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Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA cross-talks at the host-virus interface. High-confidence quantifying models of the suppression efficacy in targeting sites by integrating bioinformatics with Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIP-seq) (Chi et al., 2009) data have been poorly developed. To accurately identify miRNA target sites and calculate the targeting efficacy of miRNA-target interactions, we developed a new bioinformatic quantitation method, AGO-CLIP-seq enrichment (ACE)-scoring algorithm (Kim et al., 2015). Inclusion of the uninfected control in our AGO-CLIP-seq analysis can significantly improve the accuracy of authentic target site identification for viral or human miRNAs and extract physiologically significant changes during productive human cytomegalovirus (HCMV) infection using our ACE-scoring method. Thus, we suggest that our new ACE-scoring-based methodology can be applied to various miRNA targetome studies, which will be performed in other kinds of temporal contexts, such as developmental stages, immune stimulation by cytokines or pathogens, and lytic infection by other viruses.

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ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq
使用AGO-CLIP测序法并基于ACE分数分析人巨细胞病毒感染过程中的暂时miRNA靶标组

微生物学 > 微生物生物化学 > RNA
作者: Sungchul Kim
Sungchul KimAffiliation 1: School of Biological Sciences, Seoul National University, Seoul, Korea; Center for RNA Research, Institute for Basic Science, Seoul, Korea
Affiliation 2: Department of BioNanoscience, Delft University of Technology, Delft, The Netherlands
Bio-protocol author page: a3079
 and Kwangseog Ahn
Kwangseog AhnAffiliation: School of Biological Sciences, Seoul National University, Seoul, Korea; Center for RNA Research, Institute for Basic Science, Seoul, Korea
For correspondence: ksahn@snu.ac.kr
Bio-protocol author page: a3080
Vol 6, Iss 8, 4/20/2016, 1836 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1791

[Abstract] Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA cross-talks at the host-virus interface. High-confidence quantifying models of the suppression efficacy in targeting sites by integrating bioinformatics with Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIP-seq) (Chi et al., 2009) data have been poorly developed. To accurately identify miRNA target sites and calculate the targeting efficacy of miRNA-target interactions, we developed a new bioinformatic quantitation method, AGO-CLIP-seq enrichment (ACE)-scoring algorithm (Kim et al., 2015). Inclusion of the uninfected control in our AGO-CLIP-seq analysis can significantly improve the accuracy of authentic target site identification for viral or human miRNAs and extract physiologically significant changes during productive human cytomegalovirus (HCMV) infection using our ACE-scoring method. Thus, we suggest that our new ACE-scoring-based methodology can be applied to various miRNA targetome studies, which will be performed in other kinds of temporal contexts, such as developmental stages, immune stimulation by cytokines or pathogens, and lytic infection by other viruses.
Keywords: CLIPseq(clipseq), ACE score(ACE得分), MicroRNA(microRNA), Human cytomegalovirus(人巨细胞病毒), Argonaute(Argonaute蛋白)

[Abstract]

Materials and Reagents

  1. 100 mm cell culture dish (Sarstedt AG & Co, catalog number: 83.3902 )
  2. Disposable serological pipettes (Orange)
  3. Filter micropipette tips (Biotix, NEPTUNE)
  4. 1.6 ml microcentrifuge tube (Biotix, NEPTUNE, catalog number: 3745X )
  5. Cell scraper (25 cm) (Sarstedt AG & Co, catalog number: 83.1830 )
  6. Plastic wrap (CLEANWRAP)
  7. BAS film (Fujifilm Corporation)
  8. Razor (Dorco, model: Pace Single Edge Blades DN52 )
  9. Costar Spin-X centrifugation column (Sigma-Aldrich, catalog number: CLS8162-96EA )
  10. Human foreskin fibroblast (HFF) cells (ATCC, catalog number: SCRC-104 ™)
  11. Dulbecco’s modified Eagle’s medium (DMEM) (with High Glucose, with 4 mM L-Glutamine, without Sodium Pyruvate) (GE Healthcare, HyClone™, catalog number: SH30022.FS )
  12. Fetal bovine serum (FBS) (Enhance growth of cells with U.S. sourced GE Healthcare HyClone characterized) (GE Healthcare, HyClone™, catalog number: SH30071.03HI )
  13. GlutaMAX-I (100x) (100 ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061 )
  14. Penicillin/Streptomycin solution (P/S) (100x, 10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15-140-122 )
  15. Ice
  16. Towne strain of human cytomegalovirus (HCMV) (ATCC, model: VR-977 ™; NCBI accession number: FJ616285.1)
  17. Dynabeads® Sheep-Anti Mouse IgG (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11031 )
  18. Dynabeads® Pan Mouse IgG (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11041 )
  19. Pan anti-AGO mAb 2A8 (Diagenode, catalog number: C15200167-100 )
  20. Protease inhibitor cocktail (Cell Signaling Technology, catalog number: 5871 )
  21. Recombinant DNase I (Takara Bio Company, catalog number: 2270B )
  22. RNase A (1 MG, 20 units/μl; 5 mg/ml) (Thermo Fisher Scientific, Affymetrics, catalog number: 70194Y )
  23. Alkaline Phosphatase, Calf Intestinal (CIP) (New England Biolabs, catalog number: M0290S )
  24. 3’-Adaptor (RA3) (5’-rApp-TGGAATTCTCGGGTGCCAAGG-3’-ddC, 5’-Adenylation, 3’-Dideoxy-C) (Integrated DNA Technologies, RNase Free HPLC Purification)
  25. T4 RNA Ligase 2, truncated K227Q (New England Biolabs, catalog number: M0351L )
  26. SUPERase.In RNase Inhibitor (Thermo Fisher Scientific, Ambion™, catalog number: AM2696 )
  27. ATP, [γ-32P], 6,000 Ci/mmol (222 TBq/mmol) (PerkinElmer, catalog number: NEG502Z500UC )
  28. T4 Polynucleotide Kinase (PNK) (Takara Bio Company, catalog number: 2021A )
  29. 10 mM Adenosine 5’-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S )
  30. NuPAGE® LDS Sample Buffer (4x) (Thermo Fisher Scientific, Novex™, catalog number: NP0007 )
  31. NuPAGE® Sample Reducing Agent (10x) (Thermo Fisher Scientific, Novex™, catalog number: NP0004 )
  32. NuPAGE® Antioxidant (Thermo Fisher Scientific, Novex™, catalog number: NP0005 )
  33. NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 10-well (Thermo Fisher Scientific, Invitrogen™, catalog number: NP0321BOX )
  34. NuPAGE® MOPS SDS Running Buffer (20x) (Thermo Fisher Scientific, Novex™, catalog number: NP0001 )
  35. NuPAGE® Transfer Buffer (20x) (Thermo Fisher Scientific, Novex™, catalog number: NP0006 )
  36. Amersham Protran Supported 0.45 NC (GE Healthcare Life Sciences, catalog number: 10600016 )
  37. Bovine serum albumin (BSA) (life science grade powder) (Merck Millipore Corporation, Probumin®, catalog number: 821006 )
  38. Anti-AGO mAb 21D2 (Made in laboratory of Narry V. Kim, School of Biological Sciences, Seoul National University, Seoul, Republic of Korea)
  39. Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Inc., catalog number: 115-035-003 )
  40. EMD Millipore Immobilon™ Western Chemiluminescent HRP Substrate (ECL) (Thermo Fisher Scientific, catalog number: WBKLS0050 )
  41. Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) (RNA phenol/chloroform) (Thermo Fisher Scientific, Ambion™, catalog number: AM9720 )
  42. Linear acrylamide (Thermo Fisher Scientific, Invitrogen™, catalog number: AM9520 )
  43. 3 M Sodium acetate (pH 5.5) (Thermo Fisher Scientific, Ambion™, catalog number: AM9740 )
  44. Ethanol (Merck Millipore Corporation, catalog number: 100983 )
  45. Isopropanol (2-propanol) (Merck Millipore Corporation, catalog number: 109634 )
  46. UltraPure™ DNase/RNase-Free Distilled Water (D.W.) (Thermo Fisher Scientific, Invitrogen™, catalog number: 10977-015 )
  47. 2x RNA loading buffer (Thermo Fisher Scientific, Ambion™, catalog number: AM8546G )
  48. mirVana™ Probe & Marker Kit (Thermo Fisher Scientific, Ambion™, catalog number: AM1554 )
  49. 10% Novex® TBE-Urea Gels, 10 well (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC6875BOX )
  50. T4 RNA ligase (Takara Bio Company, catalog number: 2050B )
  51. Proteinase K (PK) (Thermo Fisher Scientific, Macherey-Nagel, catalog number: 740506 )
  52. 5’-adaptor oligonucleotide (5’-Solexa adaptor)
    5’-rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC-3’ (Integrated DNA Technologies, RNase Free HPLC Purification)
  53. RNA RT primer (RTP)
    5’-GCCTTGGCACCCGAGAATTCCA-3’ (Integrated DNA Technologies, RNase Free HPLC Purification)
  54. 5’-Primer (Integrated DNA Technologies, RNase Free HPLC Purification)
    1. RNA PCR Primer (RP1)
      5’-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3’
  55. 3’-Primers (Integrated DNA Technologies, RNase Free HPLC Purification)
    1. RNA PCR Primer, Index 1 (RPI1)
      5’-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3’
    2. RNA PCR Primer, Index 2 (RPI2)
      5’-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3’
    3. RNA PCR Primer, Index 3 (RPI3)
      5’-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3’
    4. RNA PCR Primer, Index 4 (RPI4)
      5’-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3’
  56. Phusion® High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Thermo Scientific™, catalog number: F-530L )
  57. dNTP mixture (Enzynomics, catalog number: N001L )
  58. SuperScript™ III reverse transcriptase (RT) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-044 )
  59. Novex® TBE Gels, 6%, 10 Well (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC6265BOX )
  60. Low Molecular Weight DNA Ladder (New England Biolabs, catalog number: N3233L )
  61. Ethidium bromide (EtBr) (Sigma-Aldrich, catalog number: E7637 )
  62. Dynabeads® mRNA DIRECT™ Kit (Thermo Fisher Scientific, AmbionTM, catalog number: 61011 )
  63. NEBNext® Magnesium RNA Fragmentation Module (New England Biolabs, catalog number: E6150S )
  64. Sodium chloride (NaCl) (BioXtra, ≥99.5%) (Sigma-Aldrich, catalog number: S7653 )
  65. Potassium chloride (KCl) (BioXtra, ≥99.0%) (Sigma-Aldrich, catalog number: P9333 )
  66. Sodium phosphate dibasic (Na2HPO4) (BioXtra, ≥99%) (Sigma-Aldrich, catalog number: S7907 )
  67. Potassium phosphate monobasic (KH2PO4) (≥99.0%) (Sigma-Aldrich, catalog number: P5655 )
  68. Trizma® base (An high-quality Tris base) (BioXtra, ≥99.9%) (Sigma-Aldrich, catalog number: T6791 )
  69. NP-40 Alternative (Merck Millipore Corporation, catalog number: 492018 )
  70. Sodium dodecyl sulfate (SDS) [BioXtra, ≥99.0% (GC)] (Sigma-Aldrich, catalog number: L6026 )
  71. Sodium deoxycholate (BioXtra, anhydrous, ≥98%) (Sigma-Aldrich, catalog number: 30970 )
  72. Ethylenediaminetetraacetic acid (EDTA) (BioUltra, anhydrous, ≥98%) (Sigma-Aldrich, catalog number: EDS )
  73. Magnesium chloride (MgCl2) (anhydrous, ≥98%) (Sigma-Aldrich, catalog number: M8266 )
  74. Tween-20 (Sigma-Aldrich, catalog number: P7949 )
  75. NEBuffer3 (New England Biolabs, catalog number: B7003S )
  76. 10x T4 RNA ligase buffer (New England Biolabs, catalog number: B0216L )
  77. 50% PEG8000 (supplied with 10x T4 RNA ligase buffer)
  78. 10 mM Adenosine 5’-Triphosphate (ATP) (supplied with 10x T4 RNA ligase buffer)
  79. Bromophenol blue (ACS reagent) (Sigma-Aldrich, catalog number: 114391 )
  80. Xylene cyanol FF (BioReagent) (Sigma-Aldrich, catalog number: X4126 )
  81. Glycerol (BioXtra, anhydrous, ≥99%) (Sigma-Aldrich, catalog number: G6279 )
  82. Boric acid (BioReagen, ≥99.5%) (Sigma-Aldrich, catalog number: B6768 )
  83. Trizol® reagent (Thermo Fisher Scientific, Ambion™, catalog number: 15596018 )
  84. Dynabeads® mRNA DIRECT™ Purification Kit (Thermo Fisher Scientific, Ambion™, catalog number: 61011)
  85. NEBNext® Magnesium RNA Fragmentation Module (New England Biolabs, catalog number: E6150S)
  86. Antarctic phosphatase (New England Biolabs, catalog number: M0289S )
  87. Phosphate-buffered saline (PBS) (see Recipes)
  88. Lysis buffer (see Recipes)
  89. High-salt buffer (see Recipes)
  90. Wash buffer (see Recipes)
  91. CIP mix (see Recipes)
  92. 3’-ligation mix (see Recipes)
  93. Hot PNK mix (see Recipes)
  94. 0.1% PBS-T (see Recipes)
  95. PK buffer (see Recipes)
  96. PK mix (see Recipes)
  97. PK/Urea buffer (see Recipes)
  98. 5’-ligation mix (see Recipes)
  99. PCR mix (see Recipes) (see Recipes)
  100. 6x DNA Loading buffer (see Recipes)
  101. TBE (Tris-borate-EDTA) (see Recipes)

