GSM9229880: AGO HITS-CLIP of MDA-MB-231HM cells with miR-342-3p rep 1; Homo sapiens; RIP-Seq
After 48 hours, cells were cross-linked using a UV Stratalinker-1800 (Agilent) and lysed in 500 mL of 1 X PXL (1 X PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% Igepal) + EDTA-free Complete protease inhibitor cocktail (Roche). Following DNA and partial RNA digestion, AGO-RNA complexes were immunoprecipitated using mouse IgA2 monoclonal anti-Ago2 antibody 4F970 conjugated to protein L Dynabeads (ThermoFisher) with a mouse IgA antibody (GeneTex S107) used as a control. AGO-RNA complexes were ligated with RNA linkers on-bead, labelled with 32P-ATP, eluted in 1 X Bolt LDS sample buffer (ThermoFisher) + 1% β-mercaptoethanol. 3' adapter NEBNext 3'SR adaptor for Illumina; /5rApp/AGA TCG GAA GAG CAC ACG TCT /3AmMO/. 5' adapter; /5AmMC6/GUUCAGAGUUCUACAGUCCGACGAUCNNNNNNNNNN. Ago-RNA complexes were separated on Bolt 8% Bis-tris Plus gels (ThermoFisher) then transferred to nitrocellulose and 120-180 kDa regions (corresponding to RNA tags 30-70 nt) were excised followed by proteinase K digestion and RNA extraction using acid phenol (ThermoFisher) with 1:1 isopropanol:ethanol precipitation. AGO bound RNA was pelleted by centrifugation then separated on a 15% denaturing polyacrylamide gel where gel slices corresponding to the expected size of the cross-linked RNA were cut out and eluted by the ''crush and soak'' method. Reverse transcription and amplification were performed essentially as previously described (Ngo et al 2024) using SR-RT primer for reverse transcription (IDT, AGACGTGTGCTCTTCCGATCT) with SuperScript IV. Products were amplified using a common forward primer (NEBNext SR primer for Illumina) and barcoded reverse primers for each sample (NEBNext Index primers for Illumina). PCR products were purified using 1.8 volumes of Axygen AxyPrep magneticbeads (MAG-PCR-CL), separated on an 8% acrylamide (29:1) TBE non- denaturing gel, stained with SYBR Gold nucleic acid gel stain (ThermoFisher) and imaged on a ChemiDoc (BioRad). Products corresponding to an insert size of ~30 – 70 nt were excised from the gel and extracted by the “crush and soak” method as previously described (Jensen and Darnell 2006). Library quality and quantity was assessed by Bioanalyzer (Agilent) and qPCR, pooled and sequenced on an Illumina NextSeq 500 (1 x 75bp).