title: GSM6638350: GFP-only transfection_rep2; Homo sapiens; RIP-Seq
library_construction_protocol: Cells were rinsed once with ice cold PBS and 6 mL of PBS was added to each plate before crosslinking. Cells were then irradiating once with 150mJ/cm2 in a Spectroline UV Crosslinker at 254nm. Irradiated cells were scraped into Eppendorf tubes, spun at 500g for 1 minute, and snap frozen. Crosslinked cell pellets were lysed in iCLIP lysis buffer (50mM Tris-HCl pH7.4, 100mM NaCl, 1% Igepal CA-630 (Sigma I8896), 0.1% SDS, 0.5% sodium deoxycholate), sonicated with the Bioruptor Pico for 10 cycles 30 seconds ON/30 seconds OFF, and supplemented with 0.5U of RNase I per 1mg/ml lysate for RNA fragmentation. Lysates were pre-cleared by centrifugation at 21,000xG at 4oC. A mix of Protein A/G Dynabeads (50ul of each per sample, Life Technologies) were coupled to 10ug of rabbit anti-GFP antibody (Abcam ab290). TIS11B protein-RNA complexes were immunoprecipitated from 1ml of crosslinked lysate and washed with high salt and PNK buffer. RNA was repaired by 3' dephosphorylation and ligated to L3-IR adaptor (Zarnegar et al. 2016) on beads. Excess adaptor was removed by incubation with 5' deadenylase and RecJf. TIS11B protein-RNA complexes were eluted from the beads by heating at 70oC for 1 minute. The complexes were then visualized via the infrared-labelled adaptor, purified with SDS-PAGE, and transfer to nitrocellulose membrane. cDNA was synthesized with Superscript IV Reverse Transcriptase (Life Technologies) and circularized by CircLigase II. Circularized cDNA was purified with AmPURE bead-based purification (A63880, Beckman Coulter) and amplified by PCR.