Equipment

  1. CO2 incubator (LabX, Sanyo, model: CO2 Incubator MCO-18AIC )
  2. Micropipettes (Gilson, PIPETMAN Classic™ P10, P20, P200 and P1000, Ordering reference: F144802 , F123600 , F123601 and F123602 )
  3. Rocker (FINEPCR, model: CR300 )
  4. UV crosslinker (Spectronics Corporation, model: XL1500 )
  5. Vortexer (Scientific Industries, model: Vortex-Genie® 2 and catalog number: SI-0256 )
  6. DynaMag™-2 Magnet (Thermo Fisher Scientific, catalog number: 12321D )
  7. Rotator (FINEPCR, model: AG) with an accessary Roller Drum (FINEPCR, model: RD25-42 )
  8. Eppendorf® Thermomixer® R, dry block heating and cooling shaker (Sigma-Aldrich, catalog number: T3317 )
  9. Refrigerated tabletop microcentrifuge (Hanil BioMed Inc., model: Centrifuge Micro 17TR )
  10. XCell SureLock® Mini-Cell (Thermo Fisher Scientific, Novex™, catalog number: EI0001 )
  11. Chemidoc (Vilber Lourmat, model: Fusion Spectra )
  12. BAS 2500 (Fujifilm Corporation)
  13. Deep freezer (-80 °C freezer)
  14. PCR cycler (MyCycler™ Thermal Cycler System) (Bio-Rad Laboratories, catalog number: # 1709703 )
  15. Illumina HiSeq2000

Software

  1. CutAdap software
  2. Human genome (hg19) BLAST+ (v. 2.2.27) software (Camacho et al., 2009)

Procedure


Figure 1. Scheme of the entire procedure (A) and AGO-CLIP-seq (B)

Note: Throughout the entire AGO-CLIP-seq protocol (Figure 1), all work must be performed in RNase-free environments and only sterile and DNase-/RNase-free solutions and materials should be used.


  1. AGO-CLIP-seq
    1. Cell culture and HCMV infection
      Note: To propagate HCMV and prepare the HCMV stock, we have followed the previous instruction (Britt, 2010).
      1. HFF cells are obtained from the American Type Culture Collection and cultured in five 100-mm culture dishes with complete DMEM supplemented with 10% FBS, 2 mM GlutaMAX-I, and 1x P/S at 37 °C in the presence of 5% CO2.
      2. Wash twice with PBS.
      3. Add HCMV-containing serum-free DMEM at an MOI of 3 with a total volume of 2 ml/dish and incubate at 37 °C for 1 h rocking by hand gently but thoroughly at an interval of 15 min.
      4. Decant the virus media, wash twice with PBS and add 10 ml 10% FBS, P/S, L-Glu DMEM/dish.
      5. Incubate for the indicated time (24, 48 and 72 h).
    2. UV irradiation
      1. Wash once with 3 ml/dish of Cold PBS.
      2. Add 2 ml/dish of Cold PBS in four of five dishes and extract total RNAs from one of five dish to construct libraries for mRNA-seq and smallRNA-seq (go to step B1a).
      3. Irradiate with 254-nm UV at 250 mJ/cm2 on ice in Spectrolinker.
      4. Decant PBS completely.
      5. Prepared cells were stored at -80 °C until subsequent steps.
    3. Preparation of antibody-conjugated Dynabeads
      1. Transfer 50 µl of Sheep α-mouse immunoglobulin G (IgG) conjugated Dynabead per sample into 1.5 ml tube. To collect the magnetic Dynabeads the DynaMeg-2 magnet rack is used. Pan α-mouse IgG conjugated Dynabead can be used as an alternative for Sheep α-mouse immunoglobulin G (IgG) conjugated Dynabead.
      2. Wash twice with 0.1 M NaHPO4 (pH 8.1) and resuspend beads in 200 µl 0.1 M NaHPO4 (pH 8.1).
      3. Add 30 μg of mAb 2A8 as a pan-anti-AGO antibody per sample.
      4. Rotate the tubes at room temperature for 2 h (at least 1 h).
      5. Wash twice with Lysis buffer.
      6. Leave in ice until the next step.
    4. Cell lysis and partial RNA digestion
      1. Add 1 ml Lysis buffer (with Protease inhibitor cocktail) into the sample dish, scrap the cell lysate, transfer the lysate into the next dish and repeat until the last dish.
      2. Finally, transfer the lysate into a 1.5 ml tube.
      3. Add 10 µl recombinant DNase I and incubate at 37 °C and 1,400 rpm in Eppendorf® Thermomixer® R for 10 min to make the lysate less viscous.
      4. Add 10 µl of diluted (1:1,000) RNase A.
      5. Incubate at 37 °C and 1,400 rpm for 10 min.
      6. Immediately transfer the tubes into ice.
      7. Centrifuge at 4 °C and 28,000 x g for 30 min.
      8. Carefully collect the supernatant and transfer the supernatant, except for 80 µl which will be used for immunoblotting, into a new tube to be combined with the prepared beads in the next step.
    5. Immunoprecipitation
      1. Combine prepared Ab-conjugated beads with cell lysates.
      2. Rotate at 4 °C for 3 h.
      3. Decant the supernatant and wash the beads twice with 1 ml High-salt buffer.
      4. Wash twice with 1 ml wash buffer.
    6. Dephosphorylation
      1. Prepare the CIP mix.
      2. Add 100 µl CIP mix to the washed beads.
      3. Incubate at 37 °C and 1,400 rpm in Eppendorf® Thermomixer® R for 10 min.
      4. Immediately transfer the tubes into ice.
      5. Wash the beads twice with 1 ml High-salt buffer and twice with 1 ml Wash buffer.
    7. 3′-adaptor ligation
      1. Prepare 3′-ligation mix.
      2. Add 100 µl 3′-ligation mix to the washed beads.
      3. Incubate at 16 °C and 1,400 rpm in Eppendorf® Thermomixer® R for overnight.
      4. Wash the beads twice with 1 ml High-salt buffer and twice with 1ml Wash buffer.
    8. RNA 5′-end labeling
      1. Prepare Hot PNK mix.
      2. Add 80 µl Hot PNK mix to the washed beads.
      3. Incubate at 37 °C and 1,400 rpm for 15 min.
      4. Immediately add 5 µl of 10 mM ATP.
      5. Incubate at 37 °C and 1,400 rpm in Eppendorf® Thermomixer® R for 5 min.
      6. Wash the beads twice with 1 ml High-salt buffer and twice with 1 ml Wash buffer.
    9. NuPAGE and membrane transfer
      1. The AGO-RNA complexes are eluted from the beads by incubating at 75 °C and 1,400 rpm for 10 min in 30 µl 1.5x NuPAGE LDS Sample Buffer.
      2. 13 µl of the lysate are mixed with 13 µl of NuPAGE LDS Sample Buffer (4x) and 2 µl of NuPAGE® Sample Reducing Agent (10x), and incubate at 75 °C for 10 min.
      3. Load the samples on a 4-12% NuPAGE Bis-Tris gel according to the manufacturer′s instructions using 800 ml of 1x MOPS running buffer with NuPAGE® Antioxidant.
      4. Run the gel for 2 h 30 min at 150 V.
      5. Autoradiograph the gel for about 5~30 min.
      6. Mark the size bands in a printed figure.
      7. Transfer the protein-RNA complexes from the gel to a nitrocellulose membrane for 70 min at 130 V using NuPAGE Transfer buffer according to the manufacturer′s instructions.
      8. After the transfer, rinse the membrane with D. W., then wrap it with plastic wrap.
      9. Autoradiograph the gel for about 5~30 min.
      10. Mark the size bands in a printed figure (Figure 2).
    10. Western blotting
      1. Take out the transferred membrane part for Western blotting and place it into 50-100 ml of 0.1% (v/v) PBS-T with 5% (w/v) BSA.
      2. Incubate the membrane for 1 h with gentle shaking at room temperature.
      3. Wash the membrane briefly with 0.1% PBS-T twice.
      4. Place each membrane into a small boat and pour primary antibody solution (20 ml of 0.1% PBS-T with 5% BSA and 20 µl of anti-AGO mAb 21D2) into the boat.
      5. Incubate them for at least 4 h with gentle shaking at 4 °C.
      6. Wash the membrane briefly with 0.1% PBS-T twice.
      7. Place the membrane into a small boat and pour secondary antibody solution (20 ml of 0.1% PBS-T with 5% BSA and 4 µl of HRP-conjugated Goat Anti-Mouse IgG) into the boat.
      8. Incubate the membrane for 1 h with gentle shaking at room temperature.
      9. Wash the membrane with 0.1% PBS-T at least five times for 10 min each with gentle shaking.
      10. Expose using Chemidoc for ECL detection (Figure 2B).


