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A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria
小RNA分离和测序方法及其在研究细菌CRISPR RNA生物合成中的应用   

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eLIFE
Aug 2017

Abstract

Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis and treated with T4 polynucleotide kinase. This allows for 3’ adapter ligation to CRISPR RNAs, which don’t have pre-existing 3’-OH ends. Pre-adenylated adapters are then ligated using T4 RNA ligase 1 in the absence of ATP and with a high concentration of polyethylene glycol (PEG). The 3’ capture step enables precise determination of the 3’ ends of diverse RNA molecules. Additionally, a random hexamer in the ligated adapter helps control for potential downstream amplification bias. Following reverse-transcription, the cDNA product is circularized and libraries are prepared by PCR. We show that the amplified library need not be visible by gel electrophoresis for efficient sequencing of the desired product. Using this method, we routinely prepare RNA sequencing libraries from minute amounts of purified small RNA. This protocol is tailored to assay for CRISPR RNA biogenesis in bacteria through sequencing of mature CRISPR RNAs, but can be used to sequence diverse classes of small RNAs. We also provide a fully worked example of our data processing pipeline, with instructions for running the provided scripts.

Keywords: CRISPR (CRISPR), Small RNA (小RNA), High throughput sequencing (高通量测序), Guide RNA (向导RNA), CRISPR RNA (CRISPR RNA), crRNA processing (crRNA加工), crRNA biogenesis (crRNA生物合成), crRNA maturation (crRNA成熟)

Background

Genetic modules associated with Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) confer adaptive immunity in diverse prokaryotic hosts (Barrangou et al., 2007). Memories of invasive elements (such as viruses, plasmids, and other mobile elements) are stored interspersed between directed repeats of the CRISPR arrays in the host genome in the form of ‘spacers’ comprising the nucleic acid sequence of the molecular parasite (Brouns et al., 2008; Jackson et al., 2017). In order to identify subsequent infections by the same invader, the information contained in CRISPR spacers must be communicated to CRISPR-associated (Cas) endonucleases (Plagens et al., 2015). For the vast majority of CRISPR-Cas systems (phylogenetically grouped as ‘type I’ and ‘type III’ [Makarova et al., 2011 and 2015]), this occurs through the activity of a family of CRISPR-associated endoribonucleases known as Cas6 (Charpentier et al., 2015; Hochstrasser and Doudna, 2015). The entire CRISPR array is transcribed as a precursor CRISPR RNA (pre-crRNA) molecule from the genome, and the Cas6 protein domain helps to process this transcript into a collection of mature CRISPR RNAs (crRNA) consisting of one CRISPR spacer each, flanked by portions of the CRISPR repeat sequence (Carte et al., 2008 and 2010; Haurwitz et al., 2010). This mechanism is known as crRNA biogenesis. Cas6 endoribonucleases promote crRNA biogenesis through site-specific cleavage of the CRISPR repeat sequence, which generates 5’-OH and 2’3’-cyclic phosphate termini (Charpentier et al., 2015; Hochstrasser and Doudna, 2015). Site-specific cleavage at every CRISPR repeat results in the pre-crRNA molecule being chopped at regular intervals into almost equal-length crRNAs, each with a different spacer sequence (Charpentier et al., 2015; Hochstrasser and Doudna, 2015). Mature crRNAs are then loaded onto Cas effector complexes and serve as molecular guides that direct Cas enzymes to target DNA or RNA parasites based on sequence complementarity (Deveau et al., 2008; Marraffini and Sontheimer, 2008). The presence or absence of mature crRNAs isolated from bacterial cell populations can be used as a proxy for Cas6 activity. While biochemical methods have been developed to detect crRNAs (Carte et al., 2008 and 2010; Haurwitz et al., 2010), high-throughput RNA sequencing can be used to assay for Cas6 activity unambiguously (Heidrich et al., 2015). Whole transcriptome sequencing is expensive and can be biased against specific classes of RNAs depending on the specific method of library preparation. Therefore, various small RNA sequencing protocols have been developed to preferentially detect mature crRNAs (Juranek et al., 2012; Richter et al., 2012; Heidrich et al., 2015).

Here, we present a multiplexed small RNA sequencing method to enable facile and reproducible comparisons of crRNA maturation between many different biological conditions at once, such as mutations in the Cas6 protein to assess the mechanism of Cas6 activity. This protocol builds on previous work on small RNA sequencing and ribosome profiling (Lau et al., 2001; Ingolia et al., 2009; Guo et al., 2010; Kwon, 2011; Kivioja et al., 2011). The assay features high sensitivity and dynamic range without expending a lot of sequencing bandwidth on other cellular RNAs, with the caveat that the full-length precursor transcript is not observed by small RNA sequencing.

Materials and Reagents

  1. Gel-Loading Pipette tips 0.5-200 μl (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC1001 )
  2. 0.6 ml microcentrifuge tubes (Sigma-Aldrich, catalog number: T5149 )
  3. Razor blade
  4. Siliconized 1.5 ml microcentrifuge tubes (VWR, catalog number: 22179-004)
    Manufacturer: BIO PLAS, catalog number: 4165SL .
  5. Plastic dish
  6. Clear plastic film (Saran wrap, or equivalent)
  7. Corning Costar Spin-X sterile 0.45 μm cellulose acetate centrifuge tube filters (Corning, catalog number: 8162 )
  8. 0.2 ml PCR tubes, MicroAmp (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8010540 ) or equivalent
  9. Gel-Excision Pipette tips (Corning, Axygen®, catalog number: TGL-1165-R )
  10. Heavy Phase Lock Gel in 2 ml tubes (Quantabio, catalog number: 2302830 )
  11. Corning tube top vacuum filtration system (Corning, catalog number: 430320 )
  12. Trizol reagent (Thermo Fisher Scietific, InvitrogenTM, catalog number: 15596026 )
  13. Pre-Cast Novex 6% TBE-Urea polyacrylamide gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC6865BOX )
  14. 10x TBE running buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9863 )–dilute to 1x before use
  15. GeneRuler Ultra Low Range DNA Ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM1211 )
  16. 2x formamide gel loading dye (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM8546G )
  17. SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S11494 )
  18. UltraPure glycogen (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10814010 )
  19. 200 Proof molecular biology grade ethanol (Sigma-Aldrich, catalog number: E7023 )
  20. UltraPure DNase/RNase-free distilled water (Thermo Fisher Scientific, catalog number: 10977035 )
  21. Polynucleotide Kinase (PNK) enzyme and buffer (New England Biolabs, catalog number: M0201S )
  22. Ammonium acetate solution 7.5 M molecular biology grade (Sigma-Aldrich, catalog number: A2706 )
  23. 50% PEG 8000 (supplied with NEB T4 RNA ligase I)
  24. Pre-adenylated 3’ adapter oligo: /5rApp/NNNNNNAGATCGGAAGAGCACACGTCT/3ddC/
  25. T4 RNA ligase I (New England Biolabs, catalog number: M0204S )
  26. NEB buffer 2 (New England Biolabs, catalog number: B7002S )
  27. 5’ Deadenylase (New England Biolabs, catalog number: M0331S )
  28. RecJf (New England Biolabs, catalog number: M0264S )
  29. Acidified phenol:chloroform 1:1 mixture (Thermo Fisher Scientific, catalog number: AM9720 )
  30. Chloroform (Sigma-Aldrich, catalog number: 496189 )
  31. 5x First Strand Buffer (supplied with SuperScript II Reverse Transcriptase)
  32. 0.1 M dithiothreitol (supplied with SuperScript II Reverse Transcriptase)
  33. 10 mM dNTP mix (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0191 )
  34. SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
  35. Reverse transcription primer:
    /5Phos/AGATCGGAAGAGCGTCGTGT/iSp18/CACTCA/iSp18/GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
  36. Pre-Cast Novex 10% TBE-Urea polyacrylamide gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC6875BOX )
  37. 1 N sodium hydroxide solution (Merck, catalog number: SX0607H )
  38. CircLigase ssDNA ligase and 10x reaction buffer (Lucigen, catalog number: CL4111K )
  39. 1 mM ATP solution (supplied with circLigase)
  40. UltraPure Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  41. 10x TAE (Thermo Fisher Scientific, catalog number: AM9869 )–dilute to 1x before use
  42. Ethidium bromide solution 10 mg/ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17898 )
  43. Phusion High-Fidelity PCR master mix (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F531S )
  44. Indexing primers:
    CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCG where the X6 barcodes correspond to Illumina TruSeq LT indexes AD001 to AD008 [ATCACG, CGATGT, TTAGGC, TGACCA, ACAGTG, GCCAAT, CAGATC, ACTTGA]
    Note: More indexing primers may be added as needed.
  45. Universal PCR primer:
    AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
  46. DNA gel loading dye 6x (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0611 )
  47. 25 bp DNA ladder
  48. MinElute Gel extraction kit (QIAGEN, catalog number: 28604 )
  49. 5 M sodium chloride solution BioUltra for molecular biology (Sigma-Aldrich, catalog number: 71386 )
  50. 0.1 M EDTA solution, pH 7.5 (Merck, catalog number: EX0546A )
  51. 1 M HEPES solution BioPerformance certified and 0.2 μm filtered (Sigma-Aldrich, catalog number: H3537 )
  52. 8 N potassium hydroxide solution (Sigma-Aldrich, catalog number: P4494 )
  53. 50 mM manganese chloride solution (supplied with circLigase)
  54. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
  55. 1 M Dithiothreitol solution BioUltra for molecular biology (Sigma-Aldrich, catalog number: 43816 )
  56. Glycerol for molecular biology (Sigma-Aldrich, catalog number: G5516 )
  57. 1 M magnesium chloride solution for molecular biology (Sigma-Aldrich, catalog number: M1028 )
  58. 20 mg/ml Acetylated bovine serum albumin (Thermo Fisher Scientific, catalog number: AM2614 )
  59. Polyacrylamide gel elution buffer (see Recipes)
  60. 1 M HEPES/KOH buffer pH 8.3 (see Recipes)
  61. 60% glycerol solution (see Recipes)
  62. 5x adenylation buffer (see Recipes)

Equipment

  1. Scissors
  2. XCell SureLock Mini-Cell (Thermo Fisher Scientific, InvitrogenTM, model: XCell SureLockTM Mini-Cell, catalog number: EI0001 )
  3. Heated-lid thermocycler, Veriti 96-well (Thermo Fisher Scientific, Applied BiosystemsTM, model: VeritiTM 96-well, catalog number: 4375786 ) or equivalent
  4. Transilluminator with 365 nm wavelength UV bulb (VWR, catalog number: 89131-464 or equivalent)
  5. Tabletop microcentrifuge (Eppendorf, model: 5424 or equivalent, for use at room temperature and 4 °C)
  6. Freezer capable of reaching -80 °C
  7. Programmable water bath/heat block
  8. Rotisserie tube rotator (VWR, catalog number: 10136-084 or equivalent)
  9. 10 μl pipette
  10. Gel electrophoresis power supply (Thermo Fisher Scientific, model: OwlTM EC1000XL or equivalent)
  11. Owl EasyCast Mini Gel electrophoresis system (Thermo Fisher Scientific, Thermo ScientificTM, model: OwlTM EasyCastTM B2 ) or equivalent
  12. 100-1,000 μl pipette
  13. Qubit 3.0 Fluorometer (Thermo Fisher Scientific, InvitrogenTM, model: QubitTM 3 , catalog number: Q33216)

Procedure

Duration: The protocol can be performed comfortably in 4 days (including RNA isolation from bacteria) as follows: Steps A1-A16 on day 1, A17-C5 on day 2, D1-E12 on day 3, and E13 onwards on day 4. The flowchart below summarizes the major steps in the protocol (Figure 1).


