title: GSM3537468: Abl B wild type biological Replicate 1; Mus musculus; RIP-Seq
library_construction_protocol: Abelson-virus transformed B cell progenitors were labeled with 500 µM 4-thiouridine (Carbosynth 13957-31-8) for 5 hours and crosslinked at a wavelength of 365 nm with a total dose of 2000 µJ/cm2 (UV Stratalinker 2400). Crosslinked cell pellets were flash frozen in liquid nitrogen and stored at -80ºC. PAR-CLIP was performed as described in Hafner et al. 2010 Cell 141, 129–141 with the following modifications. Briefly, UV-irradiated cells were lysed in NP-40 lysis buffer (50 mM HEPES-KOH at pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail). Freshly prepared lysates were incubated with protein G magnetic beads (Invitrogen 100-03D), pre-coupled to anti-mouse Ago2 antibody (Wako Chemicals 018-22021) for 1 hour at 4ºC. Beads were washed with IP wash buffer (50 mM HEPES-KOH, pH 7.5, 150 mM KCl, 0.05% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail) and subjected to RNase T1 (Fermentas EN0541) treatment (10U/µl) for 10 minutes at 37ºC. Immunoprecipitations were washed in IP wash buffer and incubated with calf intestinal phosphatase (New England BioLabs M0290), followed by radioactive end-labeling using T4 polynucleotide kinase (New England BioLabs M0201). The crosslinked protein-RNA complexes were resolved on a 4-12% NuPAGE gel (Invitrogen WG1402BOX). The SDS-PAGE gel was transferred to a nitrocellulose membrane and the protein-RNA complex migrating at the expected molecular weight was excised. RNA was isolated by Proteinase K (Roche 03115801001) treatment and phenol-chloroform extraction. Sequencing libraries were constructed as described in Hafner et al. 2010 Cell 141, 129–141.
identifiers.namespace: GEO
identifiers.id: GSM3537468