        Figure 2. Schematic representation of autoradiography and western blots of CLIPed AGO-RNA complexes and lysates transferred to nitrocellulose membrane from NuPAGE gel. A. Grahical representation. The region that should be excised by razor and Proteinase K treatment is indicated by red-dotted rectangle. To evaluate the IP efficiency of AGO-CLIP-seq procedures and the exact unconjugated AGO protein position in the gel, western blotting should be performed in the same gel with autoradiographs. B. Representative data with various conditions of RNase A and UV treatment and antibody used. As viewed, the higher concentration of RNase A is used, the less autoradiographic intensity of AGO-RNA complexes is observed. UV-untreated and using IgG control for IP produces no signal of intact complexes. AGO, Argonaute protein; Ab HC, antibody heavy chain; Ab LC, antibody light chain.

    11. RNA isolation and size fractionation
      1. Prepare PK mix.
      2. Cut the piece of 110~160 kDa Band with a razor and place it into a 1.5 ml tube. (Figure 2)
      3. Add 200 µl PK mix into the membrane-containing tube and incubate at 50 °C and 1,400 rpm for 1 h.
      4. Add 200 µl PK/Urea buffer and incubate at 50 °C and 1,400 rpm in Eppendorf® Thermomixer® R for 1 h.
      5. Add 400 µl RNA phenol/chloroform and incubate at 30 °C and 1,400 rpm in Eppendorf® Thermomixer® R for 5 min.
      6. Centrifuge at 20,000 x g for 5 min at room temperature.
      7. Transfer the upper (aqueous) phase into a new 1.5 ml tube carefully.
      8. Add 1 µl linear acrylamide and 40 µl 3 M Sodium acetate pH 5.5 and vortex.
      9. Add 1 ml of ethanol (EtOH)/isopropanol (1:1), vortex thoroughly and incubate overnight at -20 °C or -80 °C.
      10. Centrifuge at 4 °C and 28,000 x g for 30 min.
      11. Wash the pellet once with cold 75% EtOH and subsequently once with 100% EtOH.
      12. Air-dry the pellet at room temperature for about 10 min.
      13. Resuspend the pellet with 5 µl Nuclease-free D.W. and add 5 µl 2x RNA Loading buffer.
      14. Prepare the radiolabeled decade RNA marker using mirVana™ Probe & Marker Kit.
      15. Prepare Pre-cast 6% TBE-7M Urea gel.
      16. Incubate the sample tube at 95 °C for 5 min.
      17. Load the samples and marker into the prepared gels and run at 200 V until the Bromophenol blue (lower) dye has migrated 75% of the gel length.
      18. Autoradiograph the gel.
      19. Cut the gel ranged by 40~77 nt, transfer into a new 1.5 ml tube and crush the gel into small pieces (Figure 3A).
      20. Add 400 µl 0.3 M Sodium acetate pH 5.5 and rotate overnight at 4 °C.
      21. Filter the elution supernatant at 4 °C, 20,000 x g for 15 min using a Costar Spin-X centrifugation column.
      22. Transfer 400 µl of eluate into a new tube.
      23. Add 1 µl linear acrylamide and 1ml EtOH/isopropanol (1:1), vortex, and incubate for over 1 h at -80 °C.
      24. Centrifuge at 4 °C and 28,000 x g for 30 min.
      25. Wash the pellet once with cold 75% EtOH and subsequently once with 100% EtOH.
      26. Air-dry the pellet at room temperature for about 10 min.


        Figure 3. Schematic representation of autoradiographs of 3'-adaptor-labeled CLIPed RNAs (A) and 5'-adaptor/3'-adaptor-labeled AGO-CLIPed RNAs (B). A. Steps of gel excision, RNA extraction from the gel, 5′-adaptor ligation, and phenol extraction of the ligated product are shown; B. Procedure of gel running, gel excision, RNA extraction from the gel, reverse transcription and PCR steps are shown. (A, B)The region that should be excised by razor is indicated by red-dotted rectangle.

    12. 5′-adaptor ligation and ligate isolation
      1. Resuspend the pellet in 6.5 µl Nuclease-free D. W. and transfer the resuspended RNA into a new tube.
      2. Prepare 5′-ligation mix.
      3. Incubate at 95 °C for 2 min and immediately place on ice (denaturation).
      4. Add 1 µl T4 RNA ligase and 0.5 µl SUPERase•In RNase Inhibitor for total 20 µl.
      5. Incubate overnight at 16 °C.
      6. Add 1 µl linear acrylamide, 20 µl 3 M sodium acetate (pH 5.5), 165 µl Nuclease-free D. W. and 800 µl EtOH:Isopropanol (1:1).
      7. Vortex thoroughly and incubate for at least 1 h at -80 °C.
      8. Centrifuge at 4 °C and 28,000 x g for 30 min.
      9. Wash the pellet once with cold 75% EtOH and subsequently once with 100% EtOH.
      10. Air-dry the pellet at room temperature for about 10 min.
      11. Resuspend the pellet with 5 µl Nuclease-free D. W. and add 5 μl 2x RNA Loading buffer.
      12. Prepare Pre-cast 6% TBE-7M Urea gel and incubate the sample tube at 95 °C for 5 min.
      13. Load the samples and decade marker into the prepared a pre-cast gel and run at 200 V until the Bromophenol blue (lower) dye has migrated 75% of the gel length.
      14. Autoradiograph the gel.
      15. Cut the gel ranged by 65~102 nt, transfer into a new 1.5 ml tube and crush the gel into small pieces (Figure 3B).
      16. Add 400 µl 0.3 M Sodium acetate pH 5.5 and rotate at 4 h at 4 °C.
      17. Filter the elution supernatant at 4 °C, 20,000 x g for 15 min using Costar Spin-X centrifugation column.
      18. Transfer 400 µl of eluate into a new tube.
      19. Add 1 µl linear acrylamide and 1 ml EtOH/isopropanol (1:1), vortex, and incubate for over 1 h at -80 °C.
      20. Centrifuge at 4 °C and 28,000 x g for 30 min.
      21. Wash the pellet once with cold 75% EtOH and subsequently once with 100% EtOH.
      22. Air-dry the pellet at room temperature for about 10 min.
    13. Reverse transcription
      1. Resuspend the pellet in 7 µl nuclease-free D. W. and transfer the resuspended sample into a new tube.
      2. Mix 7 µl 5′/3′adaptor-ligated AGO-CLIPed RNA with 1 µl 10 μM RNA RT Primer, 5 µl of 2 mM dNTP.
      3. Heat mixture to 65 °C for 5 min and incubate on ice for at least 1 min.
      4. Collect the contents of the tube by brief centrifugation.
      5. Add 4 µl of 5x First-Strand Buffer, 1 µl of 0.1 M DTT, 1 µl of SUPERase•In RNase Inhibitor, 1 µl of SuperScript™ III RT to total 20 µl.
      6. Mix by pipetting gently up and down.
      7. Incubate at 55 °C for 1 h.
      8. Inactivate the reaction by heating at 70 °C for 15 min.
    14. PCR
      1. Prepare PCR mix.
      2. Run PCR reaction with the following thermal program:
        1. 98 °C, 30 sec
        2. 98 °C, 10 sec / (3) 55 °C, 30 sec / (4) 72 °C, 10 sec (N cycles)
        3. 72 °C, 10 min
        4. 4 °C, ∞
        Note: To optimize the cycle number, prepare a sample for each: N = 15, 20, and 25.
      3. Add 1 µl linear acrylamide, 20 µl 3 M sodium acetate (pH 5.5), 130 µl D. W. and 800 µl EtOH:Isopropanol (1:1).
      4. Vortex thoroughly and incubate for at least 1 h at -80 °C.
      5. Centrifuge at 4 °C and 28,000 x g for 30 min.
      6. Wash the pellet once with cold 75% EtOH and subsequently once with 100% EtOH.
      7. Air-dry the pellet at room temperature for about 10 min.
      8. Prepare Pre-cast 6% TBE gel.
      9. Resuspend the pellet in 10 µl D.W. and add 2 µl 6x DNA Loading buffer (with bromophenol blue + xylene cyanol).
      10. Load the sample and Low Molecular Weight DNA Ladder into prepared gel and run at 100 V until bromophenol blue (lower) dye reaches the bottom.
      11. Stain the gel in EtBr-containing 1x TBE by gentle rocking for 10 min.
      12. Identify and cut the gel of 136~173 bp amplicon (Figure 4). It should contain 18~55 nt CLIPed inserts flanked by 5′- and 3′-primer sequences. And generally, if the quantity of substrates for PCR is enough, it can be visualized clearly at 20 cycles in a size of 136~173 bp region in the gel.
      13. Add 400 µl 0.3 M Sodium acetate pH 5.5 and rotate overnight at 4 °C.
      14. Filter the elution supernatant at 4 °C, 20,000 x g for 15 min using Costar Spin-X centrifugation column.
      15. Transfer 400 µl of eluate into a new tube.
      16. Add 1 µl Linear acrylamide and 1ml EtOH/isopropanol (1:1), vortex, and incubate for over 1 h at -80 °C.
      17. Centrifuge at 4 °C and 28,000 x g for 30 min.
      18. Wash the pellet once with cold 75% EtOH and subsequently once with 100% EtOH.
      19. Air-dry the pellet at room temperature for about 10 min.
      20. Resuspend the pellet with an appropriate volume of water (~20 µl).


        Figure 4. Visualization of PCR products. The region that should be excised by razor is indicated by red-dotted rectangle. Over-amplified PCR (Over PCR) products are frequently found in >22 PCR cycles if AGO-CLIP-seq is well-performed. We recommend collecting at least five PCR reactions to gather a large enough quantity for library construction and high-throughput sequencing.

