of Trizma-base
    20 ml of EDTA 0.5 M, pH 8
    Nuclease free water to 1 L
  13. Agarose 1%
    1 g of agarose powder
    100 ml of TAE 1x
    Melt the agarose in a microwave oven
  14. Phe-CHISAM solution
    25 ml of phenol acid pH 4
    24 ml of chloroform
    1 ml of isoamyl alcohol
    Mix well, store at room temperature in the chemical hood 
  15. CHISAM
    96 ml of chloroform
    4 ml of isoamyl alcohol
    Mix well, store at room temperature in the chemical hood

Acknowledgments

A short version of this protocol was published in Cervantes-Rivera et al. (2020). We gratefully acknowledge funding from the Kempe Foundation (JCK-1528), the Knut and Alice Wallenberg Foundation (KAW 2015.0225), Umeå Centre for Microbial Research (UCMR), Umeå University and The Laboratory for Molecular Infection Medicine Sweden (MIMS). We are grateful for travel support and courses organized by The National Doctoral Program in Infections and Antibiotics (NDPIA). The computations were performed on resources provided by SNIC through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under Project SNIC 2017-7-258.

Competing interests

The authors declare no conflict of interest.

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简介

[摘要] 细菌中的基因转录通常起始于起始密码子上游的一些核苷酸。识别SPE cific Ť ranscriptional 小号挞小号ITE (TSS)为遗传操作必需的,因为在许多情况下,起始密码子上游有中涉及的基因表达调控序列元件。考虑到经典的基因结构,我们能够鉴定出两种转录起始位点:一级和二级。主要转录起始位点位于翻译起始位点上游的一些核苷酸上,而次要转录起始位点位于基因编码序列内。

这里,我们提出一步步协议全基因组吨ranscriptional 小号馅饼小号ITES d etermination通过差RNA测序(DRNA 使用肠道病原体-SEQ)福氏痢疾杆菌血清型菌株5A作为M90T模型。但是,该方法可以用于选择的任何其他细菌物种。第一步,使用热酚法从细菌培养物中纯化总RNA。核糖体RNA(rRNA)是使用商业试剂盒通过杂交探针特异性去除的。然后准备一个富含5'- 一磷酸依赖性核酸外切酶(TEX)处理的,富含初级转录本的RNA文库,用于与未进行TEX处理的文库进行比较,然后连接已知序列的RNA接头衔接子,从而确定具有单核苷酸精度的TSS。最后,对RNA进行处理以制备Illumina测序文库,并按购买的服务进行测序。通过内部生物信息学分析鉴定TSS。

我们的协议具有成本效益,因为它最大程度地减少了商业套件的使用,并使用了免费提供的软件。

[背景] 细菌中的转录是由RNA聚合酶全酶开始的,该酶可识别启动子区域内与σ因子结合的DNA上的特定序列元素(Feklistov 等人,2014)。该RNA聚合酶全酶结合位点定义了转录起始位点和转录方向。例如,最常见的管家σ因子,命名 70 在大肠杆菌中,可识别两种元素为中心约10〜35个碱基的TSS上游(Feklistov 等人,2014) 。RNA聚合酶全酶使双链DNA在TSS(+1)上游11 nt (位置-11)至下游3 nt (+3)之间融化,然后单链DNA可以用作模板添加三磷酸核糖核苷酸。引发主要在特定位置开始,但有时会遇到一个或多个碱基在上或下游的“摆动” (Murakami和Darst ,2003; Robb 等,2013; Vvedenskaya 等,2015)。长期以来,人们一直认为TSS周围的DNA序列对于细菌中的基因调控至关重要(Jacob和Monod,1961)。根据基因结构中以起始密码子(通常为ATG)开始并以三个最高密码子之一结束的正离子,我们可以确定两种类型的转录起始位点:一级和二级。初级转录起始位点(pTSS )位于翻译起始位点上游的一些核苷酸,而次级转录起始位点(sTSS )位于基因编码序列内(图1)。

图1.主要和次要翻译起始站点定义的示意图

在下一代测序技术问世之前,为了定位特定RNA的TSS,有必要使用S1保护分析,引物延伸或5'RACE方法单独检查每个转录本(Sharma and Vogel ,2014年) )。由于日益普及和高通过成本的降低PU 吨测序,2010年差动RNA-SEQ(DRNA -SEQ)的开发是为了同时映射使用基因组的所有TSS 幽门螺杆菌作为第一模型生物体(夏尔马等人。,2010) 。此后,该方法被广泛用于确定几种细菌的TSS (Berghoff 等,2009; Jager 等,2009; Albrecht 等,2010和2011; Bohn 等,2010; Irnov 等人,2010;施吕特尔等人,2010;夏尔马。等人,2010;贝克曼等人,2011; Deltcheva 。等人,2011; Filiatrault 。等人,2011; Mitschke 。等人,2011A 和2011 b ; 罗格等人,2012和2013;马杜吉里。等人,2012;德兰等人,2012和2014;萨赫尔。等人,2012; Schmidtke就等人,2012;肾母细胞等,2012; Cortes的等。,2013; Dugar 等人,2013; Mentz 等人,2013;镍等人,2013;法尔福-桑贾尔。等人,2013; Porcelli 等人,2013;施吕特尔等人,2013;沃斯等等人,2013; Wiegand 等人,2013; Zhang 等人,2013; Voigt 等人,2014; Cervantes-Rivera 等人,2020)。