Figure 1. The flowchart of the major steps in the protocol

RNA isolation from bacteria: RNA extraction methods will depend on the bacteria under study. The extraction method must avoid any column-based or size-dependent purification steps that could lead to preferential loss of small RNAs. We follow the manufacturer’s instructions provided with Trizol reagent for our model system Marinomonas mediterranea, a gamma-proteobacterium (like E. coli). We use no more than 200-500 μl of saturated M. mediterranea culture in Marine Broth 2216 for RNA isolation.

Sequencing library preparation:

  1. Small RNA isolation by denaturing polyacrylamide gel electrophoresis (PAGE)
    1. Assemble a pre-cast Novex 6% TBE-Urea denaturing polyacrylamide gel in the XCell SureLock Mini-Cell Electrophoresis System.
      Note: Remember to remove the gel comb and the green tape at the bottom of the gel cassette before assembling the electrophoresis cell.
    2. Fill the inside and outside chambers with 1x TBE running buffer, and pre-run the gel at 180 V for at least 30 min.
    3. Prepare samples of at least 5-10 μg total intact RNA and 0.1 μg Ultra Low Range DNA ladder in 2x formamide gel loading dye at a final concentration of 1x. We suggest keeping the total volume of each sample < 15 μl.
    4. Denature the samples and ladder by heating in a thermocycler with a pre-heated lid at 94 °C for 5 min, then immediately place in an ice-water slurry.
    5. While the samples are denaturing, thoroughly flush urea out of each gel well using a 100 μl pipette with the running buffer from the inner chamber several times.
    6. Load samples carefully with gel-loading pipette tips.
      Note: Leave 1-2 lanes between different RNA samples to reduce the amount of cross-contamination between experiments. We recommend including no more than 4-5 RNA samples (and one lane for the approximate sizing ladder) in a 10-lane gel.
    7. Run at 180 V until the bromophenol blue dye front (bottom band ~25 nt) reaches close to the end of the gel (about 35 min).
    8. While the gel is running, prepare gel elution tubes by making a small cross-shaped incision at the bottom of a 0.6 ml tube with a clean razor blade (see diagram for details) and placing it inside a 1.5 ml siliconized centrifuge tube. Do not remove the caps of either the 0.6 ml or 1.5 ml tubes (Figure 2).


      Figure 2. Longitudinal and transverse view of 0.6 ml tube for pulverizing polyacrylamide gel fragments. A cross-shaped incision is made at the bottom of 0.6 ml centrifuge tube using a clean razor blade. The polyacrylamide gel fragment is pulverized as it is forced through the incision and into a 1.5 ml siliconized centrifuge tube by centrifugation.
      Note: Use of siliconized tubes is critical to avoid the loss of RNA due to non-specific binding to tube walls.

    9. Carefully disassemble the cassette and remove the gel. Stain with SYBR Gold diluted 1:5,000 in 1x TBE running buffer.
      Note: We typically use 3 μl of SYBR Gold in 15 ml running buffer and stain on a slowly rocking nutator in a small plastic dish for about 5 min at room temperature. Wear appropriate protective equipment to prevent exposure to SYBR Gold, and also to prevent contamination of samples with extraneous biological material.
    10. Transfer the gel onto a clear plastic film and place on a UV transilluminator (set at 365 nm wavelength).
    11. Carefully excise out gel fragments for each sample from the 25-nt marker upto the 75-nt marker, which should be just below a bright band corresponding to cellular tRNAs (Figure 3).
      Note: For a non-degraded RNA sample, there will most likely be no visible RNA in the excised gel fragment. We often include a small portion of the lowest visible tRNA band to serve as a carrier in subsequent steps. Intact tRNAs typically do not reverse transcribe efficiently and should not result in overwhelming contamination in the final dataset.


      Figure 3. Small RNA size selection. A. Four intact total RNA samples (6% denaturing TBE-Urea PAGE). Two biological replicates for each experiment were run side-by-side, with approximate DNA sizing ladders. Images cropped and brightness/contrast adjusted in Microsoft Word. B. Size selection of 25- to 75-nt RNAs, including lowest tRNA band. The Invitrogen 10 bp DNA ladder was used in this gel but has since been discontinued by the manufacturer.

    12. Place each gel fragment in a separate elution tube.
    13. Centrifuge each elution tube at 20,000 x g at room temperature in a tabletop microcentrifuge for 1-3 min to force the gel fragment through the incision in the 0.6 ml tube and into the 1.5 ml siliconized tube. Carefully remove any leftover gel pieces in the 0.6 ml tube with a clean pipette tip, and place in the corresponding 1.5 ml siliconized tube. Discard the 0.6 ml tube.
    14. Add 300 μl of polyacrylamide gel elution buffer (see Recipes) into each 1.5 ml siliconized tube containing pulverized gel fragments, and vortex vigorously to make a uniform slurry.
    15. Place the tubes at -80 °C to freeze, then in a 37 °C water bath for 2 min to thaw. Vortex vigorously, and then repeat this step 2-3 times.
    16. Place the samples on ice for 1 min to cool, then incubate at 4 °C with shaking in a rotisserie tube rotator overnight to elute RNA from gel fragments.
    17. Centrifuge briefly to collect gel slurry at the bottom of the tube.
    18. Prepare filtration tubes by placing 0.45 μm sterile cellulose acetate filters in new 1.5 ml siliconized tubes.
    19. Widen the bore of 1,000 μl pipette tips using clean scissors, and transfer gel slurry to the filtration tubes.
    20. Collect RNA eluate by centrifugation at 16,000 x g for 2 min at room temperature, discard the filters, and add (in order) 1 μl (20 mg/ml) glycogen and 1 ml 100% ethanol to each sample.
    21. Precipitate nucleic acids by placing the tubes at -80 °C for 30 min.
    22. Centrifuge at 20,000 x g at 4 °C for 30 min, and discard the ethanol while taking care not to dislodge the pellet.
    23. Wash with 1 ml freshly prepared 70% ethanol, taking care to flush out the cap by inverting several times.
      Note: It is not necessary to vortex aggressively at this step. Vortexing can be helpful in dislodging the pellet, but excessive agitation, as well as use of more concentrated ethanol for washing will lead to pellet fragmentation and reduction in yield.
    24. Centrifuge at 20,000 x g for 2 min at room temperature to collect the pellet, pour off the ethanol and repeat the wash.
    25. Centrifuge briefly at room temperature to collect residual ethanol after the second wash step is complete. Remove remaining ethanol using a 10 μl pipette, taking care not to touch the pellet. Air dry for 3 min.
      Note: After removing residual ethanol with a pipette, 3 min is sufficient to dry the pellet. We typically dry under a flame to prevent dust from accidentally settling in the tubes.
    26. Resuspend pellet in 17 μl RNase-free water at room temperature.
      Note: The added glycogen from Step A20 should result in a clearly visible pellet that may become translucent upon drying. The pellet will be easy to resuspend provided it has not been over-dried.

  2. Polynucleotide kinase (PNK) treatment
    1. Denature RNA at 90 °C for 1 min in a heated-lid thermocycler, then plunge in ice for 1 min.
      Note: We use the entire RNA sample from the previous step, and do not attempt to measure its concentration since the amount of RNA is often below the detection limit of commercial assay kits.
    2. To 17 μl of the RNA sample, add (in order) 2 μl 10x PNK buffer and 1 μl PNK enzyme, and mix well by pipetting.
    3. Incubate at 37 °C for 1 h.
    4. Add (in order) 80 μl RNase-free water, 50 μl (7.5 M) ammonium acetate, and 500 μl 100% ethanol.
    5. Precipitate RNA as in Steps A21-A25.
    6. Resuspend pellet in 4.5 μl RNase-free water at room temperature.
      Note: The added glycogen from Step A20 should result in a clearly visible pellet that may become translucent upon drying. The pellet will be easy to resuspend provided it has not been over-dried.

  3. 3’ adapter ligation (without ATP)
    1. Pre-mix equal volumes of 5x adenylation buffer (see Recipes) and 50% PEG 8000 to make 4 μl mixture per sample (+ 20% extra to account for pipetting error).
    2. Transfer RNA samples to 0.2 ml PCR tubes, and add 4 μl of the mixture to each RNA sample. Mix well by pipetting.
      Note: PEG 8000 is viscous and pre-mixing with 5x adenylation buffer helps to reduce viscosity and make dispensing to sample tubes easier. Mix by pipetting for as long as necessary until the solution appears uniform.
    3. Heat sample to 98 °C for 1 min, plunge in ice for 1 min, then place at room temperature for the next step.
    4. Add (in order) 0.5 μl (100 μM) pre-adenylated 3’ adapter oligo, and 1 μl T4 RNA ligase I. Mix well by pipetting.
      Note: We keep the pre-adenylated 3’ adapter oligo at -80 °C and thaw on ice before use.
    5. Incubate in a thermocycler at 22 °C for 6 h. The reaction can be stored at 4 °C if performing this step overnight.

  4. Excess adapter digestion
    1. Pre-mix 78 μl RNase-free water and 10 μl NEB buffer 2 for each sample.
    2. Incubate RNA samples at 95 °C for 1 min, allow to cool and then add 88 μl of buffer mixture.
    3. Add 1 μl 5’ deadenylase, mix, and incubate at 30 °C in a thermocycler for 30 min.
    4. Add 1 μl RecJf, mix, and incubate at 37 °C in a thermocycler for 30 min.
      Note: The 5’ deadenylase removes the /5rApp/ group from the free 5’ ends of un-ligated pre-adenylated adapters, thereby exposing the excess adapter molecules to digestion by the single-stranded-DNA-specific 5’ → 3’ exonuclease RecJf.
    5. During the digestion step, pre-spin a heavy phaselock gel tube for each sample at 16,000 x g for 2 min at room temperature.
    6. Add 100 μl RNase-free water to each RNA sample, mix well, and transfer to a pre-spun phaselock tube.
    7. Add 200 μl acid-phenol:chloroform to each sample and mix by shaking vigorously by hand.
    8. Centrifuge at 16,000 x g at room temperature for 5 min.
    9. Add 200 μl chloroform to each sample in the same tube and mix gently by inversion.
    10. Centrifuge at 16,000 x g at room temperature for 5 min.
    11. Transfer the aqueous phase to a new 1.5 ml siliconized tube, and add (in order) 0.5 μl (20 mg/ml) glycogen, 100 μl (7.5 M) ammonium acetate and 1 ml 100% ethanol to each sample.
    12. Precipitate RNA as in Steps A21-A25.
    13. Resuspend pellet in 5.75 μl RNase-free water at room temperature.
      Note: The added glycogen from Step D11 should result in a clearly visible pellet that may become translucent upon drying. The pellet will be easy to resuspend provided it has not been over-dried.