  2. Library construction for mRNA-seq and smallRNA-seq
    1. Library for mRNA-seq
      Note: The mRNAseq libraries can be prepared as previously described (Guo et al., 2010) with the following modifications.
      1. With one of five dishes (from step A2b), extract total RNA using Trizol® reagent according to manufacturer’s instruction.
      2. Enrich poly(A)+ RNAs using Dynabeads® mRNA DIRECT™ Purification Kit according to manufacturer’s instruction with 5 μg of total RNA.
      3. 0.2 μg of oligo (dT)-enriched RNAs are fragmented using the NEBnext Magnesium RNA Fragmentation module.
      4. Dephosphorylate the fragmented RNAs using Antarctic phosphatase at 37 °C for 1 h and heat-inactivate at 70 °C for 5 min.
      5. Label the 5'-end of dephosphorylated RNAs using [γ-32P] ATP and PNK.
      6. Labeled 35–55-nt RNA fragments are purified from a 10% TBE-Urea Gel.
      7. Ligate 3'-adaptor and gel-purify the ligated products.
      8. Ligate 5'-adaptor and gel-purify the ligated products.
      9. Perform reverse transcription (RT) and PCR.
      Note: Ligation of adaptors, RT primer, and PCR primers are the same as for AGO-CLIP-seq library preparation to minimize potential biases and to permit loading on the same sequencing platform with AGO-CLIP-seq. Adaptor-ligated RNAs are reverse-transcribed and PCR-amplified following the protocol described for AGO-CLIP-seq, except that cDNAs are amplified for 17 cycles in PCR.
    2. Library for smallRNA-seq:
      1. The smallRNA-seq libraries are prepared using the method described for mRNA-seq libraries except for differences in the initial RNA harvest steps: 5 μg of total RNAs are electrophoresed in a 10% TBE-Urea Gel, and 18–25-nt RNA species are purified for further steps.

  3. High-throughput sequencing
    Amplified cDNA libraries are sequenced by using Illumina HiSeq2000 for 1 x 51 cycles at single read/quadruplexed.

  4. Data processing and bioinformatics
    1. Preprocessing:
      1. Sequencing reads are preprocessed before reads alignment by using CutAdap (v. 1.2.1) to remove adapter sequences, fastx_artifacts_filter in FASTX-Toolkit (v. 0.0.13.2, http://hannonlab.cshl.edu/fastx_toolkit/) to filter artifact reads, and then remove low quality read sequences. The options used in CutAdap software are "-m 17 --match-read-wildcards -O 10 -e 0.10".
      2. To calculate the relative fraction of reads mapped to human rRNA or tRNA, apply Bowtie2 (v. 2.1.0) aligning software to the rRNA reference sequences from Rfam (http://rfam.samger.ac.ur/) (Burge et al., 2013) and the tRNA reference sequences from GtRNAdb (http://gtrnadb.ucsc.edu/) (Chan and Lowe, 2009).
      3. To be as comprehensive as possible, expand these sequence sets to include homologous sequences found in the human genome (hg19) with BLAST+ (v. 2.2.27) software (Camacho et al., 2009).
    2. Genomes and databases:
      1. All alignments and analyses are conducted based on human hg19 reference sequences and the HCMV Towne genome.
      2. The genome annotations for genes, lincRNAs, miscRNAs, mt tRNAs, snoRNAs, and snRNAs are downloaded from the UCSC genome table browser and the BioMart in Ensembl for human and NCBI for HCMV, respectively. The miRNA annotations of human and HCMV miRNAs are downloaded from miRBase21 (http://mirbase.org/).
      3. Because most of HCMV-encoded miRNAs are annotated by studies using other kinds of HCMV strains, their annotated locations are not concordant to Towne genome.Blast searching is conducted with miRNAs whose annotation is based on AD169 genome to HCMV Towne and miRNAs to obtain the annotation of HCMV miRNA annotations (Stark et al., 2012).
    3. Gene expression measurement:
      1. Calculate the expression levels of mRNAs and miRNAs using the Tuxedo suite.
      2. To determine the gene expression level, apply Tophat2 (v. 2.0.9) with "--no-coverage-search --b2-very-sensitive --no-nevel-junc" options and cufflinks (v. 2.1.1) with "-q --no-update-check" options for the calculation of RPKM (reads per kilo base of transcript per million fragments mapped) scores.
      3. Apply Bowtie2 (v. 2.1.0) with "-k 101, --score-min C,0,0 (no mismatch) or --score-min C,-8,0 (for 1 mismatch) -mp 8,8 --np 8 --very-sensitive" options for the determination of miRNA expression levels with up to one mismatch to calculate RPM (reads per million reads mapped) scores.
      4. Quantify expression levels of miRNAs from AGO-CLIP-seq data similarly to smallRNA sequencing data. An additional mismatch (--score-min C, -16,0 option in Bowtie2) for AGO-CLIP-seq alignments can be allowed, because AGO-CLIP-seq is more error-prone.
    4. AGO binding site detection:
      1. As AGO-CLIP-seq reads indicate AGO-RNA interactions, a cluster of AGO-CLIP-seq reads (“peak”) suggests an AGO binding site.
      2. To exclude random AGO-RNA interactions, a peak detection method is devised to measure significant enrichment of AGO-CLIP-seq reads compared to mRNA-seq reads using Fisher's exact test.
        Note: This test compares the expression level of a given 60-bp and 1-Mbps local background region in AGO-CLIP-seq and mRNA-seq, respectively.
      3. The resulting P-value is used to define a peak region and to evaluate the significance of the peak.
        1. Read preparation and alignment
          1.) Non-miRNA reads are collected and filtered from AGO-CLIP-seq data.
          2.) Align AGO-CLIP-seq reads and mRNA-seq reads onto the human genome using TopHat2 with a less stringent mode (added --b2-score-min L, -0.6, 0.9 in mRNAseq options) to allow for slightly more sequencing errors in AGO-CLIP-seq.
          3.) For each genomic position, calculate the aligned reads for AGO-CLIP-seq and mRNA-seq to identifying the peak region.
          4.) Since mRNA sequencing has a coverage bias across the transcript, the average over the transcript length is taken for each transcript to identify peaks.
        2. Peak detection
          1.) To detect significant peaks from AGO-CLIP-seq data, apply Fisher's exact test using mapped reads in 60-bp candidate peak regions and in the surrounding 1-Mbp background region.
          2.) Window size is determined by the AGO footprint spans about 60 bp (Chi et al., 2009), and the background was set to be large enough.
          3.) Adjust p-values using Q-VALUE (v. 1.1) from the Storey lab (http://genomics.princeton.edu/storeylab/qvalue) with default settings for multiple comparison correction.
          4.) Define peaks with false discovery rates (FDRs) less than 0.01 as significant.
          5.) Combine overlapping peaks in a merged peak site to avoid redundancy.
          6.) Use peaks located in human exon regions for the analysis of AGO binding sites on human mRNAs.
        3. Increased CLIPseq constraint:
          1.) To detect target sites of a specific miRNA on 3'-UTR in mRNAs, examine whether a peak site includes a seed pairing site with canonical seeds sites (8mer, 7mer-A1, 7merm8, and 6mer) of both human and viral miRNAs by searching in the defined peak region (60 bp, longer for merged peaks). Based on this search, peaks in case that there is no seed pairing site in its region can be excluded.
          2.) To identify more robust targets of viral miRNA, “Increased CLIP-seq” constraint is applied, representing as the level of enriched AGO-CLIP-seq reads relative to the previous time of viral infection.
          3.) To measure the enrichment score for a given microRNA target site, we introduce a relative score called, the “ACE-score (AGO-CLIP-seq Enrichment score)”. ACE-score is defined as the Log ratio of normalized counts at a given time point to the normalized counts at the previous time after viral infection (Figure 5).


          Figure 5. Scheme of ACE-score calculation. Formula of ACE-scoring and examples of peak cluster sites are illustrated and adapted from Reference (Kim et al., 2015). RPKM, reads per kilo base of transcript per million fragments mapped.

Recipes

  1. Phosphate-buffered saline (PBS)
    137 mM
    NaCl
    2.7 mM
    KCl
    10 mM
    Na2HPO4
    2 mM
    KH2PO4
  2. Lysis buffer
    50 mM
    Tris-HCl (pH 7.4)
    100 mM
    NaCl
    1%
    NP-40
    0.1%
    SDS
    0.5%
    sodium deoxycholate
  3. High-salt buffer
    50 mM
    Tris-HCl (pH 7.4)
    1 M
    NaCl
    1 mM
    EDTA
    1%
    NP-40
    0.1%
    SDS
    0.5%
    sodium deoxycholate
  4. Wash buffer
    20 mM
    Tris-HCl (pH 7.4)
    10 mM
    MgCl2
    0.2%
    Tween-20
  5. CIP mix
    10 µl
    10x NE buffer 3
    5 µl
    Alkaline Phosphatase, Calf Intestinal (CIP)
    3 µl
    SUPERase.In RNase Inhibitor
    82 µl
    nuclease-free distilled water (D. W.) to 100 µl final volume
  6. 3'-ligation mix
    100 pmol
    3′-Adaptor (RA3) (1 µl 100 µM adaptor)
    10 µl
    10x T4 RNA ligase buffer (no ATP)
    3 µl
    T4 RNA ligase truncated K227Q
    3 µl
    SUPERase•In RNase Inhibitor
    30 µl
    50% PEG8000
    53 µl
    Nuclease-free D. W. to 100 µl final volume
  7. Hot PNK mix
    5 µl
    10 mCi/ml [γ-32P] ATP
    8 µl
    10x T4 PNK buffer
    3 µl
    T4 PNK buffer
    2 µl
    SUPERase.In RNase Inhibitor
    62 µl
    Nuclease-free D. W. to 80 µl final volume
  8. 0.1% PBS-T
    137 mM
    NaCl
    2.7 mM
    KCl
    10 mM
    Na2HPO4
    2 mM
    KH2PO4
    0.1% (v/v)
    Tween-20
  9. PK buffer
    100 mM
    Tris-HCl (pH 7.4)
    50 mM
    NaCl
    10 mM
    EDTA
  10. PK mix
    100 µl
    PK buffer
    100 µl
    20 mg/ml Proteinase K
  11. PK/Urea buffer
    100 mM
    Tris-HCl (pH 7.4)
    50 mM
    NaCl
    10 mM
    EDTA
    7 M
    Urea
  12. 5'-ligation mix
    8.5 µl
    Resuspended sample
    1 µl
    5′-adaptor oligonucleotide
    2 µl
    10x T4 RNA ligase buffer (with ATP)
    6 µl
    50% PEG 8000 (NEB)
    1 µl
    0.1% BSA (supplied in TaKaRa T4 RNA ligase kit)
  13. PCR mix
    2 µl
    RT product (template)
    2 µl
    5′-Primer (10 µM)
    2 µl
    3′-Primer (10 µM)
    10 µl
    5x HF buffer
    5 µl
    2 mM dNTPs mix
    0.5 µl
    phusion polymerase
    28.5 µl
    D. W. to total 50 µl
  14. 6x DNA loading buffer
    10 mM
    Tris-HCl (pH 7.6)
    0.03%
    bromophenol blue (w/v)
    0.03%
    Xylene cyanol FF (w/v)
    60%
    glycerol
    60 mM
    EDTA
  15. TBE (Tris-borate-EDTA)
    1. Prepare a 5x stock solution in 1 L of D. W.:
      54 g
      Tris base
      27.5 g
      boric acid
      20 ml
      0.5 M EDTA (pH 8.0)
    2. The 0.5x working solution is 45 mM Tris-borate/1 mM EDTA.