原核生物的初级转录本在其5'端带有一个三磷酸酯。相反,加工或降解的RNA仅在其5'末端带有一磷酸。核糖体RNA(rRNA)也是如此(Schoenberg,2007)。此处使用的dRNA- seq方法利用5'-单磷酸依赖的核酸外切酶(TEX)的特性来选择性降解加工的转录本,从而富集携带天然5'-三磷酸的未加工RNA (Schoenberg,2007)。然后可以通过比较TEX处理和未处理的RNA-seq文库来鉴定TSS,其中TSS表现为在TEX处理后丰富的覆盖范围内的局部最大值(Sharma 等,2010)。

直到2013年,TSS注释都是手动执行的,但是这种方法既费时又费力。如今,许多计算工具可用于使用dRNA -seq数据进行自动TSS注释。这些包括TSSPredator (Dugar 等,2013),TSSAR (Amman 等,2014),TruHMM (Li 等,2013),TSSer (Jorjani和Zavolan,2014)和ReadXplorer2 (Hilker 等,2016)。。

在这里,我们通过比较最初在(Cervantes-Rivera 等人,2020)中进行的弗氏志贺氏菌血清型5a菌株M90T 中TEX处理和未处理的RNA库的比较,提供了一步一步确定TSS的方案。总体工作流程如图2所示。


图2. dRNA -seq用于全基因组转录起始位点鉴定的工作流程

关键字:dRNA-seq, 转录起始位点, TSS, RNA纯化, 热酚RNA提取, rRNA去除, 5′-单磷酸依赖核酸外切酶(TEX), 苯酚氯仿:异戊醇RNA提取, 细菌基因调控, RNA沉淀, RNA磷酸化

材料和试剂
无菌注射器过滤器,孔径0.22 μ米(默克,目录号:小号LGV033RK)
丁腈手套
13 ml 培养管(TPP,目录号:91016)
1.5 ml微量离心管(Eppendorf,安全锁定管,货号:0030120086)
注意:原则上,市场上所有预包装的试管均不含RNase,可以使用。
50 ml离心管(Sarstedt ,目录号:62.547.254)
移液器吸头(VWR,目录号:89041-404、89041-412、89041-400)
注意:原则上,市场上所有预包装的吸头均不含RNase,可以使用。
半微量比色杯(Sarstedt,目录号:67.742)
弗氏志贺氏菌5a M90T (Sansonetti et al。,1982),也可以从ATCC购买,也可以应作者要求提供。
胰蛋白tone大豆肉汤(TSB)即用型粉末(Merck,目录号:105459)
胰蛋白tone大豆琼脂(TSA)即用型粉末(Merck,目录号:105458)
刚果红(西格玛,目录号:C6277)
焦碳酸二乙酯(DEPC)(Sigma,目录号:40718)
十二烷基硫酸钠(SDS)(Sigma,目录号:L3771)
溴酚蓝钠盐(西格玛,目录号:B8026)
二甲苯氰FF(Sigma,目录号:X4126)
甘油(Sigma,目录号:G5516)
甲酰胺(Sigma,目录号:47671)
绝对乙醇(VWR C hemicals,目录号:20821.558)
乙酸钠3 M,pH 5.5(Ambion ,目录号:AM9740)。
EDTA,二钠盐,二水合物(Sigma,目录号:324503)
氢氧化钠(Sigma,目录号:S8045)
Trizma基座(Sigma,目录号:T1503)
醋酸(西格玛(Sigma),目录号:695092)
琼脂糖(VWR Life Science,目录号:35-1020)
DNase I 10 U / ml(罗氏(Roche),货号:04716728001)
RNaseOUT 40 U / ml(Invitrogen,货号:10777019)
氯仿(Sigma,目录号:C2432)
苯酚溶液pH 4.3±0.2(Sigma,目录号:P4682-400ML)
异戊醇(Sigma,目录号:W205702)
乙酸钠pH 5.5(Ambion ,目录号:AM9740)
糖原20毫克/毫升(Invitrogen公司,Ç atalog号:R0551)
寡聚物SF-HFQ-F 5 ' - ACGATGAAATGGTTTATCGAG-3 ' 和SF-HFQ-R 5 ' -ACTGCTTTACCTTCACCTACA-3 ' (Sigma 公司,按订单生产)
GeneRuler 1 kb DNA阶梯(Thermo Scientific,目录号:SM0211)
接头-RNA-适配器5 ' -GACCUUGGCUGUCACUCA-3 ' (Sigma 公司,按订单生产)
用于细菌的Ribo- Zero rRNA去除试剂盒(Illumina,目录号:MRZB12424 )
注意:该产品不再在市场上出售,但可以用Pan-Prokaryote riboPOOL 试剂盒(siTOOLS BIOTECH,Pan-Prokaryote v2),RiboCop rRNA细菌耗竭试剂盒(Lexogen ,目录号:125-127)或任何其他产品代替其他基于杂交探针的rRNA消耗方法。依赖于基于酶的rRNA耗竭的替代产品(例如Ribo- Zero Plus rRNA耗竭套件(Illumina,目录号:20037125))不适用。
终结者-5 ' 磷酸依赖性外切核酸酶(TEX)(Lucigene ,目录号:TER51020)
5'-焦磷酸水解酶5,000单位/毫升(RppH )(New England BioLabs ,目录号:M0356S)
T4 RNA连接酶10,000 U / ml(New England BioLabs ,目录号:M0204L)
T4多核苷酸激酶(PNK)10,000 U / ml (New England BioLabs ,目录号:M0201L)
RNaseZap (Ambion ,目录号:AM9780)
Dream Taq DNA聚合酶(Thermo Scientific,目录号:EP0701)
dNTP混合,每个2 mM(Thermo Scie ntific,目录号:R0241)
GelRed 核酸凝胶染料(Biotium ,目录号:41003)
ATP溶液(100 mM)(Thermo Scientific,目录号:R0441)
胰蛋白so大豆肉汤(TSB)培养基(请参阅食谱)
胰蛋白so大豆琼脂(TSA)板(请参阅食谱)
刚果红溶液(请参阅食谱)
无核酸酶水(小号EE 食谱)
十二烷基硫酸钠(SDS)10%溶液(请参阅食谱)
EDTA 0.5 M,pH 8(请参见配方)
6 x DNA上样缓冲液(请参阅食谱)
2 x RNA上样缓冲液(s 配方)
停止解决方案(s 配方)
乙醇80%(请参阅食谱)
裂解液(请参见食谱)
10 x TAE(请参阅食谱)
1%琼脂糖(s 配方)
Phe -CHISAM解决方案(请参阅食谱)
CHISAM(请参阅食谱)

