title: GSM8577588: UV-crosslinked replicate 2; Homo sapiens; RIP-Seq
library_construction_protocol: Cells were lysed in iCLIP lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1% Igepal, 0.1% SDS, 0.5% sodium deoxycholate and complete protease inhibitor cocktail), sonicated using a bioruptor (Digenode) for 3 cycles with alternating 30 sec on/off at high intensity, and cleared by centrifugation (10 min, 15000 rpm, 4ºC). eIF2A was immunoprecipitated by incubation with a-eIF2A coupled to Dynabeads A for 2h at 4ºC. Beads were washed twice with High Salt wash buffer (50 mM Tris-HCl, 1M NaCl, 1 mM EDTA, 1% Igepal, 0.1% SDS and 0.5% sodium deoxycholate), once with PNK wash buffer (20 mM Tris-HCl, 10 mM MgCl2, 0.2% Tween-20) and resuspended in PNK buffer pH 6.5 (350 mM Tris-HCl pH 6.5, 40 mM MgCl2, 4 mM DTT) containing 10 U PNK enzyme (NEB, #M0201L), 0.5 µl RNasin (Promega, #N2111) and 0.5 µl of FastAP alkaline phosphatase (ThermoFisher, #EF0654), and subsequently incubated for 40 min at 37ºC with shaking (1100 rpm) to phosphorylate the 3'end. After washing with ligation buffer (50 mM Tris-HCl, 10 mM MgCl2) 3'-end adaptor ligation was performed by resuspending the beads in 25 µl of ligation mix (50 mM Tris-HCl, 10 mM MgCl2, 0.8 µl DMSO, 2.5 µl T4 RNA ligase I – high concentration (NEB, #M0437M), 0.4 µl RNasin, 0.5 µl PNK (NEB, #M0201L), 9 µl 50% PEG800) containing 2.5 µl of 1 µM preadenylated adaptor L3-App (/5Phos/AG ATC GGA AGA GCG GTT CAG AAA AAA AAA AAA/iAzideN/AA AAA AAA AAA A/3Bio/) which had been previously conjugated to an infra-red-dye (IRdye-800CW-DBCO (Licor)). This mixture was incubated for 75 min at RT with flickering every 10 min. Beads were washed twice with High Salt buffer and once with PNK buffer prior to adding loading buffer and being resolved on a NuPage 4-12% BisTris Gel, from which they were transferred to a nitrocellulose membrane. Protein-infrared-RNA complexes were visualized using an Odyssey machine, isolated, and RNA was extracted with phenol-chloroform and precipitated in ethanol. The RNA pellet was resuspended in 7 µl RNA/primer mix (5.5 µl water, 0.5 µl 10 mM dNTP mix, 1 µl of irCLIP_ddRT primer) (table S8). RNA was reverse-transcribed, cDNA gel-size selected, isolated, circularized and amplified iCLIP