Acknowledgments

We want to thank the Group of Daehyun Baek (School of Biological Sciences, Seoul National University, Seoul, Republic of Korea) for the development and technical support of bioinformatic analysis. This protocol has been adapted from our previous study (Kim et al., 2015). This work was supported by IBS-R008-D1 of Institute for Basic Science from the Ministry of Science, ICT and Future Planning of Korea (K. A.).

References

  1. Burge, S. W., Daub, J., Eberhardt, R., Tate, J., Barquist, L., Nawrocki, E. P., Eddy, S. R., Gardner, P. P. and Bateman, A. (2013). Rfam 11.0: 10 years of RNA families. Nucleic Acids Res 41(Database issue): D226-232.
  2. Britt, W. J. (2010). Human cytomegalovirus: propagation, quantification, and storage. Curr Protoc Microbiol Chapter 14: Unit 14E 13.
  3. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K. and Madden, T. L. (2009). BLAST+: architecture and applications. BMC Bioinformatics 10: 421.
  4. Chan, P. P. and Lowe, T. M. (2009). GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res 37(Database issue): D93-97.
  5. Chi, S. W., Zang, J. B., Mele, A. and Darnell, R. B. (2009). Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460(7254): 479-486.
  6. 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.
  7. Kim, S., Seo, D., Kim, D., Hong, Y., Chang, H., Baek, D., Kim, V. N., Lee, S. and Ahn, K. (2015). Temporal landscape of microRNA-mediated host-virus crosstalk during productive human cytomegalovirus infection. Cell Host Microbe 17(6): 838-851.
  8. Stark, T. J., Arnold, J. D., Spector, D. H. and Yeo, G. W. (2012). High-resolution profiling and analysis of viral and host small RNAs during human cytomegalovirus infection. J Virol 86(1): 226-235.

材料和试剂

  1. 100mm细胞培养皿(Sarstedt AG& Co,目录号:83.3902)
  2. 一次性血清移液器(橙色)
  3. 过滤微量吸头(Biotix,NEPTUNE)
  4. 1.6ml微量离心管(Biotix,NEPTUNE,目录号:3745X)
  5. 细胞刮刀(25cm)(Sarstedt AG& Co,目录号:83.1830)
  6. 塑料包装(CLEANWRAP)
  7. BAS膜(Fujifilm Corporation)
  8. 剃刀(Dorco,型号:Pace Single Edge Blades DN52)
  9. Costar Spin-X离心柱(Sigma-Aldrich,目录号:CLS8162-96EA)
  10. 人包皮成纤维细胞(HFF)细胞(ATCC,目录号:SCRC-104 TM)
  11. Dulbecco's改良的Eagle培养基(DMEM)(具有高葡萄糖,具有4mM L-谷氨酰胺,不含丙酮酸钠)(GE Healthcare,HyClone TM,目录号:SH30022.FS)
  12. 胎牛血清(FBS)(增强美国GE Healthcare HyClone特征的细胞生长)(GE Healthcare,HyClone TM,目录号:SH30071.03HI)
  13. GlutaMAX-I(100×)(100ml)(Thermo Fisher Scientific,GibcoTM,目录号:35050-061)
  14. 青霉素/链霉素溶液(P/S)(100x,10,000U/ml)(Thermo Fisher Scientific,GibcoTM,目录号:15-140-122)

  15. 人巨细胞病毒(HCMV)的Towne株(ATCC,型号:VR-977 TM; NCBI登录号:FJ616285.1)
  16. 羊抗鼠IgG(Thermo Fisher Scientific,InvitrogenTM,目录号:11031)
  17. Dynabeads Pan Mouse IgG(Thermo Fisher Scientific,InvitrogenTM,目录号:11041)
  18. Pan抗AGO mAb 2A8(Diagenode,目录号:C15200167-100)
  19. 蛋白酶抑制剂混合物(Cell Signaling Technology,目录号:5871)
  20. 重组DNA酶I(Takara Bio Company,目录号:2270B)
  21. RNA酶A(1MG,20单位/μl; 5mg/ml)(Thermo Fisher Scientific,Affymetrics,目录号:70194Y)
  22. 碱性磷酸酶,小牛肠(CIP)(New England Biolabs,目录号:M0290S)
  23. (RA3)(5'-rApp-TGGAATTCTCGGGTGCCAAGG-3'-ddC,5'-腺苷酸化,3'-二脱氧-C)(Integrated DNA Technologies,RNase Free HPLC Purification)
  24. T4 RNA连接酶2,截短的K227Q(New England Biolabs,目录号:M0351L)
  25. 在RNA酶抑制剂(Thermo Fisher Scientific,Ambion TM,目录号:AM2696)中
  26. ATP,[γ-32 P],6,000Ci/mmol(222TBq/mmol)(PerkinElmer,目录号:NEG502Z500UC)
  27. T4多核苷酸激酶(PNK)(Takara Bio Company,目录号:2021A)
  28. 10mM腺苷5'-三磷酸(ATP)(New England Biolabs,目录号:P0756S)
  29. NuPAGE LDS样品缓冲液(4x)(Thermo Fisher Scientific,Novex TM,目录号:NP0007)
  30. NuPAGE样品还原剂(10x)(Thermo Fisher Scientific,Novex TM,目录号:NP0004)
  31. NuPAGE抗氧化剂(Thermo Fisher Scientific,Novex TM,目录号:NP0005)
  32. NuPAGE TM Novex TM 4-12%Bis-Tris蛋白凝胶,1.0mm,10孔(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0321BOX)
  33. NuPAGE MOPS SDS运行缓冲液(20x)(Thermo Fisher Scientific,Novex TM,目录号:NP0001)
  34. NuPAGE传输缓冲液(20x)(Thermo Fisher Scientific,Novex TM,目录号:NP0006)
  35. Amersham Protran支持0.45NC(GE Healthcare Life Sciences,目录号:10600016)
  36. 牛血清白蛋白(BSA)(生命科学级粉末)(Merck Millipore Corporation,Probumin ,目录号:821006)
  37. 抗AGO mAb 21D2(韩国首尔国立大学生物科学学院Narry V. Kim实验室制造)
  38. 过氧化物酶AffiniPure山羊抗小鼠IgG(H + L)(Jackson ImmunoResearch Inc.,目录号:115-035-003)
  39. EMD Millipore Immobilon TM Western化学发光HRP底物(ECL)(Thermo Fisher Scientific,目录号:WBKLS0050)
  40. 酸 - 苯酚:氯仿,pH 4.5(使用IAA,125:24:1)(RNA苯酚/氯仿)(Thermo Fisher Scientific,Ambion TM,目录号:AM9720)
  41. 线性丙烯酰胺(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9520)
  42. 3 M乙酸钠(pH 5.5)(Thermo Fisher Scientific,Ambion TM,目录号:AM9740)
  43. 乙醇(Merck Millipore Corporation,目录号:100983)
  44. 异丙醇(2-丙醇)(Merck Millipore Corporation,目录号:109634)
  45. UltraPure TM DNase/RNase-Free蒸馏水(D.W.)(Thermo Fisher Scientific,Invitrogen TM,目录号:10977-015)
  46. 2x RNA上样缓冲液(Thermo Fisher Scientific,Ambion TM,目录号:AM8546G)
  47. mirVana TM探针& Marker Kit(Thermo Fisher Scientific,Ambion TM,目录号:AM1554)
  48. 10%Novex TBE-尿素凝胶,10孔(Thermo Fisher Scientific,InvitrogenTM,目录号:EC6875BOX)
  49. T4 RNA连接酶(Takara Bio公司,目录号:2050B)
  50. 蛋白酶K(PK)(Thermo Fisher Scientific,Macherey-Nagel,目录号:740506)
  51. 5'-连接寡核苷酸(5'-Solexa衔接子)
    5'-rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC-3'(Integrated DNA Technologies,RNase Free HPLC Purification)
  52. RNA RT引物(RTP)
    5'-GCCTTGGCACCCGAGAATTCCA-3'(Integrated DNA Technologies,RNase Free HPLC Purification)
  53. 5'-引物(Integrated DNA Technologies,RNase Free HPLC Purification)
    1. RNA PCR引物(RP1)
      5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3'
  54. 3'-引物(Integrated DNA Technologies,RNase Free HPLC Purification)
    1. RNA PCR引物,Index 1(RPI1)
      5'-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'
    2. RNA PCR引物,Index 2(RPI2)
      5'-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'
    3. RNA PCR引物,Index 3(RPI3)
      5'-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'
    4. RNA PCR Primer,Index 4(RPI4)
      5'-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'
  55. Phusion 高保真DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:F-530L)
  56. dNTP混合物(Enzynomics,目录号:N001L)
  57. SuperScript TM III逆转录酶(RT)(Thermo Fisher Scientific,Invitrogen TM,目录号:18080-044)
  58. (Thermo Fisher Scientific,Invitrogen TM,目录号:EC6265BOX)的6%,10%孔
  59. 低分子量DNA梯(新英格兰Biolabs,目录号:N3233L)
  60. 溴化乙锭(EtBr)(Sigma-Aldrich,目录号:E7637)
  61. Dynabeads mRNA DIRECT TM试剂盒(Thermo Fisher Scientific,Ambion TM,目录号:61011)
  62. NEBNext Magnesium RNA Fragmentation Module(New England Biolabs,目录号:E6150S)
  63. 氯化钠(NaCl)(BioXtra,≥99.5%)(Sigma-Aldrich,目录号:S7653)
  64. 氯化钾(KCl)(BioXtra,≥99.0%)(Sigma-Aldrich,目录号:P9333)
  65. 磷酸二氢钠(Na 2 HPO 4)(BioXtra,≥99%)(Sigma-Aldrich,目录号:S7907)
  66. 磷酸二氢钾(KH 2 PO 4)(≥99.0%)(Sigma-Aldrich,目录号:P5655)
  67. (高质量Tris碱)(BioXtra,≥99.9%)(Sigma-Aldrich,目录号:T6791)。
  68. NP-40 Alternative(Merck Millipore Corporation,目录号:492018)
  69. 十二烷基硫酸钠(SDS)[BioXtra,≥99.0%(GC)](Sigma-Aldrich,目录号:L6026)
  70. 脱氧胆酸钠(BioXtra,无水,≥98%)(Sigma-Aldrich,目录号:30970)
  71. 乙二胺四乙酸(EDTA)(BioUltra,无水,≥98%)(Sigma-Aldrich,目录号:EDS)
  72. 氯化镁(MgCl 2)(无水,≥98%)(Sigma-Aldrich,目录号:M8266)
  73. Tween-20(Sigma-Aldrich,目录号:P7949)
  74. NEBuffer3(New England Biolabs,目录号:B7003S)
  75. 10×T4 RNA连接酶缓冲液(New England Biolabs,目录号:B0216L)
  76. 50%PEG8000(与10×T4 RNA连接酶缓冲液一起提供)
  77. 10mM腺苷5'-三磷酸(ATP)(用10×T4 RNA连接酶缓冲液提供)
  78. 溴酚蓝(ACS试剂)(Sigma-Aldrich,目录号:114391)
  79. 二甲苯Cyanol FF(BioReagent)(Sigma-Aldrich,目录号:X4126)
  80. 甘油(BioXtra,无水,≥99%)(Sigma-Aldrich,目录号:G6279)
  81. 硼酸(BioReagen,≥99.5%)(Sigma-Aldrich,目录号:B6768)
  82. Trizol试剂(Thermo Fisher Scientific,Ambion TM,目录号:15596018)
  83. Dynabeads mRNA DIRECT TM纯化试剂盒(Thermo Fisher Scientific,Ambion TM,目录号:61011)
  84. NEBNext Magnesium RNA Fragmentation Module(New England Biolabs,目录号:E6150S)
  85. 南极磷酸酶(New England Biolabs,目录号:M0289S)
  86. 磷酸盐缓冲盐水(PBS)(见配方)
  87. 裂解缓冲液(见配方)
  88. 高盐缓冲液(见配方)
  89. 洗涤缓冲液(见配方)
  90. CIP混合(参见配方)
  91. 3'-连接混合物(参见配方)
  92. 热PNK混合(参见配方)
  93. 0.1%PBS-T(参见配方)
  94. PK缓冲区(参见配方)
  95. PK混合(参见配方)
  96. PK /尿素缓冲液(参见配方)
  97. 5'-连接混合物(参见配方)
  98. PCR混合(参见配方)(参见配方)
  99. 6x DNA加载缓冲液(参见配方)
  100. TBE(Tris-硼酸盐-EDTA)(参见配方)