设备
冷冻微型离心机(VWR,型号:Micro Star 17R)
分光光度计(Amersham ,型号:Ultrospec 2100 pro)
移液器(Eppendorf Research plus)
微波炉(惠而浦,型号:MD101)
离心机(Eppendorf,型号:5810R)
NanoDrop ND-1000分光光度计(Saveen &Werner AB)
轨道摇床(埃德蒙· 比勒,型号:SWIP SM25)
整体加热器(VWR,型号:460-0353)
DynaMag-2磁铁(Thermo Fisher Scientific,目录号:12321D)
T100热循环仪(Bio-Rad,目录号:186-1096)
化学罩
卧式凝胶电泳系统(VWR,型号:700-0015)
电泳电源(VWR,型号:E0202)

小号oftware

程式
该协议中使用的所有程序均可免费获得。
bcl2fastq(https://emea.support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html)
FastQC /0.11.8(https://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc)(Wingett和安德鲁斯,2018)
MultiQC / 1.8(https://multiqc.info/)(Ewels 等,2016)
trimmomatic / 0.36(http://www.usadellab.org/cms/?page=trimmomatic)(Bolger 等人,2014年)
samtools / 1.9(http://www.htslib.org/doc/samtools.html)(Li 等,2009)
bowtie2 / 2.3.5.1(http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)(Langmead和Salzberg,2012年)
IGV / 2.3.82(https://software.broadinstitute.org/software/igv/)(Thorvaldsdottir 等人,2013年)
ReadXplorer /2.2.3(https://www.uni-giessen.de/fbz/fb08/Inst/bioinformatik/software/ReadXplorer)(Hilker 等人,2014; Hilker 。等人,2016)
 
资料库
GenBank(https://www.ncbi.nlm.nih.gov/genome/182?genome_assembly_id=493862)
以RefSeq (https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP037923.1,https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP037924.1)
RegulonDB (http://regulondb.ccg.unam.mx/)
DRNA -SEQ原始数据可在所述下登录号SRA数据库:SRR8921222(DRNA-Seq_TEX_Positive ),SRR8921223(DRNA-Seq_TEX_Negative )(https://dataview.ncbi.nlm.nih.gov/)

