GSM6784482: FL bio rep 1; Homo sapiens; RIP-Seq
Cells were treated with 800 mM 4sU 16 hours before crosslinking by 312 nm ultraviolet exposure using Stratagene Stratalinker UV 1800. Crosslinked cells were lysed in a native lysis buffer with 50 mM HEPES, pH 7.5, 150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.1% (v/v) Nonidet P-40, 1 mM DTT, 25 ml/ml protease inhibitor cocktail for mammalian tissues (Sigma-Aldrich), and 1U/ml RNase T1. Immunoprecipitation was performed using anti-DYKDDDDK magnetic agarose (ThermoFisher) followed by on-bead RNA digestion with 5U/ml RNase T1, on-bead dephosphorylation with calf intestinal alkaline phosphatase (CIP), and on-bead phosphorylation with T4 PNK and 32[P]-ATP or non-radioactive ATP. Radio-labeled RNA-protein complexes were extracted from beads by boiling in 70 ml of SDS loading buffer (Bio-Rad), separated on a 4-12% Bis-Tris gel, and imaged with phosphorimager. Bands corresponding to the protein size were cut off from the gel, subjected to proteinase K digestion and RNA purification by phenol-chloroform. The purified non-radioactive RNAs were run on 15% denaturing polyacrylamide gel and purified for deep sequencing. Sequencing libraries were constructed using the NEBNext® Small RNA Library Prep Set following the manufacturer's instructions. PAR-CLIP-seq