设备

  1. CO 2培养箱(LabX,Sanyo,型号:CO 2)培养箱MCO-18AIC)
  2. 微量移液器(Gilson,PIPETMAN Classic TM P10,P20,P200和P1000,订购参考:F144802,F123600,F123601和F123602)
  3. Rocker(FINEPCR,型号:CR300)
  4. UV交联剂(Spectronics Corporation,型号:XL1500)
  5. Vortexer(Scientific Industries,型号:Vortex-Genie 2和目录号:SI-0256)
  6. DynaMag TM -2磁体(Thermo Fisher Scientific,目录号:12321D)
  7. 旋转器(FINEPCR,型号:AG)和辅助滚筒(FINEPCR,型号:RD25-42)
  8. 干燥块加热和冷却振荡器(Sigma-Aldrich,目录号:T3317)的微波加热器
  9. 冷冻桌面微量离心机(Hanil BioMed Inc.,型号:Centrifuge Micro 17TR??)
  10. XCell SureLock小室(Thermo Fisher Scientific,Novex TM,目录号:EI0001)
  11. Chemidoc(Vilber Lourmat,型号:Fusion Spectra)
  12. BAS 2500(Fujifilm Corporation)
  13. 深冷冻(-80℃冰箱)
  14. PCR循环仪(MyCycler TM Thermal Cycler System)(Bio-Rad Laboratories,目录号:#1709703)
  15. Illumina HiSeq2000

软件

  1. CutAdap软件
  2. 人类基因组(hg19)BLAST +(v.2.2.27)软件(Camacho et al。,2009)

程序


图1.整个程序(A)和AGO-CLIP-seq(B)的方案

注意:在整个AGO-CLIP-seq方案(图1)中,所有工作都必须在无RNA酶的环境中进行,并且只能使用无菌和无DNA酶/RNase的溶液和材料。


  1. AGO-CLIP-seq
    1. 细胞培养和HCMV感染
      注意:为了传播HCMV并准备HCMV股票,我们遵循前面的指令(Britt,2010)。
      1. HFF细胞获自美国典型培养物保藏中心(American Type Culture Collection) ?在补充有完全DMEM的5个100-mm培养皿中培养 用10%FBS,2mM GlutaMAX-1和1×P/S在37℃在5% ?CO 2
      2. 用PBS洗两次。
      3. 添加含HCMV的无血清 DMEM,MOI为3,总体积为2ml /皿,并在37℃下孵育 °C下1小时摇动,轻轻地,但以15的间隔彻底 min。
      4. 倾析病毒培养基,用PBS洗涤两次,加入10 ml 10%FBS,P/S,L-Glu DMEM /培养皿。
      5. 孵育指定的时间(24,48和72小时)。
    2. 紫外线照射
      1. 用3ml /盘的冷PBS洗一次。
      2. 在5个皿中的4个中加入2ml /盘的冷PBS并总提取 来自五个培养皿之一的RNA构建用于mRNA-seq的文库 smallRNA-seq(转到步骤B1a)。
      3. 在Spectrolinker中在250mJ/cm 2下使用254nm UV在冰上照射。
      4. 彻底清除PBS。
      5. 将制备的细胞储存在-80℃直至随后的步骤。
    3. 抗体结合Dynabeads的制备
      1. 转移50微升羊α-小鼠免疫球蛋白G(IgG)共轭 Dynabead每个样品到1.5ml管中。收集磁性Dynabeads 使用DynaMeg-2磁体机架。 Panα-小鼠IgG缀合的Dynabead 可用作羊α-小鼠免疫球蛋白G(IgG)的替代物 共轭Dynabead。
      2. 用0.1M NaHPO 4(pH8.1)洗涤两次并将珠再悬浮于200μl0.1M NaHPO 4(pH8.1)中。
      3. 每个样品加入30μgmAb 2A8作为泛抗AGO抗体。
      4. 在室温下旋转试管2小时(至少1小时)。
      5. 用裂解缓冲液洗涤两次
      6. 离开冰直到下一步。
    4. 细胞裂解和部分RNA消化
      1. 加入1ml裂解缓冲液(用蛋白酶抑制剂混合物) 样品盘,废细胞裂解物,将裂解物转移到下一个 并重复,直到最后一道菜
      2. 最后,将裂解物转移到1.5ml管中
      3. 加入10μl重组DNA酶I,并在37°C和1,400 rpm下孵育 ?Eppendorf ? Thermomixer ? R培养10分钟,使裂解液粘度降低。
      4. 加入10μl稀释(1:1,000)核糖核酸酶。
      5. 在37℃和1,400rpm孵育10分钟。
      6. 立即将管转移到冰上。
      7. 在4℃和28,000×g离心30分钟
      8. 小心收集上清液并转移上清液, 除了80μl,将用于免疫印迹,到一个新的管 以在下一步骤中与制备的珠结合。
    5. 免疫沉淀
      1. 将制备的Ab缀合的珠与细胞裂解物组合
      2. 在4℃下旋转3小时。
      3. 滗析上清液,用1ml高盐缓冲液洗涤珠两次
      4. 用1ml洗涤缓冲液洗涤两次。
    6. 脱磷酸化
      1. 准备CIP混合。
      2. 向洗涤的珠子中加入100μlCIP混合物
      3. 在37℃和1400rpm下在Eppendorf Thermomixer R中孵育10分钟。
      4. 立即将管转移到冰上。
      5. 用1ml高盐缓冲液洗涤珠子两次,用1ml洗涤缓冲液洗涤两次。
    7. 3'-衔接子连接
      1. 制备3'-连接混合物
      2. 向洗涤的珠子中加入100μl3'-连接混合物
      3. 在Eppendorf Thermomixer R中在16℃和1,400rpm温育过夜。
      4. 用1ml高盐缓冲液洗涤珠子两次,用1ml洗涤缓冲液洗涤两次。
    8. RNA 5'-末端标记
      1. 准备热PNK混合物。
      2. 向洗涤的珠子中加入80μl热PNK混合物
      3. 在37℃和1,400rpm孵育15分钟
      4. 立即加入5μl10mM ATP。
      5. 在37℃和1,400rpm下在Eppendorf Thermomixer R中孵育5分钟。
      6. 用1ml高盐缓冲液洗涤珠子两次,用1ml洗涤缓冲液洗涤两次。
    9. NuPAGE和膜转移
      1. AGO-RNA复合物通过在75℃温育从珠子洗脱 °C和1,400 rpm在30μl1.5x NuPAGE LDS样品缓冲液中10分钟。
      2. 将13μl裂解物与13μlNuPAGE LDS样品缓冲液混合 ?(4x)和2μlNuPAGE样品还原剂(10x),并在 75℃,10分钟
      3. 将样品装载在4-12%NuPAGE Bis-Tris凝胶上 ?根据制造商的说明使用800ml的1x MOPS 运行缓冲液与NuPAGE ?抗氧化剂。
      4. 在150 V下运行凝胶2小时30分钟。
      5. 放射自显影凝胶约5?30分钟。
      6. 在打印的图中标记尺寸带。
      7. 将蛋白质-RNA复合物从凝胶转移到硝酸纤维素 膜在130V使用根据的NuPAGE转移缓冲液70分钟 制造商的说明。
      8. 转移后,用D.W冲洗膜,然后用塑料包装包裹
      9. 放射自显影凝胶约5?30分钟。
      10. 在打印的图中标记尺寸带(图2)。
    10. Western印迹
      1. 取出转移的膜部分进行Western印迹 将其置于50-100ml含有5%(w/v)BSA的0.1%(v/v)PBS-T中。
      2. 孵育膜1小时,在室温下轻轻摇动
      3. 用0.1%PBS-T短暂清洗膜两次
      4. 将每个膜放入一个小船和倾倒一抗 溶液(20ml含有5%BSA的0.1%PBS-T和20μl抗AGO mAb 21D2)到船上
      5. 孵育至少4小时,在4°C轻轻摇动
      6. 用0.1%PBS-T短暂清洗膜两次
      7. 将膜放入一个小船,并倒入第二抗体 溶液(20ml含有5%BSA的0.1%PBS-T和4μlHRP缀合的) 山羊抗小鼠IgG)到船上
      8. 孵育膜1小时,在室温下轻轻摇动
      9. 用0.1%PBS-T至少洗涤膜5次,每次10分钟,轻轻摇动
      10. 使用Chemidoc进行ECL检测(图2B)

        图2.放射自显影和Western印迹的示意图 ?的剪切的AGO-RNA复合物和裂解物转移到硝酸纤维素 膜从NuPAGE凝胶。 A.形象表示。地区 应由剃刀切除,蛋白酶K处理由 红色虚线矩形。评估AGO-CLIP-seq的IP效率 程序和凝胶中确切的非共轭AGO蛋白位置, 免疫印迹应在同一凝胶中进行 放射自显影。 B.具有各种RNase条件的代表性数据 ?A和UV处理和使用的抗体。正如所看到的,更高 使用核糖核酸酶A的浓度,放射自显影强度越低 ?观察到AGO-RNA复合物。 UV-未处理和使用IgG对照 IP不产生完整复合物的信号。 AGO,Argonaute蛋白; Ab HC,抗体重链; Ab LC,抗体轻链。