程序
在开始RNA 纯化之前,请确保已使用RNaseZap 清洁了移液器,离心机,工作台,化学罩,凝胶设备和标记笔,以确保所有内容均不含RNase。在操作规程的每一步,您都应戴上丁腈/乳胶手套,以免污染RNase。
使用热酚法纯化总RNA,并通过电泳控制RNA质量
通过接种5毫升TSB培养基的制备预培养用的单个菌落福氏5A M90T 从刚果红的胰蛋白胨大豆琼脂平板上并在37℃下搅拌(150rpm)下生长过夜(〜16小时)。
注意:确保使用的菌落具有分泌能力(由于刚果红吸收而在平板上形成红色菌落)(Sharma and Puhar,2019)。
通过稀释100传代培养μ 升(:100稀释1)在10毫升TSB的预培养物。
摇动生长至OD 600 = 0.3。如果介质已预热,则大约需要2小时。
将培养物与2 ml冷终止溶液混合。
将混合的培养液与终止溶液在冰上孵育30分钟。
注意:为稳定RNA并防止降解,将其在冰上孵育至少30分钟,但不要超过2小时。
将培养物转移到15 ml管中。
在4°C下以16,200 x g 离心5分钟。
倾析除去上清液。保留约2 ml上清液以重悬沉淀。
将重悬的细胞转移到两个1.5 ml试管中。
在4°C下以16,200 x g 离心5分钟。
倾析弃去上清液。
注意:此时,样品可用液氮冷冻并保存在-80°C。样品稳定数月。
如下使用热酚法纯化总RNA。
重悬细胞沉淀在500 μ 升室温下的裂解溶液。
下的化学通风橱中添加500 μ 升苯酚pH为4预热至65℃的。颠倒混合至少20次。
注意:在开始RNA纯化之前,先加热加热苯酚。
剧烈涡旋20 s。
在加热块中于65°C孵育5分钟。
在室温下以13,800 x g 离心3分钟。
注意:如果离心步骤是在4°C下完成的,则SDS将会沉淀并且溶液变成白色,这不会影响该过程的效率,但是很难区分这两个阶段。经过这一离心时间后,您应该看到两个阶段,上一个阶段应该发白(当SDS冷却时,它变得不溶)。RNA在水相(上层相)中,而在白色盘状中间相中则存在蛋白质层和DNA,而脂质和其他一些细胞成分则溶解在有机相的下层透明相中。
传送上层相(〜500 μ 升化学罩下)到一个新的无RNase的管。
加入1毫升冷(4°C)无水乙醇。
涡旋5秒钟彻底混合,直到看到均匀的混合物。
在4°C下以16,200 x g 离心5分钟。
倾析掉含有乙醇溶液的上清液。
注意:在此步骤中,您可以在底部看到白色/透明颗粒。
在室温下加入1 ml 80%的乙醇。剧烈摇动试管10秒钟。
注意:不要使用的漩涡,它可以分离从底部的沉淀物。
在4°C下以16,200 x g 离心2分钟。
倾析弃去上清液。
离心(短旋转)试管,并通过移液将剩余的液体丢弃。
在室温下干燥样品,方法是将试管放在工作台上盖好盖子。
注意:一旦样品完全干燥,它将变成透明的。可能需要10分钟才能获得完全干燥的样品。
重悬100中的RNA样品μ 升的无核酸酶的水。
如下在TAE 1x 中的1%琼脂糖凝胶中检查纯化的RNA的质量。
在1.5 ml试管中准备要分析的样品。加入1 μ 升样品的加5 μ 升RNA装载的缓冲液2 X 和4 μ 升的无核酸酶的水。
将样品在65°C下加热5分钟,以使二级和三级结构变性。
在冰上冷却管5分钟。
旋转2秒钟并加载凝胶。
在TAE 1 x 缓冲液中以100 V 运行凝胶40分钟。
用3 x 浓缩的凝胶红将凝胶染色10分钟。
用20 ml无核酸酶的水在室温下振摇洗涤凝胶10分钟。
在紫外光下可视化凝胶(图3A)。如果总RNA纯化成功,则可见23S和16S为清晰的重条带,而在凝胶下部则观察到较弱的tRNA条带。无法看到mRNA。如果发生RNA降解,条带表现为涂片。
在NanoDrop 分光光度计中对R NA进行定量(图3B)。
注意:至少有20 μ 克(10 μ 克的每两个条件)的总RNA是必要继续与协议。典型浓度应在500-900 ng / ml左右。非常成功的纯化产生高达1,500 ng / ml的RNA。

图3.总RNA质量控制。A.在志贺氏志贺氏菌5a M90T的TAE的1%琼脂糖凝胶中,从三个重复样本中分离出总RNA,作为总RNA质量控制。B.从NanoDrop中总RNA定量获得的典型值的示例。260/280的比例应在1.88-2.00左右。

在-20°C下冷冻RNA样品。RNA样品可稳定保存数月。
 
DNase处理可从总RNA中去除污染的基因组DNA
协议量是针对一个反应的,但是每个步骤都可以扩展到两个或更多反应。

稀10 μ 克总RNA至30的最终体积的μ 升用无核酸酶的水。
在加热块中于65°C下在无核酸酶的水中使RNA变性5分钟。
在冰上冷却5分钟。
制备含有所指示的数量是足够的在表1中列出的组分的反应混合物为含有10一个采样μ 克RNA组成。