  5. Reverse-transcription
    1. Prepare a reverse-transcription master mix, with 2 μl 5x First-strand buffer, 1 μl (0.1 M) dithiothreitol, and 0.5 μl (10 mM) dNTPs for each RNA sample (+ 20% extra to account for pipetting error).
    2. Transfer RNA samples to 0.2 ml PCR tubes, and add 0.25 μl (100 μM) reverse-transcription primer to each tube.
    3. Heat samples to 90 °C for 1 min, then plunge on ice for 1 min.
    4. Add 3.5 μl of reverse-transcription master mix to each sample.
      Note: Also maintain a ‘no-template’ control, which will allow for visualization of the reverse-transcription primer during the subsequent gel purification step.
    5. Add 0.5 μl SuperScript II reverse transcriptase to each reaction and mix well by pipetting.
    6. Incubate at 42 °C for 30 min in a heated-lid thermocycler to synthesize complementary DNA (cDNA).
    7. During this incubation step, set up and pre-run a Novex 10% TBE-Urea denaturing polyacrylamide gel in the XCell SureLock Mini-Cell Electrophoresis System at 180 V for at least 30 min as described in Steps A1-A2.
    8. Add 2 μl (1 N) sodium hydroxide to each reaction.
    9. Incubate at 70 °C for 15 min in a heated-lid thermocycler to hydrolyze RNA.
    10. Add 12 μl 2x formamide gel loading dye (i.e., at a final concentration of 1x) to each sample.
    11. Prepare 0.1 μg of Ultra Low Range DNA ladder in 2x formamide gel loading dye at a final concentration of 1x.
    12. Denature and run cDNA on pre-run gels as in Steps A4-A25, with the following modifications:
      1. In Step A7, run the gel until the Xylene Cyanol dye front (top band ~55 nt) reaches close to the bottom of the gel (about 45-60 min).
      2. In Step A11, excise gel fragments in the 100- to 160-nt range (processed CRISPR RNAs are generally in the ~50-100-nt range and the reverse transcription primer adds ~65-nt to the size of the desired small RNAs). Use the no-template control as a visual guide during gel excision, and avoid the bright bands formed in this lane (typically no higher than 90-nt) (Figure 4).
      3. In step A16, cDNA elution should be carried out at room temperature.
    13. Resuspend the cDNA pellet in 17 μl water. Reserve half the sample and store at -20 °C as a backup.


      Figure 4. Purification of cDNA following reverse transcription. A. Four cDNA sample (10% denaturing TBE-Urea PAGE). The first lane is the no-template control, followed by an approximate DNA sizing ladder. Brightness/contrast adjusted in Microsoft Word. B. Size selection of 100- to 160-nt cDNAs, avoiding bright high-molecular-weight bands. The Invitrogen 10 bp DNA ladder was used in this gel but has since been discontinued by the manufacturer.

  6. cDNA circularization
    1. Add 1 μl 10x circLigase reaction buffer, 0.5 μl (1 mM) ATP, and 0.5 μl (50 mM) manganese chloride solution to 8 μl cDNA in 0.2 ml PCR tubes.
    2. Add 0.5 μl circLigase enzyme. Mix well by pipetting.
    3. Incubate at 60 °C for 75 min in a heated-lid thermocycler.
    4. While the circularization reaction is proceeding, prepare enough 3-3.5% agarose gels (in 1x TAE with 0.5 μg/ml ethidium bromide) to accommodate 5 PCR lanes plus 1 DNA sizing ladder per cDNA sample.
      Note: We use the 1.5 mm thick 12-well combs supplied with Owl EasyCast B2 gel electrophoresis systems to make enough lanes for two cDNA samples.
    5. Stop the reaction by heating to 80 °C for 15 min. Use this circularized cDNA (ccDNA) sample directly as a template for PCR.

  7. PCR amplification and purification of sequencing libraries
    1. Prepare a PCR mix with 100 μl 2x Phusion Master Mix, 1 μl (100 μM) Universal PCR primer, and 100 μl water for each cDNA sample.
    2. Add 200 μl PCR mix to 5 μl ccDNA from Step F5. Add 1 μl of a different indexing primer for each sample.
    3. Split each reaction mixture into 5 separate 0.2 ml PCR tubes (40 μl each).
    4. Perform a PCR titration for each ccDNA sample by running each sub-reaction for a different number of cycles according to the following program:
      1. 98 °C for 30 sec
      2. N cycles of
        98 °C for 10 sec
        60 °C for 10 sec
        72 °C for 10 sec
      3. hold at 10 °C
      Note: We typically perform titrations with N = 12, 15, 18, 21, and 24 cycles for each ccDNA sample.
    5. Add 8 μl of 6x DNA gel loading dye to each reaction.
    6. Load all 5 titrations for each sample side-by-side on the agarose gel. We suggest using the Ultra Low Range DNA ladder (~0.5 μg/lane) to demarcate sets of titrations of different ccDNA samples.
    7. Run the gel in agarose gel running buffer (1x TAE with 0.5 μg/ml ethidium bromide) at 3.6-3.7 V/cm for 1-2 h.
    8. Place the gels on a UV transilluminator (set at 365 nm wavelength). Choose the appropriate number of PCR cycles for each ccDNA sample by visually assessing the PCR titration (see Note below) and excise a gel slice containing the PCR amplicon corresponding to the size of the desired product using a 100-1,000 μl pipette fitted with gel excision tips. Expel each gel slice into a separate 1.5 ml centrifuge tube.
      Note: A bright band corresponding to the ‘empty’ circularized ccDNA product (i.e., without a small RNA insert) should be visible in each lane. This may appear as a doublet as the number of PCR cycles (N) is increased. For most small RNA sequencing applications, the desired product will be ~50 bp above this bright band/doublet. For CRISPR RNA sequencing, we rarely ever see a visible smear at this size range, and cut ‘blindly’ using the DNA ladder and the location of the bright band/doublet (~125 bp) as a visual guide. We typically aim for the highest number of PCR cycles for each ccDNA sample while still safely avoiding the upward-smear from the bright ~125 bp band/doublet (Figure 5).


      Figure 5. Library preparation by ‘blind’ gel excision. A. PCR titrations of amplified sequencing libraries for two ccDNA samples (3-3.5% native agarose gel electrophoresis). 5 titrations for each ccDNA sample were run side-by-side along with a DNA sizing ladder. Brightness/contrast adjusted in Microsoft Word. B. Size selection of DNA at the 175 bp marker, above the bright band/doublet formed by amplification from empty ccDNA (i.e., without a small RNA insert). The Invitrogen 25 bp DNA ladder was used in this gel but has since been discontinued by the manufacturer.

    9. Extract DNA from the gel slices according to manufacturer’s instructions using the QIAGEN MinElute Gel Extraction kit.
    10. Quantify each purified DNA sample according to manufacturer’s instructions using the high-sensitivity double stranded DNA quantification kit accompanying the Qubit fluorometer.
    11. Calculate the approximate concentration of each sample according to the following formula:

    12. Pool the samples in equimolar amounts. The pooled library can be sequenced according to the specifications of your Illumina high-throughput sequencing services provider. We typically use the single-read configuration for 80 cycles for small RNA sequencing applications. For assessing pre-crRNA processing in Marinomonas mediterranea, we sequence no more than 1 million reads per sample, but this will depend on the level of expression of pre-crRNA in the species of interest.

Data analysis

We include a worked example with sample data, which requires the following programs to be installed:

cutadapt (tested on v1.14; likely compatible with most other versions)
Python 2.7 (with numpy, matplotlib for plotting)

The usage formats of the provided python scripts are in bold italics, followed by the specific commands in bold for the worked example with sample data. Start by downloading the worked example, and navigating to the worked_example/ directory in a unix terminal.

  1. Demultiplex reads: Obtain the high-throughput sequencing data in ‘fastq’ format.
    1. Sample and index reads will be in files Undetermined_S0_L001_R1_001.fastq and Undetermined_S0_L001_I1_001.fastq respectively, with the first read corresponding to the first index, the second read corresponding to the second index, and so on. A sample dataset is provided in the sample_data directory.
    2. To segregate reads corresponding to each index, prepare a demultiplexing ‘key’–a tab separated text file with the first column containing the desired sample name, and the second containing the reverse complement of the corresponding TruSeq LT index (AD001-8). A sample file deMultiplexKey_sample.dat is provided.
    3. Now run the deMultiplexer.py file as follows:

      python deMultiplexer.py <path_to_directory> <key>
      e.g., python deMultiplexer.py sample_data/ deMultiplexKey_sample.dat

      This generates a FASTQ file for each index provided in the key. Note how the files for samples 1-4 in the provided example are empty. The example dataset only contains reads corresponding to the index reads provided for samples 5-8.
      Move demultiplexed data (samples 5-8) to a separate directory

      mkdir sample_demultiplexed
      cd sample_data/
      mv sample[5-8]*.fastq ../sample_demultiplexed/
      cd ../

  2. Trim adapters: The high-throughput sequencing data will contain Illumina adapter sequences. These are parts of the molecule that were necessary for sequencing on the Illumina flowcell.
  3. Collapse reads to eliminate amplification bias: The assay design includes a random hexamer (NNNNNN) in the 3’ adapter sequence, which is ligated to every RNA molecule before reverse-transcription. This helps eliminate amplification bias in downstream steps and helps ensure that every read corresponds to a distinct RNA molecule in the biological sample.
    Both Steps 2 and 3 (trimming and collapsing) are performed in the provided example with the dirRNAseqAnalyse.py script (using the readCollapser2.py function, which must be in the same directory as the script) as follows:

    python dirRNAseqAnalyse.py <path_to_directory> <maximum_read_length>
    e.g., python dirRNAseqAnalyse.py sample_demultiplexed/ 80