    11. RNA分离和大小分离
      1. 准备PK混合物。
      2. 用剃刀切成110?160 kDa的条带,放入1.5 ml的试管中。 (图2)
      3. 加入200μlPK混合物到含膜管中,并在50°C和1,400 rpm孵育1小时
      4. 加入200μlPK /尿素缓冲液并在Eppendorf Thermomixer R R中在50℃和1,400rpm温育1小时。
      5. 加入400μlRNA苯酚/氯仿,并在Eppendorf Thermomixer R中在30℃和1,400rpm温育5分钟。
      6. 在室温下以20,000×g离心5分钟
      7. 小心地将上层(水相)转移到新的1.5ml管中
      8. 加入1μl线性丙烯酰胺和40μl3 M醋酸钠pH 5.5,涡旋
      9. 加入1ml乙醇(EtOH)/异丙醇(1:1),彻底涡旋并在-20℃或-80℃下孵育过夜。
      10. 在4℃和28,000×g离心30分钟
      11. 用冷的75%EtOH洗涤沉淀一次,然后用100%EtOH洗涤一次
      12. 在室温下将沉淀物风干约10分钟
      13. 用5μl无核酸酶的D.W.重悬沉淀。并加入5μl2x RNA上样缓冲液
      14. 使用mirVana TM Probe& Gibco公司制备放射性标记的十年RNA标记物。标记工具包。
      15. 准备预制6%TBE-7M尿素凝胶
      16. 将样品管在95°C孵育5分钟
      17. 将样品和标记物装入制备的凝胶中并在200V下运行 直到溴酚蓝(较低)染料迁移了75%的凝胶 长度
      18. 放射自显影凝胶。
      19. 切割凝胶范围为40?77 nt,转移到一个新的1.5 ml管中,将凝胶粉碎成小块(图3A)。
      20. 加入400μl0.3M醋酸钠pH5.5,在4℃下旋转过夜
      21. 使用Costar Spin-X离心柱在4℃,20,000xg下过滤洗脱上清液15分钟。
      22. 将400μl洗脱液转移到新试管中。
      23. 加入1μl线性丙烯酰胺和1ml EtOH /异丙醇(1:1),涡旋,并在-80℃下孵育1小时以上。
      24. 在4℃和28,000×g离心30分钟
      25. 用冷的75%EtOH洗涤沉淀一次,然后用100%EtOH洗涤一次
      26. 在室温下将沉淀物风干约10分钟

        图3.放射自显影的示意图 3'-衔接子标记的剪接的RNA(A)和5'-衔接子/3'-衔接子标记 AGO-CLIPed RNA(B)。 A.凝胶切除步骤,从RNA提取 凝胶,5'-衔接子连接,以及连接产物的苯酚提取 显示; B.凝胶运行,凝胶切除,RNA提取的程序 从凝胶中,显示了逆转录和PCR步骤。 (A,B) 应该由剃刀切除的区域由红点表示 长方形。

    12. 5'-衔接子连接和连接隔开
      1. 将沉淀重悬在6.5μl无核酸酶的D.W中,并将重悬的RNA转移到新管中。
      2. 准备5'-连接混合物
      3. 在95℃孵育2分钟,立即置于冰上(变性)
      4. 加入1μlT4 RNA连接酶和0.5μlSUPERase?在RNase抑制剂中总共20μl
      5. 在16℃下孵育过夜。
      6. 加入1μl线性丙烯酰胺,20μl3M乙酸钠(pH5.5),165μl无核酸酶D.W.和800μlEtOH:异丙醇(1:1)。
      7. 彻底涡旋并在-80℃下孵育至少1小时
      8. 在4℃和28,000×g离心30分钟
      9. 用冷的75%EtOH洗涤沉淀一次,然后用100%EtOH洗涤一次
      10. 在室温下将沉淀物风干约10分钟
      11. 用5μl无核酸酶的D.W.重悬沉淀,加入5μl2×RNA上样缓冲液
      12. 准备预制6%TBE-7M尿素凝胶,并在95°C孵育样品管5分钟。
      13. 将样品和十年标记物装入制备的预制凝胶中 并在200V下运行,直到溴酚蓝(低级)染料迁移75% ?的凝胶长度
      14. 放射自显影凝胶。
      15. 切割凝胶范围65?102 nt,转移到一个新的1.5毫升管,将凝胶粉碎成小块(图3B)。
      16. 加入400μl0.3M乙酸钠pH5.5,并在4℃下4小时转动
      17. 使用Costar Spin-X离心柱在4℃,20,000xg下过滤洗脱上清液15分钟。
      18. 将400μl洗脱液转移到新试管中。
      19. 加入1μl线性丙烯酰胺和1ml EtOH /异丙醇(1:1),涡旋,并在-80℃下孵育1小时以上。
      20. 在4℃和28,000×g离心30分钟
      21. 用冷的75%EtOH洗涤沉淀一次,然后用100%EtOH洗涤一次
      22. 在室温下将沉淀物空气干燥约10分钟。
    13. 反转录
      1. 将沉淀重悬在7μl无核酸酶的D.W中,并将重悬的样品转移到新管中。
      2. 将7μl5'/3'接头连接的AGO-Cliped RNA与1μl10μMRNA RT引物,5μl2 mM dNTP混合。
      3. 将混合物加热至65℃5分钟,并在冰上孵育至少1分钟
      4. 通过短暂离心收集管的内容物。
      5. 加入4μl的5x First-Strand缓冲液,1μl的0.1 M DTT,1μl的 SUPERase?在RNase抑制剂中,1μlSuperScript?III RT,总体积为20μl
      6. 通过轻轻地上下轻轻混合。
      7. 在55℃孵育1小时
      8. 通过在70℃加热15分钟使反应失活。
    14. PCR
      1. 准备PCR混合物。
      2. 使用以下热程序运行PCR反应:
        1. 98℃,30秒
        2. 98℃,10秒/(3)55℃,30秒/(4)72℃,10秒(N个循环)
        3. 72℃,10分钟
        4. 4℃,∞
        注意:要优化循环次数,请为每个样品准备一个样品:N = 15,20和25。
      3. 加入1μl线性丙烯酰胺,20μl3 M乙酸钠(pH 5.5),130μlD. W.和800μlEtOH:异丙醇(1:1)。
      4. 彻底涡旋并在-80℃下孵育至少1小时
      5. 在4℃和28,000×g离心30分钟
      6. 用冷的75%EtOH洗涤沉淀一次,然后用100%EtOH洗涤一次
      7. 在室温下将沉淀物空气干燥约10分钟。
      8. 准备预制6%TBE凝胶
      9. 将沉淀重悬于10μlD.W。并加入2μl6x DNA上样缓冲液(含溴酚蓝+二甲苯蓝)
      10. 将样品和低分子量DNA梯子装入制备 凝胶并在100V下运行,直到溴酚蓝(较低)染料达到 底部
      11. 通过温和摇动10分钟,将凝胶在含EtBr的1x TBE中染色
      12. 识别并切割136?173 bp扩增子的凝胶(图4)。它 应包含侧翼为5'-和3'-引物的18?55nt的剪接插入片段 序列。通常,如果PCR的底物的量是 足够,它可以在20个周期可视化清楚在136?173的大小 bp区域。
      13. 加入400μl0.3M醋酸钠pH5.5,在4℃下旋转过夜
      14. 使用Costar Spin-X离心柱在4℃,20,000xg下过滤洗脱上清液15分钟。
      15. 将400μl洗脱液转移到新试管中。
      16. 加入1μl线性丙烯酰胺和1ml EtOH /异丙醇(1:1),涡旋,并在-80℃下孵育1小时以上。
      17. 在4℃和28,000×g离心30分钟
      18. 用冷的75%EtOH洗涤沉淀一次,然后用100%EtOH洗涤一次
      19. 在室温下将沉淀物风干约10分钟
      20. 用适当体积的水(?20μl)重悬沉淀

        图4. PCR产物的可视化。应该是的区域 由剃刀切除由红色虚线矩形表示。过度放大 在≥22个PCR循环中经常发现PCR(过度PCR)产物 AGO-CLIP-seq执行良好。我们建议至少收集五个 PCR反应收集足够大的量用于文库构建 ?和高通量测序。

  2. mRNA-seq和smallRNA-seq的文库构建
    1. mRNA-seq的文库
      注意:mRNAseq文库可以按照先前描述的(Guo 等人 ,2010)进行修改。
      1. 使用五个培养皿之一(来自步骤A2b),根据制造商的说明使用Trizol试剂提取总RNA。
      2. 使用Dynabeads mRNA富集poly(A)+ RNA DIRECT?纯化试剂盒 根据制造商的说明用5μg总RNA
      3. 使用NEBnext Magnesium RNA Fragmentation模块将0.2μg富含寡(dT)的RNA片段化。
      4. 使用南极磷酸酶在37℃下使断裂的RNA脱磷酸化1小时,并在70℃热灭活5分钟。
      5. 使用[γ- 32 P] ATP和PNK标记去磷酸化RNA的5'-末端。
      6. 从10%TBE-尿素凝胶纯化标记的35-55nt RNA片段
      7. 连接3'-衔接子并凝胶纯化连接的产物
      8. Ligation 5'-adapter并凝胶纯化结合的产物
      9. 进行逆转录(RT)和PCR。
      注意:衔接子,RT引物和PCR引物的连接是相同的 为AGO-CLIP-seq文库制备以最小化潜在偏差和 ?允许在与AGO-CLIP-seq相同的测序平台上加载。 接头连接的RNA进行逆转录和PCR扩增 ?描述的AGO-CLIP-seq的协议,除了cDNAs 在PCR中扩增17个循环。
    2. smallRNA-seq的库:
      1. 使用所述的方法制备小RNA-seq文库 mRNA-seq文库,除了初始RNA收获的差异 步骤:将5μg总RNA在10%TBE-尿素凝胶中电泳, ?纯化18-25-nt RNA种类用于进一步的步骤。