表1的DNA Ë反应混合为一个反应
注:1 1个单位DNA酶I的μ 克总RNA。

将反应混合物加到冰上的RNA样品管中。
将反应混合物在加热块中于37°C孵育60分钟。
注意:请确保加热块处于正确的温度,DNase在37°C时可有效工作。
添加到反应管60 μ 升的无核酸酶的水,以获得100 μ 升终体积。
用氯仿纯化RNA :异戊醇提取和乙醇沉淀
100添加μ 升CHISAM的到反应管中的化学通风橱下。
与涡旋剧烈混合15秒钟。
在13,800 xg (4°C)下离心2分钟。
下的化学通风橱转移〜100 μ 升从水相(上部),包含RNA至新的1.5毫升管。丢弃有机相(下部),缓冲液中溶解的蛋白质和盐会溶解在该相中。
重复步骤小号C1-C4。
添加300 μ 升冷的无水乙醇(3个样品体积),10 μ 升的小号裂果乙3 中号pH为5.5(样品体积的1/10)和1 μ 升糖原(20毫克/毫升)。
将混合物剧烈涡旋5秒钟。
将试管在-20°C下孵育30分钟。
注意:此步骤是可选的,但最好这样做以减少后续步骤中RNA的损失。
在4°C下以16,200 x g (或最大速度)离心30分钟。
注意:离心步骤可能需要更长的时间。最小时间为30分钟,但可能需要2到4小时。
轻轻倒掉上清液。
注意:在试管底部应有白色沉淀。弃去上清液而不分离沉淀。 
在室温下添加1 ml的80%乙醇洗涤RNA。
颠倒20次将其混合。
在13,800 x g (4°C)下离心5分钟。
倾析弃去上清液。
离心机(短纺),丢弃剩余的液体,具有100 μ 升移液管。
保持试管在冰上干燥以干燥样品。
注:干燥样品所需的时间取决于如何以及除去液相(û sually它需要10-15分钟)。一旦完全干燥,颗粒就完全透明。
重悬23总RNA μ 升的无核酸酶的水。
在总RNA提取后,通过目视检查1%琼脂糖凝胶上的23S和16S条带,检查不含DNase的RNA的完整性。
按照步骤A30-A37制备运行样品。
在紫外光下可视化凝胶(图4A )。
量化在所述RNA 纳米滴分光光度计(最终RNA浓度应〜600-1 ,000纳克/毫升)(图4B)。由于DNA的存在会增加样品的吸光度,因此在处理后检测较低的浓度是正常的。

图4. DNase处理后的总RNA质量控制。A. 在志贺氏志贺氏菌5a M90T的TAE 的1%琼脂糖凝胶中,从三个重复样本中分离出总RNA,作为总RNA质量控制。B.在NanoDrop中DNase处理后从总RNA定量中获得的典型值的例子。

在-20°C下冷冻RNA样品。RNA样品可稳定保存数月。

PCR验证基因组DNA的去除
为了确保RNA样品中没有DNA痕迹,通过PCR检查基因组DNA的缺失非常重要。
使用1 μ 升(〜1 μ 克的)的DNA酶处理RNA样品作为模板来核实没有基因组DNA。
作为阳性对照使用〜10纳克来自基因组DNA的弗氏志贺菌5A M90T。可以使用(He,2011)中所述的酚-氯仿方案提取基因组DNA 。
作为阴性对照,使用没有任何模板的反应。
准备PCR混合物(表2)。所示数量足以容纳一个样品。

表2. 用于PCR对照的反应混合物

注:寡核苷酸使用这里扩增〜909 bp的所述的长的产品HFQ 基因(303 个nt 从福氏5A M90T包括300bp的上游和300bp的所述的下游)HFQ 基因。


PCR反应设置(表3)。

表 3.设置为控制PCR
按以下步骤在1%琼脂糖凝胶中检查产品。
准备要分析的样品。添加6 μ 升样品的加2 μ 升DNA加载缓冲器6 X 。
在100 V下在TAE 1x 缓冲液中运行1%琼脂糖凝胶40分钟。
用Gel Red 3 x 将凝胶染色10分钟。
用无核酸酶的水洗涤凝胶。
在紫外光下可视化凝胶(图5)。

图5. 使用基因组DNA 作为阳性对照,用hfq基因加300pb弗氏志贺氏菌5a M90T的上游和下游作为靶标的hpq 基因加上基因组DNA的对照PCR 。TAE中1%的琼脂糖凝胶1 x 。在泳道2-4中不存在被视为909 bp带的PCR产物,这表明纯化的RNA没有DNA污染。
rRNA耗竭
核糖体RNA是最丰富的RNA种类。如果不去除rRNA,则测序的大多数读数将来自rRNA。结果,所比较的样品都需要具有或不具有rRNA,否则测序深度将非常不同,并且无法进行统计分析。下一部分中描述的用于富集一级RNA的TEX处理会去除所有未被三磷酸化的RNA(包括rRNA),从而使该库中的rRNA耗尽。因此,为了能够将TEX处理的样品与模拟处理的样品进行比较,需要先从总RNA中特异性去除rRNA,然后再将RNA分成两部分进行进一步处理。
  使用商购试剂盒进行rRNA消耗,该试剂盒可根据制造商的说明专门消耗细菌rRNA。细菌rRNA的特异性消耗通常依赖于与识别rRNA中保守序列的带标签泛细菌探针杂交,然后去除捕获的rRNA,例如通过用与探针标签结合的磁珠沉淀。
  我们已经使用了Illumina 的Ribo -Zero rRNA细菌去除试剂盒,但不幸的是,它在市场上不再可用。但是,可以用您选择的任何基于杂交的试剂盒替换它。我们在“材料”部分提供了一些建议。
终止子核酸外切酶(TEX)处理以丰富初级转录本
准备两个(TEX-和TEX +)1.5ml反应管s的10 μ 克(10 μ 升在无核酸酶的水),每个DNA酶I处理过的RNA(来自相同样品)。
在90°C下将RNA变性2分钟。
在冰上冷却5分钟。
添加到的Reacti每个管上组合描述于表4中指示的数量是足够的含10一个采样μ 克RNA的:

