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^TM, catalog number: SH30022.FS )
    
12. Fetal bovine serum (FBS) (Enhance growth of cells with U.S. sourced GE Healthcare HyClone characterized) (GE Healthcare, HyClone^TM, catalog number: SH30071.03HI )
    
13. GlutaMAX-I (100x) (100 ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050-061 )
    
14. Penicillin/Streptomycin solution (P/S) (100x, 10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, 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, Invitrogen^TM, catalog number: 11031 )
    
18. Dynabeads^® Pan Mouse IgG (Thermo Fisher Scientific, Invitrogen^TM, 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^TM, 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^TM, catalog number: NP0007 )
    
31. NuPAGE^® Sample Reducing Agent (10x) (Thermo Fisher Scientific, Novex^TM, catalog number: NP0004 )
    
32. NuPAGE^® Antioxidant (Thermo Fisher Scientific, Novex^TM, catalog number: NP0005 )
    
33. NuPAGE^TM Novex^TM 4-12% Bis-Tris Protein Gels, 1.0 mm, 10-well (Thermo Fisher Scientific, Invitrogen^TM, catalog number: NP0321BOX )
    
34. NuPAGE^® MOPS SDS Running Buffer (20x) (Thermo Fisher Scientific, Novex^TM, catalog number: NP0001 )
    
35. NuPAGE^® Transfer Buffer (20x) (Thermo Fisher Scientific, Novex^TM, 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^TM 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^TM, catalog number: AM9720 )
    
42. Linear acrylamide (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9520 )
    
43. 3 M Sodium acetate (pH 5.5) (Thermo Fisher Scientific, Ambion^TM, catalog number: AM9740 )
    
44. Ethanol (Merck Millipore Corporation, catalog number: 100983 )
    
45. Isopropanol (2-propanol) (Merck Millipore Corporation, catalog number: 109634 )
    
46. UltraPure^TM DNase/RNase-Free Distilled Water (D.W.) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10977-015 )
    
47. 2x RNA loading buffer (Thermo Fisher Scientific, Ambion^TM, catalog number: AM8546G )
    
48. mirVana^TM Probe & Marker Kit (Thermo Fisher Scientific, Ambion^TM, catalog number: AM1554 )
    
49. 10% Novex^® TBE-Urea Gels, 10 well (Thermo Fisher Scientific, Invitrogen^TM, 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^TM, catalog number: F-530L )
    
57. dNTP mixture (Enzynomics, catalog number: N001L )
    
58. SuperScript^TM III reverse transcriptase (RT) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 18080-044 )
    
59. Novex^® TBE Gels, 6%, 10 Well (Thermo Fisher Scientific, Invitrogen^TM, 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^TM Kit (Thermo Fisher Scientific, Ambion^TM, 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^TM, catalog number: 15596018 )
    
84. Dynabeads^® mRNA DIRECT^TM Purification Kit (Thermo Fisher Scientific, Ambion^TM, 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^TM 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. DynaMag^TM-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^TM, 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^TM 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