GSM5743370: ACT_REP1_L001; Homo sapiens; OTHER
The cells were pelleted by centrifugation at 100 x g for 15 min at 4 °C, and the pellet was resuspended in three volumes of NP40 lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% Nonidet P-40 alternative, 0.5 mM dithiothreitol (DTT), 1 Complete Mini EDTA-free Protease Inhibitor Cocktail tablet (Roche)). The cell suspension was then incubated on ice for 10 min, passed through an 18G needle five times, and centrifuged at 13,000 x g for 15 min at 4 °C and RNAs were lightly digested by treatment with MNase (Thermo Scientific) at a final concentration of 0.05 U μl-1 for 20 min at 16 °C. For each PAR-CLIP assay 1000 μL of Dynabeads (Invitrogen) and 800 μL of anti-EIF3B antibody (Bethyl A301-761A) were used. The remaining steps of the PAR-CLIP analysis were performed exactly as described in (Pubmed ID: 25849773, 26463383) with the exception of using MNase at 5 U μL-1 for the on-bead digestion step. PAR-CLIP cDNA libraries were sequenced on an Illumina HiSeq 2500 platform. To eliminate potential PCR biases during PAR-CLIP library preparation, a random bar code was introduced into the 3' adapter and all the reads that matched the random barcode were collapsed into single reads.