表4. TEX处理的反应混合物

注意:VOLU最终的反应我将是20 μ 升添加酶用于TEX +样品或水的TEX-样品之后。
添加1 μ 升的无核酸酶的水至TEX-样品。
加入1 μ 升TEX(1单位/毫升)至TEX +样品。
在热循环仪中于30°C孵育60分钟。
注意:此步骤可以在加热块中进行,但是温度变化和液体蒸发是妨碍良好结果的重要因素,并且这些影响在加热块中更强。
添加80 μ 升的无核酸酶的水,以获得100 μ 升终体积。
100添加μ 升的苯丙氨酸- CHISAM到反应管中的化学通风橱下。
与涡旋剧烈混合15秒钟。
在13,800 x g (4°C)下离心2分钟。
转印〜100 μ 升包含化学通风橱下的RNA至新管上部相。
重复步骤小号F9到F12。
添加300 μ 升冷的无水乙醇(3个样品体积),10 μ 升的小号裂果3M乙pH为5.5(样品体积的1/10 )和2 μ 升糖原(20毫克/毫升)。
剧烈涡旋样品。
将试管在-20°C下孵育过夜(约16小时),但可以更长一些,例如在周末。
注意:此步骤非常重要。它有助于完成RNA的回收。
在4°C下以16,200 x g 离心1 h。
注意:离心步骤可能需要更长的时间。最少1小时,但可能会持续2-4小时。
倾析小心弃去水相。
注意:在试管底部应有白色沉淀。丢弃水相而不分离颗粒。在的情况下看不到任何沉淀弃去上清液之前,只是一个DD 10 μ 升号第裂果乙3 中号pH为5.5(样品体积的1/10)和2 μ 升糖原(20毫克/毫升)和重复步骤s F 16至F18)。
在室温下加入1 ml 80%的冷乙醇洗涤RNA。
在13,800 x g ,4°C下离心5分钟。
通过倾析丢弃水相。
离心机(短纺),丢弃剩余的液体,具有100 μ 升移液管。
保持试管在冰上干燥以干燥样品。
注意:干燥样品所需的时间取决于去除液相的程度。大约需要10-15分钟。一旦完全干燥,颗粒将完全透明。
重悬13 RNA μ 升ö ˚F无核酸酶的水。
通过在1%琼脂糖凝胶上目视检查,检查TEX-和TEX +处理过的RNA的完整性。
按照步骤A 29- A 36 制备运行样品。
在紫外光下可视化凝胶(图6 )。

图6. 用TEX处理过的志贺氏菌5a M90T RNA(TEX + )和未处理过的(TEX- )(TEX- )
注意:正常情况下,在此凝胶中看不到mRNA的清晰条带,该条带是可变长度的不同转录本的混合物。要看到凝胶手段的治疗是successf涂抹ü 湖 如果您没有大量的RNA,也许您只会看到tRNA。

在NanoDrop 分光光度计中定量RNA (最终RNA浓度应为〜20 ng / ml)。
在-20°C下冷冻RNA样品。RNA样品可稳定保存数月。

RNA的RNA 5' 焦磷酸水解酶(RppH )处理以去除末端磷酸盐
在下面的步骤中,5 ' 的RNA的-ends中制备程序F 修饰有已知序列的接头-RNA-适配器,它允许TSS的识别单核苷酸精度。这是必需的,因为在文库制备过程中添加的用于测序的衔接子序列可能是商业秘密(Illumina衔接子的完整序列仅在最近才公开,并且存在使用未知序列的衔接子的其他平台)。在对测序结果进行生物信息学分析时,有时不完全从读物中除去衔接子的序列,如果转录的碱基恰好与DNA序列比对,则可能会将其余的核苷酸与转录的碱基混淆。但是,可以通过在衔接子和TSS 之间引入已知序列的RNA衔接子来避免此问题。而且,接头-RNA-适配器允许在文库制备期间唯一地鉴定在RNA剪切之前存在的5 ' 末端。为了在文库制备过程中将衔接子连接至接头RNA适配器的5'末端,接头RNA适配器的5 ' 末端必须具有三磷酸。

变性剩下的1 0 μ 升TEX +和TEX-的处理的RNA进行1分钟,在90℃。
在冰上冷却5分钟。
如表5所述制备RppH 混合物。所示数量足以用于一种样品:

表5. TEX处理的RNA的R ppH 反应的反应混合物

在热循环仪中于37°C孵育30分钟。
加入80 μ 升的无核酸酶的水,然后按照步骤F8-F23。
重悬13 RNA样品μ 升的无核酸酶的水。
通过在1%琼脂糖凝胶上目测检查RppH 处理的RNA 的完整性。
注意:此步骤并非严格必要,并且每一步都会丢失一些RNA。凝胶外观应类似于图6 所示。
在NanoDrop 分光光度计中定量RNA 。
连接至TEX +和TEX-文库之前,接头RNA适配器磷酸化
变性100 皮摩尔(1 0 μ 升每溶解在不含核酸酶的水中5分钟,在90℃下样品的接头-RNA-适配器)。
在冰上冷却5分钟。
注意:准备反应时,您可以将寡核苷酸在冰上放置更长的时间。
根据表6准备T4 PNK混合物。

