GSM5592067: MesHMLE_HITS-CLIP_sRNA_replicate_5; Homo sapiens; OTHER
Cells were suspended in ice-cold PBS before UV crosslinking at 254 nM (Stratalinker). Cell pellets were then lysed on ice for 10 min using lysis buffer containing 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40 and protease inhibitors (Roche) followed by RQ1 DNase (Promega) treatment at 37C for 15 min on shaker. RNase A/T1 (Ambion) was then added for a further 8 min flowed by addition of 30 mM ethylenediaminetetraacetic acid (EDTA) and a centrifuge at 30000 rpm. The lysate was then subjected to immunoprecipitation for 2 h with a pan-anti-Ago antibody (2A8) conjugated to protein-A dynabeads (Invitrogen) using bridging rabbit anti-mouse IgG (Jackson Immunoblots). After washing the pellets (0.1% SDS, 0.5% deoxycholate, 0.5% NP40 in 1 × PBS; 0.1% SDS, 0.5% deoxycholate, 0.5% NP40 in 5xPBS; 50 mM Tris pH 7.5, 10 mM MgCl2, 0.5% NP40), on-bead phosphatase treatment performed for 30 min with Antarctic phosphatase (New England Biolabs) and SUPERaseIn RNase inhibitor (Ambion). After labelling the 39 RNA linker (CAGACGACGAGCGGG) with P32 using T4-PNK (NEB), it was ligated on beads for 1 h at 16C using T4 Ligase (Fermentas). Following a wash, beads were treated with PNK to ligate the 59 RNA linker (AGGGAGGACGAUGCGGxxxG, with ''X'' representing different nucleotides for barcoding) and resuspended in 4x LDS Novex loading buffer with 4% B-mercaptoethanol, incubated at 70uC for 10 mins and the supernatant loaded on Novex NuPAGE 4-12% Bis-Tris acrylamide gels (Biorad). After the gel run, the Ago-RNA complexes were then transferred to nitrocellulose and exposed to film at 280uC for 3 days. Complexes at 110 kDa were excited and resuspended in a solution containing 100 mM Tris pH 7.5, 50 mM NaCl, 10 mM EDTA and 4 mg/ml proteinase K for 20 min at 37C. After adding 3.5 M urea the samples were incubated for 20 min and RNA was isolated by a phenol-chloroform extraction method. Samples were then run on a 10% denaturing (1:19) polyacrylamide gel and exposed to film with an intensifying screen at 280C for 5 d. The band corresponding to miRNAs was excised, crushed and eluted at 37C for 1 h in elution buffer containing 1 M NaOAc, pH 5.2 and 1 mM EDTA. After overnight ethanol precipitation, RNA was centrifuged, dried and resuspended in 8 mL RNase-free H2O. First primer added (5'-TCCCGCTCGTCGTCTG-3') and reverse transcription performed using SuperScriptIII (Invitrogen) and then PCR was performed for 25 cycles by adding the second primer (5'-ACGGAGGACGATGCGG-3'). PCR products were then run on a 10% native (1:29) polyacrylamide gel, stained with Sybr Gold (Qiagen) and bands excised over a UV light box. After DNA precipitation using with isopropanol, additional 10 cycle PCR was performed by adding 2 primers :5'-AATGATACGGCGACCACCGACTATGGATACTTAGTCAGGGAGGACGATGCGG-3' and 5'-CAAGCAGAAGACGGCATACGATCCCGCTCGTCGTCTG-3'. After running products on a 2% metaphor agarose/TBE gels and bands at 115 bp excised corresponding to the linker sequence + miRNA CLIP tag. Finally, samples were purified using quick-spin columns (Qiagen).