  3. 高通量测序
    扩增的cDNA文库通过使用Illumina HiSeq2000以单次读取/四链体进行1×51个循环而测序。

  4. 数据处理和生物信息学
    1. 预处理:
      1. 序列读取在读取之前进行预处理 通过使用CutAdap(v.1.2.1)去除衔接头序列进行比对, fastX_artifacts_filter in FASTX-Toolkit(v。0.0.13.2, http://hannonlab.cshl.edu/fastx_toolkit/)来过滤工件读取,和 然后删除低质量的读序列。 CutAdap中使用的选项 软件是"-m 17 --match-read-wildcards -O 10 -e 0.10"。
      2. 至 计算映射到人rRNA或tRNA的读数的相对分数, 将Bowtie2(v。2.1.0)比对软件应用于rRNA参考 来自Rfam的序列(http://rfam.samger.ac.ur/)(Burge等人,2013)和 ?来自GtRNAdb(http://gtrnadb.ucsc.edu/)的tRNA参考序列 (Chan和Lowe,2009)。
      3. 为了尽可能全面, 扩展这些序列集以包括在中发现的同源序列 人类基因组(hg19)与BLAST +(v.2.2.27)软件(Camacho等人, 2009)。
    2. 基因组和数据库:
      1. 所有比对和分析基于人hg19参考序列和HCMV Towne基因组进行
      2. 基因组注释,lincRNAs,miscRNAs,mt tRNAs, snoRNA和snRNA从UCSC基因组表浏览器下载 和人类的Ensembl中的BioMart和HCMV的NCBI。 人类和HCMV miRNA的miRNA注释是从中下载的 miRBase21(http://mirbase.org/)。
      3. 因为大多数HCMV编码 miRNA通过使用其他种类的HCMV毒株的研究来注释 ?注释位置不与Towne基因组一致 是用注释基于AD169基因组的miRNA进行的 HCMV Towne和miRNA获得HCMV miRNA注释的注释 ?(Stark等人,2012)。
    3. 基因表达测量:
      1. 使用Tuxedo套件计算mRNA和miRNA的表达水平。
      2. 要确定基因表达水平,应用Tophat2(v。2.0.9) 与"--no-coverage-search --b2-very-sensitive --no-nevel-junc"选项 和带有"-q - no-update-check"选项的袖扣(v。2.1.1) 计算RPKM(每百万转录物每千碱基读数) 碎片映射)分数。
      3. 应用Bowtie2(v。2.1.0)with"-k 101, --score-min C,0,0(无失配)或--score-min C,-8,0(对于1个失配) -mp 8,8 --np 8 - 非敏感性"选项来确定miRNA 表达水平与多达一个不匹配以计算RPM(读数/ 百万读取映射)分数。
      4. 量化表达水平 来自AGO-CLIP-seq数据的miRNA与小RNA测序数据类似。一个 额外的不匹配(--score-min C,-16,0选项在Bowtie2)为 可以允许AGO-CLIP-seq比对,因为AGO-CLIP-seq更多 容易出错。
    4. AGO结合位点检测:
      1. 由于AGO-CLIP-seq读数指示AGO-RNA相互作用,AGO-CLIP-seq读数簇("峰")表明AGO结合位点。
      2. 为了排除随机AGO-RNA相互作用,峰检测方法是 设计以测量显着富集的AGO-CLIP-seq读数 ?到mRNA-seq读取使用Fisher精确检验。
        注意:此测试 比较给定60-bp和1-Mbps局部的表达水平 背景区域分别在AGO-CLIP-seq和mRNA-seq。
      3. 得到的P值用于定义峰值区域并评估峰值的重要性。
        1. 阅读准备和对齐
          1.收集非miRNA读数,并从AGO-CLIP-seq数据中过滤。
          2.)将AGO-CLIP-seq读数和mRNA-seq读数对齐到人类基因组上 使用具有较不严格模式的TopHat2(加上-b2-score-min L,-0.6, 0.9在mRNAseq选项中),以允许稍微更多的测序错误 AGO-CLIP-seq。
          3.)对于每个基因组位置,计算AGO-CLIP-seq和mRNA-seq的比对读数以鉴定峰区。
          4.)由于mRNA测序在转录物上具有覆盖偏差, 对于每个转录物采用对于转录物长度的平均值 识别峰。
        2. 峰值检测
          1.检测显着 来自AGO-CLIP-seq数据的峰,应用使用映射的Fisher精确检验 读取60 bp的候选峰区域和周围的1 Mbp 背景区域。
          2.)窗口大小由AGO确定 足迹跨越大约60bp(Chi等人,2009),背景是 设置得足够大。
          3.)使用Q-VALUE(v。1.1)调整p值 来自Storey实验室(http://genomics.princeton.edu/storeylab/qvalue) 具有用于多重比较校正的默认设置。
          4.)将假发现率(FDR)小于0.01的峰定义为显着的。
          5.)在合并峰位点合并重叠峰以避免冗余。
          6.)使用位于人外显子区域的峰用于分析人mRNA上的AGO结合位点
        3. 增加CLIPseq约束:
          为了检测mRNA中3'-UTR上特异性miRNA的靶位点, 检查峰位点是否包括具有规范的种子配对位点 种子位点(8mer,7mer-A1,7merm8和6mer)的人和病毒 通过搜索定义的峰区域(60 bp,更长的合并 ?峰)。基于此搜索,在没有种子的情况下的峰 可以排除其区域中的配对位点。
          2.)识别更多 强大的病毒miRNA靶标,"增加CLIP-seq"约束是 应用,表示作为富集的AGO-CLIP-seq读数的水平 相对于先前的病毒感染时间。
          3.)测量 对于给定的microRNA靶位点的富集得分,我们引入a 相对分数,称为"ACE分数(AGO-CLIP-seq Enrichment分数)"。 ACE评分定义为给定的标准化计数的对数比 时间点到病毒后的上一时间的归一化计数 感染(图5)。


          图5. ACE评分计算方案。ACE评分公式和峰值集群站点的示例 从参考文献(Kim等人,2015)中说明和改编。 RPKM,读 每千万基因的转录物每百万片段映射。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    137 mM
    NaCl
    2.7 mM
    KCl
    10 mM
    Na HPO 4
    2 mM
    KH 2 PO 4
  2. 裂解缓冲液
    50 mM
    Tris-HCl(pH 7.4)
    100 mM
    NaCl
    1%
    NP-407
    0.1%
    SDS
    0.5%
    脱氧胆酸钠
  3. 高盐缓冲液
    50 mM
    Tris-HCl(pH 7.4)
    1兆
    NaCl
    1 mM
    EDTA
    1%
    NP-40
    0.1%
    SDS
    0.5%的
    脱氧胆酸钠
  4. 洗涤缓冲液
    20 mM
    Tris-HCl(pH 7.4)
    10 mM
    MgCl 2
    0.2%
    吐温-20
  5. CIP混合
    10微升
    10x NE缓冲区3
    5微升
    碱性磷酸酶,小牛肠(CIP)
    3微升
    在RNase抑制剂中
    82微升
    无核酸酶蒸馏水(D.W。)至100μl终体积
  6. 3'-连接混合物
    100 pmol
    3'-衔接子(RA3)(1μl100μM衔接子)
    10微升
    10×T4 RNA连接酶缓冲液(无ATP)
    3微升
    T4 RNA连接酶截短的K227Q
    3微升
    SUPERase?在RNase抑制剂中
    30微升
    50%PEG8000
    53微升
    无核酸酶D.W至100μl终体积
  7. 热PNK混合物
    5微升
    10mCi/ml [μ-γ-32P] ATP
    8微升
    10x T4 PNK缓冲区
    3微升
    T4 PNK缓冲区
    2微升
    在RNase抑制剂中
    62微升
    无核酸酶D.W至80μl终体积
  8. 0.1%PBS-T
    137 mM
    NaCl
    2.7 mM
    KCl
    10 mM
    Na HPO 4
    2 mM
    KH 2 PO 4
    0.1%(v/v)
    吐温-20
  9. PK缓冲区
    100 mM
    Tris-HCl(pH 7.4)
    50 mM
    NaCl
    10 mM
    EDTA
  10. PK混合物
    100微升
    PK缓冲区
    100微升
    20mg/ml蛋白酶K
  11. PK /尿素缓冲区
    100 mM
    Tris-HCl(pH 7.4)
    50 mM
    NaCl
    10 mM
    EDTA
    7 M
    尿素
  12. 5'-连接混合物
    8.5微升
    重悬样品
    1微升
    5'-衔接子寡核苷酸
    2微升
    10x T4 RNA连接酶缓冲液(含ATP)
    6微升
    50%PEG 8000(NEB)
    1微升
    0.1%BSA(TaKaRa T4 RNA连接酶试剂盒提供)
  13. PCR混合物
    2微升
    RT产品(模板)
    2微升
    5'-引物(10μM)
    2微升
    3'-引物(10μM)
    10微升
    5x HF缓冲液
    5微升
    2 mM dNTPs mix
    0.5μl
    phusion聚合酶
    28.5微升
    总量为50微升
  14. 6x DNA加载缓冲液
    10 mM
    Tris-HCl(pH 7.6)
    0.03%
    溴酚蓝(w/v)
    0.03%
    二甲苯蓝藻FF(w/v)
    60%
    甘油
    60 mM
    EDTA
  15. TBE(Tris-硼酸盐-EDTA)
    1. 准备一个5升储备溶液在1升的D. W .:
      54克
      Tris碱
      27.5克
      硼酸
      20ml
      0.5 M EDTA(pH 8.0)
    2. 0.5x工作溶液是45mM Tris-硼酸盐/1mM EDTA。

致谢

我们要感谢大韩民国集团(首尔国立大学生物科学学院,韩国首尔)为生物信息学分析的发展和技术支持。该协议已经从我们以前的研究(Kim等人,2015年)改编。这项工作得到科学,ICT和韩国未来规划部(K.A。)基础科学研究所的IBS-R008-D1的支持。

参考文献

  1. Burge,S.W.,Daub,J.,Eberhardt,R.,Tate,J.,Barquist,L.,Nawrocki,E.P.,Eddy,S.R.,Gardner,P.P.and Bateman, Rfam 11.0:10年的RNA家族。 Nucleic Acids Res < em> 41(数据库问题):D226-232。
  2. Britt,W. J.(2010)。 人类巨细胞病毒:繁殖,定量和储存。 Curr Protoc Microbiol 第14章:单位14E 13.
  3. Camacho,C.,Coulouris,G.,Avagyan,V.,Ma,N.,Papadopoulos,J.,Bealer,K.and Madden,T.L。(2009)。 BLAST +:体系结构和应用。 BMC Bioinformatics 10:
  4. Chan,P.P。和Lowe,T.M。(2009)。 GtRNAdb:在基因组序列中检测到的转移RNA基因的数据库核Acids Res 37(数据库问题):D93-97。
  5. Chi,S.W.,Zang,J.B.,Mele,A。和Darnell,R.B。(2009)。 Argonaute HITS-CLIP解码microRNA-mRNA相互作用图。 em> 460(7254):479-486。
  6. Guo,H.,Ingolia,N.T.,Weissman,J.S。和Bartel,D.P。(2010)。 哺乳动物微小RNA主要用于降低靶mRNA水平。 自然 466(7308):835-840。
  7. Kim,S.,Seo,D.,Kim,D.,Hong,Y.,Chang,H.,Baek,D.,Kim,V.N.,Lee,S.and Ahn,K。 生产性人巨细胞病毒感染过程中微小RNA介导的宿主 - 病毒串扰的时空格局。 em> Cell Host Microbe 17(6):838-851。
  8. Stark,T.J.,Arnold,J.D.,Spector,D.H。和Yeo,G.W。(2012)。 在人类巨细胞病毒感染期间对病毒和宿主小RNA的高分辨率分析和分析。 J Virol 86(1):226-235。
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How to cite this protocol: Kim, S. and Ahn, K. (2016). ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq . Bio-protocol 6(8): e1791. DOI: 10.21769/BioProtoc.1791; Full Text



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