表6. 接头-RNA- 接头的T4 PNK磷酸化反应混合物

在热循环仪中于37°C 孵育30分钟。
加入80 μ 升的无核酸酶的水,按照步骤F8-F23。
重悬在10 RNA样品μ 升的无核酸酶的水。
注意:此步骤后,可将RNA接头冷冻在-2 0°C并稍后使用。

接头RNA适配器与TEX +和TEX-样品的连接
接头-RNA-接头连接的效率约为80-90%。
变性磷酸化RNA适配器(10 μ 升与处理过的)和RNA的RppH (10 μ 升)在65℃下5分钟。
在冰上冷却5分钟。
如表7中所述准备T4 RNA连接酶混合物(表中描述的数量仅适用于一个样品,如果您要处理更多样品,请按比例放大)。
 
表7. 接头-RNA-接头连接的反应混合物

在热循环仪中于25°C孵育16小时。
添加6 0 μ 升的无核酸酶的水。
停止反应并按照步骤F9-F23沉淀反应。
重悬23 RNA样品μ 升的无核酸酶的水。
注意:重悬液量由您决定使用的测序设备定义。在我们的情况下,RNA浓度为23纳克150 μ 升。
通过NanoDrop 分光光度计定量RNA衔接子。
用于Illumina测序的文库制备
测序和RNA测序的文库制备可在您选择的服务平台或公司进行。必须准备链状文库以鉴定编码DNA链。应执行读取长度为150 nt的配对末端测序。我们在HiSeq 2000平台上使用了TrueSeq 库。对于微生物基因组,每个样品10-20 M读数将提供适当的测序深度,但5 M足以获得可接受的覆盖范围。

数据分析
该协议中使用的所有程序均基于Linux,并且可以在网上免费获得。我们在高性能计算机集群上远程执行所有分析。但是,如果无法访问高速远程分析,则可以在下载程序后在本地运行所有分析,但是如果计算能力较低,则可能会花费很长时间。
  将所有程序,原始数据和参考基因组下载到工作目录中。在此协议中,我们以工作目录dRNA_seq为例。

注意:如果使用Windows系统,则应安装Ubuntu(https://ubuntu.com/download/desktop)并在此环境中运行所有程序。
d Illumina公司的emultiplexing读
注意:仅当您在自己的实验室中执行测序时,才需要执行此步骤。如果是这种情况,您将获得一个名为* .bcl.gz的文件,该文件首先需要多路分解以进行下游分析。如果您使用测序服务,则无需执行此步骤,因为您将获得已处理的* .fastq.gz文件。

bcl2fastq

转到您的工作目录,您在其中拥有* .bcl.gz文件。
执行bcl2fastq
$ bcl2fastq [选项]

原始数据质量控制
FastQC (图7)
转到您的工作目录。在此示例中,工作目录:
$ cd dRNA_seq

创建一个新目录以输出FastQC 分析。
$ mkdir FastQC_1


执行FastQC 。
$ fastqc / dRNA_seq /your_file.fq.gz -o / dRNA_seq / FastQC_1

图7. 质量控制的FastQC 报告。A.成功的质量控制。对于已测序片段的整个长度,质量得分应在绿色区域中。B.质量控制失败。部分读取的质量得分很低(在橙色或红色区域中找到)。


多质量控制
运行MultiQC 以合并所有FastQC 输出。

$ multiqc / FastQC_1


从低质量序列和Illumina适配器清除原始数据
Trimmomatic

在您的工作目录中运行Trimmomatic 。

$ java -jar trimmomatic.jar PE /your_raw_data_1.fq.gz your_raw_data_2.fq.gz your_raw_data_paired_1.fq.gz your_raw_data_unpaired_1.fq.gz your_raw_data_paured_2.fq.gz your_raw_data_unpaired_2.fqCL。 2:30:10领先:20牵引:20滑窗:4:15敏伦:100

选择包含RNA适配器序列的fastq 文件
执行此步骤的最简单方法是在终端中使用命令grep。此步骤中的接头RNA适配器应为DNA格式。

$ zgrep -B 1 -A 2 GACCTTGGCTGTCACTCA your_raw_data_paired_1.fq.gz> Picked_ Up.fastq

基因组参考指数
您的参考基因组很可能具有多个复制子。我们建议将它们全部合并到一个文件中,用作参考基因组。
$ cat 染色体.fasta质粒.fasta > reference_genome.fasta

使用bowtie2创建索引。
$ bowtie2-build reference_ 基因组.fasta index_base_name

基因组参考图谱
将选择的读数与参考基因组作图。

$ bowtie2 -q -N 0 – 非-unal -x index_base_name -U your_raw_data_paured_1.fq.gz -S mapping_1.sam

按位置排序并建立BAM文件的索引
Bowtie2的输出文件应进行排序和索引以便在IGV中使用。

$ samtools 视图-Sb- o mapping_1.bam mapping_1.sam

$ samtools sort -o mapping_Sorted_1.bam mapping_1.bam


在IG V中可视化映射文件(图8)
创建一个基因组参考索引。
$ samtools 索引reference_ 基因组.fasta reference_genome