    The program produces a log file dirRNAseqAnalyseLog.txt which contains details of the adapter trimming step.
  4. Convert to fasta: The fastq2fasta.sh script has been prepared anticipating the files that will be generated in Step 3 for sample data. Each line in this script processes one input fastq file to one output fasta file. Modify this file with your input files (with .trimmed.collapsed.fastq extensions) and output files (with .fasta extensions) as desired. Convert using the following commands:
    1. Run the provided fastqtofasta.sh script:

      sh fastq2fasta.sh

      Move the trimming intermediates to a new directory:

      mkdir sample_trimmed_collapsed
      mv *.trimmed* sample_trimmed_collapsed/

    2. Move the .fasta files to a new directory:

      mkdir sample_fasta
      mv *.fasta sample_fasta/

  5. Filtering:
    1. Identify CRISPR derived reads: First, identify sequencing reads containing the 5’ end of the CRISPR direct repeat sequence. We require at least 5 contiguous bases in the sequencing read to match the first five bases of the CRISPR repeat. The CRISPR repeat of interest is supplied in the 1st line of the parameters file crRNAfigureMaker_params.txt. Any sequence upstream of the start of the CRISPR repeat is removed.
    2. Remove short matches: If the resulting processed repeat is shorter than 12 bases, also check to see if the 5 bases preceding the CRISPR repeat in the original read match one of the possible spacer endings from the CRISPR arrays in the bacterial genome. In this way, we require at least 12 bases from the CRISPR repeat, or 10 bases across the spacer-repeat junction for any read to qualify for downstream analysis. A dictionary of all possible native spacer endings from the type III-B CRISPR locus in the Marinomonas mediterranea MMB-1 genome is provided in spacerEnds.dict.
      Note: The spacerEnds.dict file can be modified in any text editor, but its formatting must be preserved to prevent parsing errors in python.
    3. Assess match fidelity: If the read passes initial filtering, the processed repeat is then matched to the expected CRISPR repeat sequence. We require the repeat to be a left-anchored substring of the CRISPR repeat (i.e., the processed repeat may be shorter than the CRISPR repeat, but it must match at the 5’ end and cannot contain mismatches).
  6. Measure levels of a reference gene: Next, count reads containing 25-nt substrings of a reference gene that is highly expressed and does not vary with the biological conditions under study. We use the isoleucine-tRNA sequence as a reference in M. mediterranea datasets, but this may need to be empirically determined based on your RNAseq data for your model. This sequence must be provided in the 2nd line of the crRNAfigureMaker_params.txt file.
  7. Plot a histogram of lengths of trimmed reads: Finally, plot a histogram of the lengths of the processed CRISPR repeats normalized to the reference gene. The 3rd line of the crRNAfigureMaker_params.txt file is an arbitrary scaling parameter that controls the height of the Y axis in the plot. It can be changed to accommodate the levels of processed crRNAs relative to the reference gene in your dataset.
    Steps 5, 6, and 7 are performed by the crRNAfigureMaker.py script as follows:

    python crRNAfigureMaker.py <path_to_fasta_files> <keyword>
    e.g., python crRNAfigureMaker.py sample_fasta/ 8


    The keyword option specifies which files should be included in the analysis. The keyword can be any part of the file name. For instance, using the keyword ‘8’ will only process sample8.fasta in the worked example, while using the keyword ‘mpl’ will include all 4 sample files for processing, and using the keyword ‘sem’ will result in no files being included.
    The crRNAfigureMaker_param.txt file must be in the same directory as the code. Running the above command (i.e., only processing sample8.fasta) should generate Figure 6 below.


    Figure 6. Expected output of code provided in the worked example. Processed crRNA levels assayed by high throughput small RNA sequencing. This dataset has been artificially supplemented with sequences matching expected CRISPR-derived RNAs. The CRISPR repeat sequence from the 1st line of the parameters file crRNAfigureMaker_params.txt is on the X-axis. The height of the bar at each base along the X-axis represents the relative proportion of crRNAs with 3’ ends at that base, normalized to the levels of the reference RNA (isoleucine tRNA; consistently the most abundant species encountered in our M. mediterranea datasets). The presence of a distinct 3’ end sequence in the population of CRISPR repeat containing RNAs indicates site-specific cleavage and processing of pre-crRNA.

    The data files in the worked example are small subsets of our experimental data and have been artificially supplemented with sequences matching expected CRISPR-derived RNAs. Please refer to our public datasets at the NCBI Short Read Archive (SRP103952) to recreate the published graphs (Silas et al., 2017a). The following table specifies the accession numbers for the experiments that correspond to each of the relevant figure panels in (Silas et al., 2017a).

Recipes

  1. Polyacrylamide gel elution buffer
    300 mM NaCl
    1 mM EDTA
    To make 50 ml:
    3 ml
    5 M sodium chloride solution
    500 μl
    0.1 M EDTA solution
    46.5 ml
    RNase free water
  2. 1 M HEPES/KOH buffer pH 8.3
    Adjust the pH of 1 M HEPES solution with 8 N potassium hydroxide to 8.3
    Sterile filter using a 0.2 μm vacuum filtration unit
  3. 60% glycerol solution
    To make 10 ml, mix 6 ml glycerol with 4 ml RNase-free water
    Sterilize by autoclaving
  4. 5x adenylation buffer
    Note: Store in 1 ml aliquots at -20 °C up to 1 year.
    41% glycerol
    250 mM HEPES/KOH pH 8.3
    50 mM MgCl2
    16.5 mM DTT
    50 μg/ml Ac-BSA
    To make 5 ml, mix:
     3.4 ml
    60% glycerol solution
    1.25 ml
    1 M HEPES/KOH buffer pH 8.3
    250 μl
    1 M magnesium chloride solution
    82.5 μl
    1 M dithiothreitol solution
    12.5 μl
    20 mg/ml acetylated BSA

Acknowledgments

S.S. was supported by a Stanford Graduate Fellowship and an HHMI International Student Research Fellowship. This protocol was developed with support from the NIH (grant R01-GM37706 to A.Z.F.). We adapted earlier RNA sequencing methods (Lau et al., 2001; Ingolia et al., 2009; Guo et al., 2010; Kwon, 2011) for RNA sequencing from C. elegans (Lamm et al., 2011), incorporated others’ work on unique molecular identifiers to remove PCR duplicates (Kivioja et al., 2011), and introduced enzymatic cleanup steps (5’ deadenylase/RecJ) to circumvent a gel purification step. We have found this protocol useful for a variety of RNA sequencing applications, such as crRNA detection (Silas et al., 2017a), prokaryotic transcriptome profiling (Silas et al., 2016), sequencing RNA from metagenomic environmental samples (Silas et al., 2017b), and ribosome footprinting (Arribere et al., 2016). We declare that we have no competing interests or conflicts of interest.

References

  1. Arribere, J. A., Cenik, E. S., Jain, N., Hess, G. T., Lee, C. H., Bassik, M. C. and Fire, A. Z. (2016). Translation readthrough mitigation. Nature 534(7609): 719-723.
  2. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A. and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819): 1709-1712.
  3. Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., Dickman, M. J., Makarova, K. S., Koonin, E. V. and van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321(5891): 960-964.
  4. Carte, J., Pfister, N. T., Compton, M. M., Terns, R. M. and Terns, M. P. (2010). Binding and cleavage of CRISPR RNA by Cas6. RNA 16(11): 2181-2188.
  5. Carte, J., Wang, R., Li, H., Terns, R. M. and Terns, M. P. (2008). Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev 22(24): 3489-96.
  6. Charpentier, E., Richter, H., van der Oost, J. and White, M. F. (2015). Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity. FEMS Microbiol Rev 39(3): 428-441.
  7. Deveau, H., Barrangou, R., Garneau, J. E., Labonte, J., Fremaux, C., Boyaval, P., Romero, D. A., Horvath, P. and Moineau, S. (2008). Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol 190(4): 1390-1400.
  8. Guo, H., Ingolia, N. T., Weissman, J. S. and Bartel, D. P. (2010). Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466(7308): 835-840.
  9. Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. and Doudna, J. A. (2010). Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329(5997): 1355-1358.
  10. Heidrich, N., Dugar, G., Vogel, J. and Sharma, C. M. (2015). Investigating CRISPR RNA biogenesis and function using RNA-seq. Methods Mol Biol 1311: 1-21.
  11. Hochstrasser, M. L. and Doudna, J. A. (2015). Cutting it close: CRISPR-associated endoribonuclease structure and function. Trends Biochem Sci 40(1): 58-66.
  12. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. and Weissman, J. S. (2009). Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924): 218-223.
  13. Jackson, S. A., McKenzie, R. E., Fagerlund, R. D., Kieper, S. N., Fineran, P. C. and Brouns, S. J. (2017). CRISPR-Cas: Adapting to change. Science 356(6333).
  14. Juranek, S., Eban, T., Altuvia, Y., Brown, M., Morozov, P., Tuschl, T. and Margalit, H. (2012). A genome-wide view of the expression and processing patterns of Thermus thermophilus HB8 CRISPR RNAs. RNA 18(4): 783-794.
  15. Kivioja, T., Vaharautio, A., Karlsson, K., Bonke, M., Enge, M., Linnarsson, S. and Taipale, J. (2011). Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods 9(1): 72-74.
  16. Kwon, Y. S. (2011). Small RNA library preparation for next-generation sequencing by single ligation, extension and circularization technology. Biotechnol Lett 33(8): 1633-1641.
  17. Lamm, A. T., Stadler, M. R., Zhang, H., Gent, J. I. and Fire, A. Z. (2011). Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome. Genome Res 21(2): 265-275.
  18. Lau, N. C., Lim, L. P., Weinstein, E. G. and Bartel, D. P. (2001). An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294(5543): 858-862.
  19. Makarova, K. S., Haft, D. H., Barrangou, R., Brouns, S. J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F. J., Wolf, Y. I., Yakunin, A. F., van der Oost, J. and Koonin, E. V. (2011). Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9(6): 467-477.
  20. Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., van der Oost, J., Backofen, R. and Koonin, E. V. (2015). An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 13(11): 722-736.
  21. Marraffini, L. A. and Sontheimer, E. J. (2008). CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322(5909): 1843-1845.
  22. Plagens, A., Richter, H., Charpentier, E. and Randau, L. (2015). DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes. FEMS Microbiol Rev 39(3): 442-463.
  23. Richter, H., Zoephel, J., Schermuly, J., Maticzka, D., Backofen, R. and Randau, L. (2012). Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis. Nucleic Acids Res 40(19): 9887-9896.
  24. Silas, S., Lucas-Elio, P., Jackson, S. A., Aroca-Crevillen, A., Hansen, L. L., Fineran, P. C., Fire, A. Z. and Sanchez-Amat, A. (2017a). Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems. Elife 6.
  25. Silas, S., Makarova, K. S., Shmakov, S., Paez-Espino, D., Mohr, G., Liu, Y., Davison, M., Roux, S., Krishnamurthy, S. R., Fu, B. X. H., Hansen, L. L., Wang, D., Sullivan, M. B., Millard, A., Clokie, M. R., Bhaya, D., Lambowitz, A. M., Kyrpides, N. C., Koonin, E. V. and Fire, A. Z. (2017b). On the origin of reverse transcriptase-using CRISPR-Cas systems and their hyperdiverse, enigmatic spacer repertoires. MBio 8(4).
  26. Silas, S., Mohr, G., Sidote, D. J., Markham, L. M., Sanchez-Amat, A., Bhaya, D., Lambowitz, A. M. and Fire, A. Z. (2016). Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein. Science 351(6276): aad4234.

