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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

SK Sungchul Kim
Kwangseog Ahn Kwangseog Ahn

发布: 2016年04月20日第6卷第8期 DOI: 10.21769/BioProtoc.1791 浏览次数: 13345

评审: Yannick DebingFeng LiVaibhav B Shah

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Cover of Cell Host & Microbe, featuring study using the protocol.

Jun 2015

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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) search ACE score (ACE得分) search MicroRNA (microRNA) search Human cytomegalovirus (人巨细胞病毒) search Argonaute (Argonaute蛋白) search

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, HyCloneTM, catalog number: SH30022.FS )
  12. Fetal bovine serum (FBS) (Enhance growth of cells with U.S. sourced GE Healthcare HyClone characterized) (GE Healthcare, HyCloneTM, 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, AmbionTM, 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, NovexTM, catalog number: NP0007 )
  31. NuPAGE® Sample Reducing Agent (10x) (Thermo Fisher Scientific, NovexTM, catalog number: NP0004 )
  32. NuPAGE® Antioxidant (Thermo Fisher Scientific, NovexTM, catalog number: NP0005 )
  33. NuPAGETM NovexTM 4-12% Bis-Tris Protein Gels, 1.0 mm, 10-well (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0321BOX )
  34. NuPAGE® MOPS SDS Running Buffer (20x) (Thermo Fisher Scientific, NovexTM, catalog number: NP0001 )
  35. NuPAGE® Transfer Buffer (20x) (Thermo Fisher Scientific, NovexTM, 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 ImmobilonTM 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, AmbionTM, catalog number: AM9720 )
  42. Linear acrylamide (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9520 )
  43. 3 M Sodium acetate (pH 5.5) (Thermo Fisher Scientific, AmbionTM, catalog number: AM9740 )
  44. Ethanol (Merck Millipore Corporation, catalog number: 100983 )
  45. Isopropanol (2-propanol) (Merck Millipore Corporation, catalog number: 109634 )
  46. UltraPureTM DNase/RNase-Free Distilled Water (D.W.) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977-015 )
  47. 2x RNA loading buffer (Thermo Fisher Scientific, AmbionTM, catalog number: AM8546G )
  48. mirVanaTM Probe & Marker Kit (Thermo Fisher Scientific, AmbionTM, 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 ScientificTM, catalog number: F-530L )
  57. dNTP mixture (Enzynomics, catalog number: N001L )
  58. SuperScriptTM 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 DIRECTTM 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, AmbionTM, catalog number: 15596018 )
  84. Dynabeads® mRNA DIRECTTM Purification Kit (Thermo Fisher Scientific, AmbionTM, 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 ClassicTM P10, P20, P200 and P1000, catalog number: 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. DynaMagTM-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, NovexTM, catalog number: EI0001 )
  11. Chemidoc (Vilber Lourmat, model: Fusion Spectra )
  12. BAS 2500 (Fujifilm Corporation)
  13. Deep freezer (-80 °C freezer)
  14. PCR cycler (MyCyclerTM 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

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

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分类

微生物学 > 微生物生物化学 > RNA

生物化学 > RNA > miRNA靶标

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