GSM7142980: QKIpan-NM, monomer, rep2; Homo sapiens; RIP-Seq
Cells grown to 90% confluency in 100mm dishes were rinsed with ice-cold PBS and UV irradiated with 600 mJ/cm2, 254 nm, in ice-cold PBS using a Stratalinker 1800. Cells were collected by scraping & centrifugation and stored at -80˚C until use. Cell pellets were lysed resuspended using 200 µl of 1 X QLB (1 X PBS, 0.3% SDS, 0.5% deoxycholate, 0.5% Igepal, EDTA-free Complete protease inhibitor cocktail (PIC; Roche, 11873580001) for 15 min, followed by addition of 400 µl of 1 X QDB (1 X PBS, 0.5% deoxycholate, 0.5% Igepal, PIC). Rep1 libraries were constructed using NEBNext Multiplex small RNA library prep kit for Illumina with substitution of the 3' adapter with 3'SRdeg_DNA; /5rApp/GTACNNNNNNNNAGATCGGAAGAGCACACGTCT/3ddC/. Rep 2 libraries were constructed using a protocol modified from eCLIP (Van Nostrand et al Nat Methods, 2016) using custom adapters: 3' adapter 3'SRdeg_DNA, RT primer SR-RT; 5'-AGACGTGTGCTCTTCCGATCT-3', 5' adapter 5'SR_DNA; 5'P-GATCGTCGGACTGTAGAACTCTGAAC-3'SpC3/. PCR amplification with a common forward primer (NEBNext SR primer for Illumina) and barcoded reverse primers for each sample (NEBNext Index primers for Illumina). CLIP-seq