简介

新一代高通量测序技术能够对细胞中的RNA群体进行敏感和明确的分析。在这里,我们描述了一种从细菌中分离和链特异性测序小RNA池的方法,所述细菌可以在单个实验中多路复用以容纳多个生物样品。小RNA通过聚丙烯酰胺凝胶电泳分离并用T4多核苷酸激酶处理。这允许3'衔接头连接至CRISPR RNA,其不具有预先存在的3'-OH末端。然后使用T4 RNA连接酶1在不存在ATP和高浓度聚乙二醇(PEG)的情况下将前腺苷酸化的衔接子连接。 3'捕获步骤能够精确测定不同RNA分子的3'末端。此外,连接适配器中的随机六聚体有助于控制潜在的下游扩增偏差。逆转录后,将cDNA产物环化并通过PCR制备文库。我们显示扩增的文库不需要通过凝胶电泳可见,以期望产物的有效测序。使用这种方法,我们通常从少量纯化的小RNA制备RNA测序文库。该协议适合于通过对成熟的CRISPR RNA进行测序来测定细菌中的CRISPR RNA生物合成,但可以用于测序不同类型的小RNA。我们还提供了一个完整的数据处理管道示例,并提供了运行所提供脚本的说明。


【背景】与聚集的经常散布的短回文重复序列(CRISPR)相关的遗传模块赋予不同的原核宿主适应性免疫(Barrangou et al。,2007)。将侵入性元件(例如病毒,质粒和其他可移动元件)的记忆以包含分子寄生虫的核酸序列的“间隔子”的形式散布在宿主基因组的CRISPR阵列的定向重复之间(Brouns 等人,2008;杰克逊等人,,2017)。为了识别相同入侵者的后续感染,CRISPR间隔区中包含的信息必须传达给CRISPR相关(Cas)核酸内切酶(Plagens et al。,2015)。对于绝大多数CRISPR-Cas系统(系统发生学分组为'I型'和'III型'[Makarova等人,<2011年和2015年]),这是通过一系列称为Cas6的CRISPR相关的内切核糖核酸酶(Charpentier等人,2015; Hochstrasser和Doudna,2015)。整个CRISPR阵列被转录为来自基因组的前体CRISPR RNA(前crRNA)分子,并且Cas6蛋白结构域有助于将该转录转化成由每个CRISPR间隔区组成的成熟CRISPR RNA(crRNA)的集合,两侧为CRISPR重复序列的部分(Carte等人,2008和2010; Haurwitz等人,2010)。这种机制被称为crRNA生物发生。 Cas6内切核糖核酸酶通过位点特异性切割CRISPR重复序列来促进crRNA生物发生,其产生5'-OH和2'3'-环状磷酸酯末端(Charpentier等人,2015; Hochstrasser和Doudna, 2015年)。每个CRISPR重复序列的位点特异性切割导致pre-crRNA分子以规则的间隔切割成几乎等长的crRNA,每个crRNA具有不同的间隔序列(Charpentier等人,2015; Hochstrasser和Doudna,2015)。然后将成熟的crRNA加载到Cas效应复合物上并作为基于序列互补性引导Cas酶以靶向DNA或RNA寄生虫的分子指导(Deveau等,2008; Marraffini和Sontheimer,2008)。从细菌细胞群分离的成熟crRNA的存在或不存在可以用作Cas6活性的代用品。虽然生物化学方法已被开发用于检测crRNA(Carte等人,2008和2010; Haurwitz等人,2010),但高通量RNA测序可用于明确测定Cas6活性(Heidrich等人,2015)。根据文库制备的具体方法,整个转录组测序是昂贵的并且可以针对特定类型的RNA进行偏倚。因此,已经开发了各种小RNA测序方案以优先检测成熟的crRNA(Juranek等人,2012; Richter等人,2012; Heidrich等人, 。,2015)。

在这里,我们提出了一种多路复用的小RNA测序方法,以便能够在许多不同的生物条件下一次比较容易和可重复地比较crRNA成熟性,例如Cas6蛋白中的突变以评估Cas6活性的机制。该协议建立在以前关于小RNA测序和核糖体分析的工作上(Lau等人,2001; Ingolia等人,2009; Guo等人, 2010; Kwon,2011; Kivioja等人,2011年)。该测定法具有高灵敏度和动态范围,而不会在其他细胞RNA上花费大量的测序带宽,同时警告小RNA测序不能观察到全长前体转录物。

关键字:CRISPR, 小RNA, 高通量测序, 向导RNA, CRISPR RNA, crRNA加工, crRNA生物合成, crRNA成熟

材料和试剂

  1. 凝胶加样移液器吸头0.5-200μl(Thermo Fisher Scientific,Invitrogen TM,目录号:LC1001)
  2. 0.6 ml微量离心管(Sigma-Aldrich,目录号:T5149)
  3. 剃刀刀片
  4. 硅化1.5毫升微量离心管(VWR,目录号:22179-004)
    制造商:BIO PLAS,目录号:4165SL。
  5. 塑料盘子
  6. 透明塑料薄膜(Saran包装或等同物)
  7. Corning Costar Spin-X无菌0.45μm醋酸纤维素离心管过滤器(Corning,目录号:8162)
  8. 0.2ml PCR管,MicroAmp(Thermo Fisher Scientific,Applied Biosystems TM,目录号:N8010540)或同等产品
  9. 凝胶切除移液器吸头(Corning,Axygen ,目录号:TGL-1165-R)

  10. 2 ml试管中的重相锁凝胶(Quantabio,目录号:2302830)
  11. Corning管顶部真空过滤系统(Corning,目录号:430320)
  12. Trizol试剂(Thermo Fisher Scietific,Invitrogen TM,目录号:15596026)
  13. 预铸Novex 6%TBE-尿素聚丙烯酰胺凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:EC6865BOX)
  14. 10X TBE运行缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9863) - 使用前稀释至1x
  15. GeneRuler超低量程DNA梯(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:SM1211)
  16. 2x甲酰胺凝胶上样染料(Thermo Fisher Scientific,Invitrogen TM,目录号:AM8546G)
  17. SYBR Gold核酸凝胶染色(Thermo Fisher Scientific,Invitrogen TM,目录号:S11494)
  18. UltraPure糖原(Thermo Fisher Scientific,Invitrogen TM,目录号:10814010)
  19. 200 Proof分子生物学级乙醇(Sigma-Aldrich,目录号:E7023)
  20. UltraPure DNase / RNase-free蒸馏水(Thermo Fisher Scientific,目录号:10977035)
  21. 多核苷酸激酶(PNK)酶和缓冲液(New England Biolabs,目录号:M0201S)
  22. 醋酸铵溶液7.5M分子生物学级(Sigma-Aldrich,目录号:A2706)
  23. 50%PEG 8000(与NEB T4 RNA连接酶I一起提供)
  24. 前腺苷酸化3'衔接子寡核苷酸:/ 5rApp / NNNNNNAGATCGGAAGAGCACACGTCT / 3ddC /
  25. T4 RNA连接酶I(New England Biolabs,目录号:M0204S)
  26. NEB缓冲液2(New England Biolabs,目录号:B7002S)
  27. 5'Deadenylase(New England Biolabs,目录号:M0331S)
  28. RecJf(新英格兰生物实验室,目录号:M0264S)
  29. 酸化的酚:氯仿1:1混合物(Thermo Fisher Scientific,目录号:AM9720)
  30. 氯仿(Sigma-Aldrich,目录号:496189)
  31. 5x第一链缓冲液(由SuperScript II逆转录酶提供)
  32. 0.1 M二硫苏糖醇(与SuperScript II逆转录酶一起提供)
  33. 10mM dNTP混合物(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0191)
  34. SuperScript II逆转录酶(Thermo Fisher Scientific,Invitrogen TM,目录号:18064014)
  35. 反转录引物:
    / 5Phos / AGATCGGAAGAGCGTCGTGT / iSp18 / CACTCA / iSp18 / GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
  36. 预铸Novex 10%TBE-尿素聚丙烯酰胺凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:EC6875BOX)
  37. 1N氢氧化钠溶液(Merck,目录号:SX0607H)
  38. CircLigase ssDNA连接酶和10x反应缓冲液(Lucigen,目录号:CL4111K)
  39. 1 mM ATP溶液(由circLigase提供)
  40. UltraPure琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:16500500)
  41. 10倍TAE(Thermo Fisher Scientific,产品目录号:AM9869) - 使用前稀释至1倍
  42. 溴化乙锭溶液10mg / ml(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17898)
  43. Phusion高保真PCR主混合物(Thermo Fisher Scientific,Thermo Scientific TM,目录号:F531S)
  44. 索引引物:
    CAAGCAGAAGACGGCATACGAGAT XXXXXX GTGACTGGAGTTCAGACGTGTGCTCTTCCG其中第X6条形码对应于Illumina TruSeq LT指数AD001至AD008 [ATCACG,CGATGT,TTAGGC,TGACCA,ACAGTG,GCCAAT,CAGATC,ACTTGA] /> 注:可根据需要添加更多索引引物。
  45. 通用PCR引物:
    AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
  46. DNA凝胶上样染料6x(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0611)
  47. 25 bp DNA梯子
  48. MinElute凝胶提取试剂盒(QIAGEN,目录号:28604)
  49. 用于分子生物学的5M氯化钠溶液BioUltra(Sigma-Aldrich,目录号:71386)
  50. 0.1M EDTA溶液,pH7.5(Merck,目录号:EX0546A)
  51. 1 M HEPES溶液BioPerformance认证,0.2μm过滤(Sigma-Aldrich,目录号:H3537)
  52. 8N氢氧化钾溶液(Sigma-Aldrich,目录号:P4494)
  53. 50mM氯化锰溶液(由circLigase提供)
  54. Qubit dsDNA HS分析试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32854)
  55. 用于分子生物学的1μM二硫苏糖醇溶液BioUltra(Sigma-Aldrich,目录号:43816)
  56. 用于分子生物学的甘油(Sigma-Aldrich,目录号:G5516)
  57. 用于分子生物学的1M氯化镁溶液(Sigma-Aldrich,目录号:M1028)
  58. 20mg / ml乙酰化牛血清白蛋白(Thermo Fisher Scientific,目录号:AM2614)
  59. 聚丙烯酰胺凝胶洗脱缓冲液(见食谱)
  60. 1 M HEPES / KOH缓冲液pH 8.3(见食谱)
  61. 60%甘油溶液(见食谱)
  62. 5倍腺苷酸化缓冲液(见食谱)

设备

  1. 剪刀
  2. XCell SureLock Mini-Cell(Thermo Fisher Scientific,Invitrogen TM,型号:XCell Sure LockTM TM Mini-Cell,目录号:EI0001) br />
  3. 加热盖热循环仪,Veriti 96孔(Thermo Fisher Scientific,Applied Biosystems TM,型号:Veriti 96孔,目录号:4375786) >
  4. 具有365纳米波长紫外灯泡的透射仪(VWR,目录号:89131-464或同等产品)
  5. 台式微量离心机(Eppendorf,型号:5424或同等产品,适用于室温和4°C)
  6. 冰箱能够达到-80°C
  7. 可编程水浴/加热块
  8. Rotisserie管旋转器(VWR,目录号:10136-084或同等产品)
  9. 10μl移液器
  10. 凝胶电泳电源(Thermo Fisher Scientific,型号:Owl TM EC1000XL或同等产品)
  11. Owl EasyCast Mini Gel电泳系统(Thermo Fisher Scientific,Thermo Scientific TM,型号:Owl TM EasyCast TM B2)或等同物。
  12. 100-1,000μl移液器
  13. Qubit 3.0荧光计(Thermo Fisher Scientific,Invitrogen TM,型号:Qubit TM 3,目录号:Q33216)。

程序

方案可以在4天内舒适地进行(包括从细菌中分离RNA)如下:第1天的步骤A1-A16,第2天的A17-C5,第3天的D1-E12,并在第4天E13开始。下面的流程图总结了协议中的主要步骤(图1)。


图1.协议主要步骤的流程图

从细菌中分离RNA:RNA提取方法将取决于研究中的细菌。提取方法必须避免任何基于柱或尺寸的纯化步骤,这可能会导致小RNA的优先丢失。我们按照制造商提供的Trizol试剂说明书为我们的模型系统
测序库准备:

