title: GSM3325742: LIN28B-Rep2-input (CLIP-seq); Homo sapiens; RIP-Seq
library_construction_protocol: NA Cells were crosslinked using 0.4 J/cm2 of 254 nm UV light, pelleted by centrifugation and washed twice with ice-cold PBS. Cell lysates were prepared as described in Hafner et al. 2010 Cell 141, 129–141. RNA was partially digested using RNase I, followed by addition of RNase inhibitor and DNA digestion. Insoluble material was removed by centrifugation and lysate was pre-cleared by incubating with Protein A/G magnetic beads. We added the pre-cleared lysates to antibody-coupled beads and incubated overnight at 4 °C. The following day, we washed the beads 4 times in IP wash buffer (50 mM HEPES pH 7.5, 150 mM KCl, 0.5% (v/v) NP40, 0.25 mM DTT, complete EDTA-free protease inhibitor cocktail). Protein-RNA complexes were resolved by SDS-PAGE, followed by transfer to a nitrocellulose membrane. RNA was isolated by proteinase K treatment and phenol-chloroform extraction. We constructed RNA sequencing libraries using the SMARTer smRNA-Seq Kit (Clontech) by following the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM3325742