在IGV中创建您的基因组文件。
IGV>基因组> 创建.genome 文件

加载您的reference_ 基因组.fasta 和reference_genome.gbk 文件

加载映射文件。
IGV>文件>从文件加载

图8.用于对齐可视化的IGV浏览器的屏幕截图。与参照基因组TEX +和TEX-库的对准福氏5A M90T。蓝色直方图显示TEX-样本的覆盖率,红色直方图显示TEX +样本的覆盖率。红色突出显示了假定的转录起始位点。

识别转录起始位点
将三个副本合并到一个文件中。
$ samtools 合并输出。sam mapping_Sorted_1.bam mapping_Sorted_2.bam mapping_Sorted_3.bam

将合并的文件加载到ReadXplorer中。
使用以下参数运行TSS分析(表8):

表8. ReadXplorer中的TSS分析设置

                  TSS 的ReadXplorer 分析的输出文件是纯文本表格,其中包含39个不同的列,可用于下游分析。作为示例,您可以看到图9中输出文件的某些列的摘录。
 
图9.从ReadXplorer 输出文件中提取的示例

根据与分析ReadXplorer ,我们确定6723 的PTS 和7328 STSS 在福氏5A M90T (塞万提斯-Rivera的等人,2020) ,它是围绕着相同数量的TSS中鉴定大肠杆菌MG1655 (Conway的等。,2014; Thomason 等人,2015)。这些结果也可以在RegulonDB 上获得,网址为http://regulondb.ccg.unam.mx/central_panel_menu/integrated_views_and_tools_menu.jsp。

菜谱
胰蛋白so大豆汤(TSB)
30克TSB中等粉末
MilliQ 瓦特亚特至1L
高压灭菌解决方案
胰蛋白so大豆琼脂(TSA)板
40克胰蛋白so大豆琼脂粉
的MilliQ 瓦特亚特至1L
高压灭菌解决方案
在水浴中冷却至〜50°C,加入10 ml 1%浓红色溶液,倒入板中
刚果红溶液1%(w / v)
1克刚果红
100毫升M illi Q 水
过滤消毒溶液中,用一个0.2 μ 米的膜过滤器。
注:在刚果红溶液在室温个月。
无核酸酶的水
1 ml 焦碳酸二乙酯(DEPC),(终浓度0.1%)
1升MilliQ 水
使用磁力搅拌棒在晚上搅拌均匀
高压釜
使用前让其冷却至室温
十二烷基硫酸钠(SDS)10%(w / v)
10克SDS粉
无核酸酶水100毫升
高压灭菌解决方案
EDTA 0.5 M,pH 8
18.61 g d 乙二胺四乙酸二钠钠(EDTA)
2克氢氧化钠
无核酸酶水100毫升
充分混合并消毒
注意:EDTA在pH 8时溶解。
DNA上样缓冲液6 x
0.125 g b 苯酚蓝(终浓度0.025%)
0.125克x亚乙基氰基(终浓度0.025%)
30毫升甘油
不含核酸酶的水至100毫升
混合均匀,在室温下稳定多年
RNA上样缓冲液2 x
0.125 g b 苯酚蓝(终浓度0.025%)
0.125克x亚乙基氰基(终浓度0.025%)
100毫升甲酰胺
混合均匀,在室温下稳定多年
停止解决方案
95毫升乙醇
5毫升的苯酚pH 4
最终体积100毫升
拌匀,储存在4℃
乙醇80%
80毫升乙醇
20毫升无核酸酶的水
充分混合并储存在4°C
裂解液
5 ml SDS 10%(终浓度0.5%)
0.6毫升的小号裂果醋酸盐pH 5.5(20mM的最终浓度)
2 ml EDTA pH 8(最终浓度10 mM)
不含核酸酶的水至100毫升(92.4毫升)
拌匀,储存在室温下。
注意:在低温下,SDS会沉淀。如果发生这种情况,请在微波炉中以最大功率预热15秒。
10 x TAE
11 。42 毫升乙酸
48.4克的的Trizma 碱基
20 ml EDTA 0.5 M,pH 8
不含核酸酶的水至1 L
琼脂糖1%
1克琼脂糖粉
100毫升TAE 1 x
将琼脂糖在微波炉中融化
Phe -CHISAM解决方案
25毫升酚酸pH 4
24毫升氯仿
1毫升异戊醇
充分混合,室温下储存在化学通风柜中
基萨姆
96毫升氯仿
4毫升异戊醇

充分混合,室温下储存在化学通风柜中


Acknowledg 发言:

该协议的简短版本发表在Cervantes-Rivera 等人的文章中。(2020年)。我们非常感谢肯普基金会(JCK-1528),克努特和爱丽丝·沃伦伯格基金会(KAW 2015.0225),于默奥微生物研究中心(UCMR),于默奥大学和瑞典分子感染医学实验室(MIMS)的资助。我们感谢国家感染与抗生素博士计划(NDPIA)组织的旅行支持和课程。在SNIC 2017-7-258项目下,由SNIC通过Uppsala高级计算科学多学科中心(UPPMAX)提供的资源进行了计算。

利益争夺

作者宣称没有利益冲突。

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引用:Cervantes-Rivera, R. and Puhar, A. (2020). Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria. Bio-protocol 10(18): e3757. DOI: 10.21769/BioProtoc.3757.
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