  1. 通过变性聚丙烯酰胺凝胶电泳(PAGE)分离小RNA
    1. 在XCell SureLock迷你细胞电泳系统中组装预制的Novex 6%TBE-Urea变性聚丙烯酰胺凝胶。
      注意:在组装电泳池之前,请记得取下凝胶盒底部的凝胶梳子和绿色胶带。
    2. 用1x TBE运行缓冲液填充内室和外室,并在180 V下预凝胶至少30分钟。
    3. 在2倍甲酰胺凝胶上样染料中制备至少5-10μg总RNA完整RNA和0.1μg超低量程DNA梯的样品,终浓度为1x。我们建议保持每个样品的总体积< 15μl。
    4. 通过加热预热盖子的热循环仪在94°C下加热5分钟使样品和梯子变性,然后立即放入冰水浆液中。
    5. 当样品变性时,使用100μl移液管和来自内室的运行缓冲液多次彻底冲洗每个凝胶孔中的尿素。

    6. 使用凝胶加样枪头小心加样 注意:在不同RNA样品之间留1-2个泳道以减少实验之间的交叉污染。我们建议在10通道凝胶中包含不超过4-5个RNA样品(以及一个通道用于近似大小的梯子)。
    7. 以180V运行,直至溴酚蓝染料前沿(底部带〜25nt)达到接近凝胶末端(约35分钟)。
    8. 在凝胶运行的同时,准备凝胶洗脱管,在0.6毫升管的底部用干净的剃须刀片(详见图)制成小十字形切口,并将其置于1.5毫升硅化离心管内。不要取下0.6毫升或1.5毫升管的盖子(图2)。


      图2.用于粉碎聚丙烯酰胺凝胶片段的0.6ml试管的纵向和横向视图。 使用干净的剃刀刀片在0.6ml离心管的底部制作十字形切口。当聚丙烯酰胺凝胶片段被迫通过切口并通过离心进入1.5ml硅化离心管时被粉碎。
      注意:硅化管的使用对于避免由于非特异性结合管壁造成的RNA损失是至关重要的。

    9. 小心拆卸盒子并取出凝胶。用1x TBE运行缓冲液1:5,000稀释SYBR Gold染色。
      注意:我们通常在15 ml运行缓冲液中使用3μlSYBR Gold,并在室温下在小塑料培养皿中缓慢摇动的章动器上染色约5 min。佩戴适当的防护设备以防止暴露于SYBR Gold,并防止样品受到外来生物材料的污染。
    10. 将凝胶转移到透明塑料膜上,并置于紫外透射仪(设置在365nm波长)。
    11. 小心地从25-nt标记到75-nt标记切除每个样本的凝胶片段,这应该恰好在对应于细胞tRNA的亮带下方(图3)。
      注意:对于未降解的RNA样品,切下的凝胶片段中很可能没有可见的RNA。我们通常包括最低可见tRNA带的一小部分,作为后续步骤中的载体。完整的tRNA通常不会有效地转录,并且不会导致最终数据集中的压倒性污染。


      图3.小RNA大小选择A.四个完整的总RNA样品(6%变性TBE-尿素PAGE)。每个实验的两个生物学重复并行运行,具有近似的DNA上浆梯。在Microsoft Word中调整图像并调整亮度/对比度。 B. 25到75nt RNA的大小选择,包括最低的tRNA带。该凝胶中使用了Invitrogen的10 bp DNA梯,但此后已被制造商停产。

    12. 将每个凝胶片段放入单独的洗脱管中。
    13. 在室温下,在桌面微量离心机中将每个洗脱管在20,000g x g离心1-3分钟以迫使凝胶片段通过0.6ml试管中的切口并进入1.5ml硅化试管。用干净的移液管小心地将0.6ml试管中的剩余凝胶块小心地取出,并放入相应的1.5ml硅化试管中。丢弃0.6毫升管。
    14. 将300μl聚丙烯酰胺凝胶洗脱缓冲液(见配方)加入每个含有粉碎凝胶碎片的1.5 ml硅化管中,并剧烈涡旋以形成均匀的浆液。
    15. 将试管置于-80°C冷冻,然后放入37°C水浴2分钟解冻。大力漩涡,然后重复此步骤2-3次。
    16. 将样品置于冰上1分钟以冷却,然后在4℃下在旋转式旋转器旋转器中摇动过夜以洗脱凝胶片段中的RNA。

    17. 短暂离心以收集管底部的凝胶浆。

    18. 通过将0.45μm无菌醋酸纤维素过滤器放入新的1.5 ml硅化管中来准备过滤管。

    19. 使用清洁剪刀扩大1,000μl移液器吸头的孔径,并将凝胶浆液转移到过滤管中。
    20. 通过在室温下以16,000xg离心2分钟收集RNA洗脱物,弃去滤器,并向每个样品(按顺序)添加1μl(20mg / ml)糖原和1ml 100%乙醇。
    21. 将管放置在-80°C下30分钟以沉淀核酸。
    22. 在4℃下以20000×g离心30分钟,弃去乙醇,同时注意不要除去沉淀。
    23. 用1毫升新鲜制备的70%乙醇洗涤,注意倒转几次来冲洗瓶盖。
      注:在这一步没有必要积极地漩涡。涡旋可能有助于排出颗粒,但过度搅动,以及使用更浓的乙醇进行清洗将导致颗粒碎裂并降低产量。
    24. 在室温下以20000×g离心2分钟以收集沉淀物,倒出乙醇并重复洗涤。
    25. 在第二次洗涤步骤完成后,在室温下简单离心以收集残留的乙醇。用10μl移液管移除剩余的乙醇,注意不要接触沉淀。空气干燥3分钟。
      注意:用移液管除去残留的乙醇后,3分钟就足以干燥颗粒。我们通常在火焰下干燥,以防止灰尘意外沉淀在管中。

    26. 在室温下将沉淀重悬于17μlRNase-free水中。
      注意:步骤A20中添加的糖原应产生清晰可见的颗粒,干燥后可变为半透明。如果没有过度干燥,颗粒很容易重新悬浮。

  2. 多核苷酸激酶(PNK)治疗
    1. 在加热盖热循环仪中于90°C变性1分钟RNA,然后在冰上浸1分钟。
      注意:我们使用上一步的整个RNA样本,并且不要尝试测量其浓度,因为RNA的量通常低于商业化验检测试剂盒的检测限。
    2. 向17μlRNA样品中加入2μl10x PNK缓冲液和1μlPNK酶,并通过移液充分混匀。

    3. 在37°C孵育1小时
    4. 加入(按顺序)80μl不含RNase的水,50μl(7.5M)乙酸铵和500μl100%乙醇。
    5. 按步骤A21-A25沉淀RNA。

    6. 在室温下将沉淀重悬于4.5μlRNase-free水中。
      注意:步骤A20中添加的糖原应产生清晰可见的颗粒,干燥后可变为半透明。
      如果颗粒没有过度干燥,颗粒很容易重新悬浮。

  3. 3'衔接子连接(无ATP)
    1. 预混合等体积的5倍腺苷酸化缓冲液(参见食谱)和50%PEG 8000以制备4μl混合物/样品(+ 20%以补偿移液误差)。
    2. 将RNA样品转移到0.2ml PCR管中,并向每个RNA样品中加入4μl混合物。通过移液很好地混合。
      注意:PEG 8000是粘稠的,并且与5x腺苷酸化缓冲液预先混合有助于降低粘度并使样品管更容易分配。
      。通过移液进行混合,直至溶液显得均匀。
    3. 将样品加热至98℃1分钟,在冰中浸1分钟,然后置于室温下进行下一步。
    4. 添加(按顺序)0.5μl(100μM)前腺苷酸化3'衔接头寡核苷酸和1μlT4 RNA连接酶I.通过移液完成混合。
      注意:我们将前腺苷酸化的3'衔接头寡核苷酸保存在-80°C并在使用前在冰上融化。
    5. 在22℃的热循环仪中孵育6小时。
      如果这一步过夜,反应可以保存在4°C
  4. 过量的适配器消化

    1. 每种样品预混合78μl无RNase的水和10μlNEB缓冲液2
    2. 将RNA样品在95°C孵育1分钟,让其冷却,然后加入88μl缓冲液混合物。
    3. 加入1μl5'脱腺苷酶,混匀,30°C温育30分钟。
    4. 加入1μlRecJ f ,混合,并在37℃下在热循环仪中孵育30分钟。
      注意:5'脱腺苷酶从未连接的预腺苷酸化衔接子的游离5'端去除/ 5rApp /组,从而暴露过量的衔接子分子以被单链DNA特异性5'→ 3'外切核酸酶RecJ f 。
    5. 在消化步骤中,在室温下,将每个样品的重的锁相凝胶管在16,000×gg下预旋转2分钟。
    6. 向每个RNA样品中加入100μl无RNase的水,充分混合,并转移到预旋转锁相管中。

    7. 在每个样品中加入200μl酸 - 酚:氯仿并通过剧烈摇动混合。

    8. 在室温下以16,000×gg离心5分钟。

    9. 加入200微升三氯甲烷到同一管中的每个样品,轻轻混合倒置。
    10. 在室温下在16,000×gg下离心5分钟。
    11. 将水相转移到新的1.5ml硅化管中,并向每个样品中加入(按顺序)0.5μl(20mg / ml)糖原,100μl(7.5M)乙酸铵和1ml 100%乙醇。
    12. 按步骤A21-A25沉淀RNA。

    13. 在5.75μlRNase-free水中重悬沉淀 注意:步骤D11中添加的糖原应产生清晰可见的颗粒,干燥后可变为半透明。
      如果颗粒没有过度干燥,颗粒很容易重新悬浮。

  5. 逆转录
    1. 准备一个逆转录主混合物,每个RNA样本加2μl5x第一链缓冲液,1μl(0.1M)二硫苏糖醇和0.5μl(10mM)dNTPs(+ 20%以补偿移液误差) br />
    2. 将RNA样品转移至0.2ml PCR管中,并向每个管中加入0.25μl(100μM)逆转录引物。
    3. 将样品加热至90°C 1分钟,然后在冰上浸1分钟。

    4. 加入3.5μl反转录混合物 注意:还要保留一个“无模板”控制,这样可以在随后的凝胶纯化步骤中对逆转录引物进行可视化。

    5. 加入0.5μlSuperScript II逆转录酶到每个反应中并充分混匀。
    6. 在热盖热循环仪中于42°C孵育30分钟以合成互补DNA(cDNA)。
    7. 在此孵育步骤中,如步骤A1-A2中所述,在XCell SureLock微型细胞电泳系统中在180V下建立并预先操作Novex 10%TBE-Urea变性聚丙烯酰胺凝胶至少30分钟。

