No Transfection Control MDA-MB-231
MDA‐MB‐231 cells were grown in 20 × 10 cm dishes and transfected with 60 nM miRNA mimic (Ambion or GenePharma) using HiPerfect transfection reagent (Qiagen). After 24 h, cells were suspended in ice‐cold PBS by scraping and subjected to UV cross‐linking at 254 nm (Stratalinker). Cell pellets were lysed (0.1% SDS, 0.5% deoxycholate, 0.5% NP‐40 with protease inhibitors, Roche) for 10 mins on ice followed by RQ1 DNase (Promega) at 37°C for 15 min with shaking. RNAse A/T1 (Ambion) was then added for further 8 min, prior to the addition of EDTA (30 mM). Pellets were then spun (92,000 g) and the lysate subjected to immunoprecipitation for 2 h with a pan‐anti‐Ago antibody (2A8, kind gift of Zissimos Mourelatos) conjugated to protein‐A dynabeads (Invitrogen) using bridging rabbit anti‐mouse IgG (Jackson Immunolabs). Pellets were then successively washed (0.1% SDS, 0.5% deoxycholate, 0.5% NP‐40 in 1× PBS; 0.1% SDS, 0.5% deoxycholate, 0.5% NP‐40 in 5× PBS; 50 mM Tris pH 7.5, 10 mM MgCl2, 0.5% NP‐40), and on‐bead phosphatase treatment performed for 30 min with antarctic phosphatase (New England Biolabs) in the presence of superasin RNAse inhibitor (Ambion). The 3′ RNA linker (CAGACGACGAGCGGG) was labeled with P32 using T4‐PNK (NEB) and ligated on‐bead for 1 h at 16°C with T4 RNA ligase (Fermentas). Beads were then washed as previous and treated with PNK to ligate the 5′ RNA linker (AGGGAGGACGAUGCGGxxxG, with ‘X’ representing different nucleotides for barcoding). Beads were resuspended in 4× LDS Novex loading buffer with 4% 2‐mercaptoethanol, incubated at 70°C for 10 min and the supernatant loaded on Novex NuPAGE 4–12% Bis‐Tris acrylamide gels (Bio‐Rad). After running, the Ago–RNA complexes were then transferred to nitrocellulose and exposed to film at −80°C for 3 days. Complexes running at ~110 kDa (AGO + miRNA) and ~130 kDa (AGO + mRNA fragments) were then excised with a scalpel and resuspended (100 mM Tris pH 7.5, 50 mM NaCl, 10 mM EDTA, 4 mg/ml proteinase K) for 20 min at 37°C. The sample was incubated for an additional 20 min in the presence of 3.5 M urea and RNA isolated by a phenol–chloroform extraction. Sample was then run on a 10% denaturing (1:19) polyacrylamide gel and exposed to film with an intensifying screen at −80°C for 5 days. Thin bands corresponding to the Ago–miRNA (~110 kD) and AGO mRNA fragments (~130 kDa) were excised, crushed, and eluted at 37°C for 1 h (1 M NaOAc, pH 5.2, 1 mM EDTA). RNA was then precipitated overnight with ethanol, centrifuged, and dried. RNA was then resuspended in 8 μl H2O, primer added (TCCCGCTCGTCGTCTG) and reverse transcription performed using SuperScriptIII (Invitrogen). PCR was then performed with the above primer and an additional primer (ACGGAGGACGATGCGG) for 25 cycles. PCR product was then run on a 10% native (1:29) polyacrylamide gel, stained with Sybr Gold (Qiagen) and bands excised over a UV light box. The DNA was then precipitated using isopropanol and a final 10 cycle PCR performed with the following primers: AATGATACGGCGACCACCGACTATGGATACTTAGTCAGGGAGGACGATGCGG, CAAGCAGAAGACGGCATACGATCCCGCTCGTCGTCTG. Reactions were then run on 2% metaphor agarose/TBE gels and bands (~115 and 135 bp) excised corresponding to the linker sequence + RNA CLIP tag. Samples were finally purified using quick‐spin columns (Qiagen) and subjected to Illumina GAII 35‐bp read‐length deep sequencing (GeneWorks).