title: GSM2474166: TIA1 rep2 (CLIP-seq); Mus musculus; RIP-Seq
library_construction_protocol: For CLIP, after UV light crosslinking (400 mJ/cm2 at 254nm, Stratalinker 1800) to covalently bind proteins to RNA molecules, cells were lysed using PLX buffer (1X PBS, 0.1% SDS, 0.5% NP40, 0.5% sodium deoxycholate). Endogenous TIA1-RNA complexes were immunoprecipitated overnight at 4°C using rabbit monoclonal TIA1 antibody (clone EPR9304) or goat anti-TIA1 (Santa Cruz, C20, sc-1751 for CLIP-seq) coupled to Dynabeads® Protein G (Novex). 10% of the initial extract was kept as input. Two extensive washes were performed with RIPA-S (50 mM Tris-HCl pH 7.5, 0.1% SDS, 0.5% NP40, 1% sodium deoxycholate, 5 mM EDTA, 1 M NaCl, 2 M Urea) and PNK buffer (50 mM Tris-HCl pH 7.5, 0.5% NP40, 5 mM MgCl2). For CLIP-seq, the RNA was radioactively labelled on beads. After gel electrophoresis and nitrocellulose membrane transfer, protein-RNA complexes were visualized by autoradiogram. The RNA was recovered by proteinase K digestion. The libraries were prepared by the TruSeq Small RNA Library prep (Illumina) according to the manufacturer’s instructions. Purified libraries were sequenced using the HiSeq 2500 machine (Illumina). 50 bp reads were obtained by single-end sequencing with P5 Illumina primers.
attributes.tag: GEO Accession
attributes.value: GSM2474166