    8. 加入2μl(1 N)氢氧化钠

    9. 在70°C孵育15分钟,在加热盖热循环仪中水解RNA。
    10. 向每个样品中加入12μl2x甲酰胺凝胶加样染料(即,最终浓度为1x)。

    11. 在2x甲酰胺凝胶上样染料中制备0.1μg超低量程DNA梯,终浓度为1x。
    12. 按照步骤A4-A25在预运行凝胶上变性并运行cDNA,并进行以下修改:
      1. 在步骤A7中,运行凝胶,直至二甲苯氰醇染料前沿(顶峰〜55 nt)接近凝胶底部(约45-60分钟)。
      2. 在步骤A11中,切割100至160nt范围内的凝胶片段(处理的CRISPR RNA通常在〜50-100-nt范围内,逆转录引物加入〜65-nt至所需小RNA的大小) 。在凝胶切除过程中使用无模板对照作为视觉引导,并避免在此泳道形成的亮带(通常不高于90-nt)(图4)。
      3. 在步骤A16中,应该在室温下进行cDNA洗脱。
    13. 用17μl水重悬cDNA沉淀。
      保留样品的一半,并储存在-20°C作为备份。


      图4.逆转录后纯化cDNA。 :一种。四个cDNA样品(10%变性TBE-尿素PAGE)。第一条泳道是无模板控制,接着是一个近似的DNA分级梯。在Microsoft Word中调整亮度/对比度。 B.大小选择100到160-nt的cDNA,避免明亮的高分子量条带。该凝胶中使用了Invitrogen的10 bp DNA梯,但此后已被制造商停产。

  6. cDNA环化

    1. 加入1μl10x circLigase反应缓冲液,0.5μl(1 mM)ATP和0.5μl(50 mM)氯化锰溶液至8μlcDNA中。
    2. 加入0.5μlcircLigase酶。通过移液很好地混合。

    3. 在60°C孵育75分钟在加热盖热循环仪。
    4. 在环化反应正在进行的同时,制备足够的3-3.5%琼脂糖凝胶(在含有0.5μg/ ml溴化乙锭的1x TAE中)以容纳5个PCR泳道加上每个cDNA样品1个DNA上浆梯。
      注意:我们使用Owl EasyCast B2凝胶电泳系统提供的1.5 mm厚的12孔梳,为两个cDNA样品制作足够的泳道。
    5. 加热到80°C 15分钟停止反应。直接使用该环化的cDNA(ccDNA)样品作为PCR的模板。

  7. PCR扩增和测序文库的纯化
    1. 准备PCR混合物,每个cDNA样本使用100μl2x Phusion Master Mix,1μl(100μM)通用PCR引物和100μl水。
    2. 将200μlPCR混合物添加到来自步骤F5的5μlccDNA中。
      为每个样本添加1μl不同的索引引物
    3. 将每个反应混合物分成5个独立的0.2ml PCR管(每个40μl)。
    4. 根据以下程序,通过对每个不同次数的循环运行每个子反应来对每个ccDNA样品进行PCR滴定:
      1. 98℃30秒
      2. N个周期的
        98°C 10秒
        60°C 10秒
        72°C 10秒
      3. 保持在10°C
      注:我们通常对每个ccDNA样品进行N = 12,15,18,21和24个循环的滴定。
    5. 每个反应添加8微升6x DNA凝胶加载染料。
    6. 在琼脂糖凝胶上并排放置每个样品的全部5滴。我们建议使用超低量程DNA梯(〜0.5μg/泳道)来标定不同ccDNA样品滴定的组合。
    7. 在琼脂糖凝胶电泳缓冲液(1×TAE和0.5μg/ ml溴化乙锭)中以3.6-3.7 V / cm运行凝胶1-2 h。
    8. 将凝胶放在紫外透射仪(设置在365nm波长)。通过肉眼评估PCR滴定(见下面的注释),为每个ccDNA样品选择适当数量的PCR循环,并使用装有凝胶切除的100-1,000μl移液管切下含有对应于所需产物大小的PCR扩增子的凝胶切片提示。
      每个凝胶切片放入一个单独的1.5 ml离心管中。
      注:对应于'空'环化ccDNA产物(即没有小RNA插入物)的亮带应在每个泳道中可见。随着PCR循环数(N)的增加,这可能表现为双峰现象。对于大多数小RNA测序应用而言,期望的产物将在该亮带/双重体上约50bp。对于CRISPR RNA测序,我们很少在这个大小范围内看到可见的涂片,并且使用DNA梯子和亮带/双峰(〜125bp)的位置作为视觉指导来“盲目”地切割。我们通常旨在为每个ccDNA样本提供最高的PCR循环次数,同时仍能安全地避免从明亮的〜125 bp的条带/双峰向上涂片(图5)。


      图5.通过“盲”凝胶切除制备文库。 :一种。用于两个ccDNA样品(3-3.5%天然琼脂糖凝胶电泳)的扩增测序文库的PCR滴定。每个ccDNA样品的5次滴定与DNA大小梯度一起并排运行。在Microsoft Word中调整亮度/对比度。 B.在175bp标记处DNA的大小选择,在由空ccDNA扩增形成的亮带/双峰之上(即,没有小RNA插入物)。该凝胶中使用了Invitrogen 25 bp DNA梯,但此后已被制造商停产。

    9. 使用QIAGEN MinElute凝胶提取试剂盒根据制造商的说明从凝胶切片中提取DNA。
    10. 根据制造商的说明使用伴随Qubit荧光计的高灵敏度双链DNA定量试剂盒对每个纯化的DNA样品进行定量。
    11. 根据以下公式计算每个样本的大致浓度:

    12. 以等摩尔量汇集样品。可以根据Illumina高通量测序服务提供商的规格对合并的文库进行测序。我们通常将80个循环的单读配置用于小RNA测序应用。为了评估地中海单胞菌中的pre-crRNA加工,我们每个样品的序列不超过100万个读数,但这取决于目的物种中前crRNA的表达水平。 >

数据分析

我们在示例数据中包含了一个示例,这需要以下要安装的程序:

cutadapt(在v1.14上测试;可能与大多数其他版本兼容)
Python 2.7(用numpy,用于绘图的matplotlib)

提供的python脚本的使用格式为 粗体斜体 ,后面紧跟粗体中具有示例数据的特定命令。首先下载工作示例,然后导航到unix终端中的processed_example /目录。

  1. 解复用读取:以'fastq'格式获取高通量测序数据。
    1. 样本和索引读取将分别位于文件
    2. 为了分离与每个索引对应的读取,准备一个多路分解“密钥” - 一个制表符分隔的文本文件,第一列包含所需样品名称,第二列包含相应TruSeq LT索引(AD001-8)的反向互补。提供了一个示例文件 deMultiplexKey_sample.dat 。
    3. 现在运行 deMultiplexer.py 文件,如下所示:

      python deMultiplexer.py&lt; path_to_directory&gt; &lt; key&gt;
      ,例如,python deMultiplexer.py sample_data /deMultiplexKey_sample.dat

      这将为密钥中提供的每个索引生成一个FASTQ文件。请注意所提供示例中样本1-4的文件是否为空。示例数据集只包含与样本5-8提供的索引读取对应的读取。
      将解复用的数据(样本5-8)移至单独的目录

      mkdir sample_demultiplexed
      cd sample_data /
      mv sample [5-8] *。fastq ../ sample_demultiplexed /
      cd ../

  2. 微调适配器:高通量测序数据将包含Illumina适配器序列。这些是Illumina流通池测序所需分子的一部分。
  3. 折叠读取以消除扩增偏差:测定设计包括3'衔接子序列中的随机六聚体(NNNNNN),其在逆转录之前与每个RNA分子连接。这有助于消除下游步骤中的扩增偏倚,并有助于确保每次读取对应于生物样品中独特的RNA分子。
    步骤2和步骤3(修剪和折叠)在提供的示例中使用 dirRNAseqAnalyse.py 脚本执行(使用 readCollapser2.py 函数,该函数必须相同目录作为脚本),如下所示:

    python dirRNAseqAnalyse.py&lt; path_to_directory&gt; &lt; maximum_read_length&gt;
    例如,python dirRNAseqAnalyse.py sample_demultiplexed / 80

    该程序会生成一个日志文件 dirRNAseqAnalyseLog.txt ,其中包含适配器修剪步骤的详细信息。
  4. 转换为fasta: fastq2fasta.sh 脚本已准备好预测将在步骤3中生成的文件以获取示例数据。此脚本中的每行都将一个输入fastq文件处理为一个输出fasta文件。使用输入文件(使用 .trimmed.collapsed.fastq 扩展名)和输出文件(使用。 fasta 扩展名)修改此文件。使用以下命令进行转换:
    1. 运行提供的 fastqtofasta.sh 脚本:

      sh fastq2fasta.sh

      将修剪中间体移动到新目录:

      mkdir sample_trimmed_collapsed
      mv * .trimmed * sample_trimmed_collapsed /

    2. 将.fasta文件移至新目录:

      mkdir sample_fasta
      mv * .fasta sample_fasta /

  5. 过滤:
    1. 鉴定CRISPR衍生的读段:首先,鉴定含有CRISPR直接重复序列的5'末端的测序读段。我们需要测序中至少5个连续的碱基进行读取以匹配CRISPR重复序列的前5个碱基。感兴趣的CRISPR重复序列在参数文件 crRNAfigureMaker_params.txt的1 st 行中提供。 CRISPR重复序列开头的任何上游序列都被删除。 >
    2. 删除短匹配:如果产生的加工重复序列短于12个碱基,还要检查原始阅读中CRISPR重复序列之前的5个碱基是否与细菌基因组中CRISPR阵列的一个可能的间隔序列末端匹配。通过这种方式,我们需要CRISPR重复中至少12个碱基,或跨越间隔区 - 重复连接处的10个碱基进行任何读取,才有资格进行下游分析。 spacerEnds.dict 中提供了来自地中海单胞菌基因组III-B CRISPR位点的所有可能的本地间隔区末端的字典。
      注意:可以在任何文本编辑器中修改spacerEnds.dict文件,但必须保留其格式以防止在Python中解析错误。
    3. 评估匹配保真度:如果读取通过初始过滤,则处理的重复然后与预期的CRISPR重复序列匹配。我们要求重复为CRISPR重复序列的左锚定子字符串( ie ,处理后的重复序列可能比CRISPR重复序列短,但它必须在5'末端匹配并且不能包含错配) 。
  6. 测量参照基因的水平:接下来,计数读数含有高度表达且不随研究中的生物学条件而变化的参照基因的25-nt子串。我们使用异亮氨酸-tRNA序列作为地中海地中海数据集中的参考,但这可能需要根据您的模型的RNAseq数据根据经验确定。该序列必须在 crRNAfigureMaker_params.txt 文件的2 nd 行中提供。
  7. 绘制修剪阅读长度的直方图:最后,绘制经过处理的CRISPR重复长度的直方图,所述长度与参考基因归一化。 crRNAfigureMaker_params.txt 文件的第3行是一个任意的缩放参数,用于控制图中Y轴的高度。它可以被改变以适应数据集中相对于参考基因的加工的crRNA的水平。
    第5步,第6步和第7步由 crRNAfigureMaker.py 脚本执行,如下所示:

    python crRNAfigureMaker.py&lt; path_to_fasta_files&gt; &lt; keyword&gt;
    例如,python crRNAfigureMaker.py sample_fasta / 8

    关键字选项指定分析中应包含哪些文件。关键字可以是文件名的任何部分。例如,使用关键字'8'将只在处理过的示例中处理sample8.fasta,而使用关键字'mpl'将包括所有4个样本文件进行处理,并且使用关键字'sem'将导致不包含任何文件。
    crRNAfigureMaker_param.txt 文件必须与代码位于同一目录中。运行上述命令(即,只处理sample8.fasta)应该生成下面的图6。


    图6.实施例中提供的代码的预期输出。通过高通量小RNA测序法分析处理的crRNA水平。该数据集已经人为补充了与期望的CRISPR衍生的RNA匹配的序列。来自参数文件 crRNAfigureMaker_params.txt 的1 st 行的CRISPR重复序列位于X轴上。沿着X轴的每个碱基处的条的高度表示在该碱基处具有3'末端的crRNA的相对比例,归一化为参考RNA(异亮氨酸tRNA;一致地是在我们的实验中遇到的最丰富的物种的水平) M. mediterranea 数据集)。在包含RNA的CRISPR重复群体中存在不同的3'末端序列表明位点特异性切割和前crRNA的加工。

    工作示例中的数据文件是我们的小型子集实验数据并且人为补充了与期望的CRISPR衍生的RNA匹配的序列。请参阅NCBI Short Read Archive(SRP103952)上的公共数据集以重新创建已发布的图表(Silas et。,2017a)。下表指定了与(Silas et。,2017a)中的每个相关图形面板相对应的实验的登录号。

食谱

  1. 聚丙烯酰胺凝胶洗脱缓冲液 300 mM NaCl
    1 mM EDTA
    制作50毫升:
    3 ml
    5 M sodium chloride solution
    500 μl
    0.1 M EDTA solution
    46.5 ml
    RNase free water
  2. 1 M HEPES / KOH缓冲液pH 8.3
    用8N氢氧化钾调节1M HEPES溶液的pH至8.3
    使用0.2μm真空过滤装置的无菌过滤器
  3. 60%甘油溶液
    为了使10毫升,混合6毫升甘油与4毫升无RNase水
    通过高压灭菌来消毒
  4. 5倍腺苷酸缓冲液
    注意:在-20°C下保存1 ml等分样品至1年。
    41%甘油
    250 mM HEPES / KOH pH 8.3
    50mM MgCl 2 2/2 16.5 mM DTT
    50μg/ ml Ac-BSA
    要制成5毫升,混合:
     3.4 ml
    60% glycerol solution
    1.25 ml
    1 M HEPES/KOH buffer pH 8.3
    250 μl
    1 M magnesium chloride solution
    82.5 μl
    1 M dithiothreitol solution
    12.5 μl
    20 mg/ml acetylated BSA

致谢

S.S.得到斯坦福研究生奖学金和HHMI国际学生研究奖学金的支持。该协议是在NIH支持下开发的(授予R01-GM37706至A.Z.F.)。我们调整了较早的RNA测序方法(Lau等人,2001; Ingolia等人,2009; Guo等人,2010; Kwon ,2011)用于来自 C的RNA测序。 (Lamm et al。,2011),并将其他人关于独特分子标识符的工作纳入其中以去除PCR重复序列(Kivioja et al。,2011),引入酶清除步骤(5'去腺苷酶/ RecJ)以规避凝胶纯化步骤。我们发现该方案适用于多种RNA测序应用,例如crRNA检测(Silas等人,2017a),原核转录组特征分析(Silas等人, 2016),来自宏基因组环境样品的RNA测序(Silas等人,2017b)和核糖体足迹(Arribere等人,2016)。我们宣布我们没有利益冲突或利益冲突。

参考

  1. Arribere,J.A.,Cenik,E.S。,Jain,N.,Hess,G.T.,Lee,C.H.,Bassik,M.C。和Fire,A.Z.(2016)。 缓解翻译通读。 534(7609): 719-723。
  2. Barrangou,R.,Fremaux,C.,Deveau,H.,Richards,M.,Boyaval,P.,Moineau,S.,Romero,D.A。和Horvath,P.(2007)。 CRISPR提供对原核生物病毒的获得性抗性。 科学 315(5819):1709-1712。
  3. Brouns,S.J.,Jore,M.M.,Lundgren,M.,Westra,E.R.Slijkhuis,R.J.,Snijders,A.P.,Dickman,M.J.,Makarova,K.S.,Koonin,E.V.and van der Oost,J.(2008)。 小型CRISPR RNA指导原核生物的抗病毒防御。 科学 321(5891):960-964。
  4. Carte,J.,Pfister,N.T.,Compton,M.M.,Terns,R.M。和Terns,M.P。(2010)。 Cas6结合和切割CRISPR RNA RNA 16(11):2181-2188。
  5. Carte,J.,Wang,R.,Li,H.,Terns,R.M。和Terns,M.P。(2008)。 Cas6是一种内切核糖核酸酶,可在原核生物中产生入侵者防御指导RNA。 Genes Dev 22(24):3489-96。
  6. Charpentier,E.,Richter,H.,van der Oost,J.和White,M.F。(2015)。 古细菌和细菌CRISPR-Cas适应性免疫中RNA指导的生物通路 FEMS Microbiol Rev 39(3):428-441。
  7. Deveau,H.,Barrangou,R.,Garneau,J.E。,Labonte,J.,Fremaux,C.,Boyaval,P.,Romero,D.A.,Horvath,P.and Moineau,S。(2008)。 噬菌体对嗜热链球菌中CRISPR编码抗性的反应。 > Bacteriol 190(4):1390-1400。
  8. Guo,H.,Ingolia,N.T。,Weissman,J.S。和Bartel,D.P.(2010)。 哺乳动物microRNA主要起降低靶mRNA水平的作用。 Nature > 466(7308):835-840。
  9. Haurwitz,R.E.,Jinek,M.,Wiedenheft,B.,Zhou,K.and Doudna,J.A。(2010)。 CRISPR内切核酸酶的序列和结构特异性RNA处理。 科学 329(5997):1355-1358。
  10. Heidrich,N.,Dugar,G.,Vogel,J。和Sharma,C.M。(2015)。 使用RNA-seq来研究CRISPR RNA的生物发生和功能方法Mol Biol 1311:1-21。
  11. Hochstrasser,M.L。和Doudna,J.A。(2015)。 切断它:CRISPR相关的内切核糖核酸酶结构和功能。 Trends Biochem Sci 40(1):58-66。
  12. Ingolia,N.T。,Ghaemmaghami,S.,Newman,J.R。和Weissman,J.S。(2009)。 利用核糖体分析技术进行核苷酸解析翻译的全基因组分析 。 科学 324(5924):218-223。
  13. Jackson,S.A.,McKenzie,R.E.,Fagerlund,R.D.,Kieper,S.N.,Fineran,P.C和Brouns,S.J。(2017)。 CRISPR-Cas:适应变化。 科学 356 (6333)。
  14. Juranek,S.,Eban,T.,Altuvia,Y.,Brown,M.,Morozov,P.,Tuschl,T。和Margalit,H。(2012)。 嗜热栖热菌(Thermus thermophilus)的表达和加工模式的全基因组视图 HB8 CRISPR RNAs。 18(4):783-794。
  15. Kivioja,T.,Vaharautio,A.,Karlsson,K.,Bonke,M.,Enge,M.,Linnarsson,S.and Taipale,J.(2011)。 使用独特的分子标识符计算分子的绝对数量 Nat Methods 9(1):72-74。
  16. Kwon,Y.S.(2011)。 通过单连接,延伸和环化技术为下一代测序准备小RNA文库。 Biotechnol Lett 33(8):1633-1641。
  17. Lamm,A.T.,Stadler,M.R.,Zhang,H.,Gent,J.I and Fire,A.Z.(2011)。 使用单链,双链和基于CircLigase的捕获的多模RNA-seq产生精制并扩展了 C的描述。 elegans 转录组。 Genome Res 21(2):265-275。
  18. Lau,N.C.,Lim,L.P.,Weinstein,E.G。和Bartel,D.P.(2001)。 一类丰富的微小RNA,在秀丽隐杆线虫中具有可能的调节作用 。 Science 294(5543):858-862。
  19. Makarova,KS,Haft,DH,Barrangou,R.,Brouns,SJ,Charpentier,E.,Horvath,P.,Moineau,S.,Mojica,FJ,Wolf,YI,Yakunin,AF,van der Oost,和Koonin,EV(2011)。 CRISPR-Cas系统的进化和分类 Nat Rev Microbiol 9(6):467-477。
  20. 马卡洛娃,KS,Wolf,YI,Alkhnbashi,OS,Costa,F.,Shah,SA,Saunders,SJ,Barrangou,R.,Brouns,SJ,Charpentier,E.,Haft,DH,Horvath,P.,Moineau, S.,Mojica,FJ,Terns,RM,Terns,MP,White,MF,Yakunin,AF,Garrett,RA,van der Oost,J.,Backofen,R.和Koonin,EV(2015)。 更新CRISPR-Cas系统的进化分类 Nat Rev Microbiol 13(11):722-736。
  21. Marraffini,L.A。和Sontheimer,E.J。(2008)。 CRISPR干扰通过靶向DNA限制葡萄球菌的水平基因转移科学322(5909):1843-1845。
  22. Plagens,A.,Richter,H.,Charpentier,E.和Randau,L.(2015)。 CRISPR-Cas监测复合物的DNA和RNA干扰机制 FEMS Microbiol Rev 39(3):442-463。
  23. Richter,H.,Zoephel,J.,Schermuly,J.,Maticzka,D.,Backofen,R.和Randau,L。(2012)。 在热纤梭菌和 Methanococcus maripaludis中表征CRISPR RNA加工。 Nucleic Acids Res 40(19):9887-9896。
  24. Silas,S.,Lucas-Elio,P.,Jackson,S.A。,Aroca-Crevillen,A.,Hansen,L.L.,Fineran,P.C.,Fire,A.Z.and Sanchez-Amat,A.(2017a)。 III型CRISPR-Cas系统可以提供冗余以抵制I型系统的病毒逃逸。 Elife 6。
  25. Silas,S.,Makarova,KS,Shmakov,S.,Paez-Espino,D.,Mohr,G.,Liu,Y.,Davison,M.,Roux,S.,Krishnamurthy,SR,Fu,BXH,Hansen ,LL,Wang,D.,Sullivan,MB,Millard,A.,Clokie,MR,Bhaya,D.,Lambowitz,AM,Kyrpides,NC,Koonin,EV和Fire,AZ(2017b)。 关于逆转录酶的起源 - 使用CRISPR-Cas系统及其多种多样的神秘间隔曲目。< / a> MBio 8(4)。
  26. Silas,S.,Mohr,G.,Sidote,D.J.,Markham,L.M.,Sanchez-Amat,A.,Bhaya,D.,Lambowitz,A.M。和Fire,A.Z。(2016)。 通过天然逆转录酶-cas1融合蛋白从RNA中直接获取CRISPR间隔物。 < 科学 351(6276):aad4234。
  • English
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Copyright Silas et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Silas, S., Jain, N., Stadler, M., Fu, B. X., Sanchez-Amat, A., Fire, A. Z. and Arribere, J. (2018). A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria. Bio-protocol 8(4): e2727. DOI: 10.21769/BioProtoc.2727.
  2. Silas, S., Lucas-Elio, P., Jackson, S. A., Aroca-Crevillen, A., Hansen, L. L., Fineran, P. C., Fire, A. Z. and Sanchez-Amat, A. (2017a). Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems. Elife 6.
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