GSM1694273: LARP4B_PARCLIP; Homo sapiens; OTHER
PAR-CLIP cDNA and small RNA cDNA preparation were prepared basically as described in Hafner et al. Cell 141, 129–141. Briefly, PAR-CLIP was performed as described (Hafner et al., 2010). UV-irradiated cells were lysed in NP-40 lysis buffer (50 mM HEPES-KOH pH 7.4, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail (Roche)). After treatment with RNaseT1 (Fermentas) at a final concentration of 2 U/ul for 7 min, immunoprecipitation was carried out with Anti-Flag Magnetic beads (SIGMA) for 1 h at 4ºC. Following an additional RNaseT1 treatment (final concentration 2 U/ul) for 7 min the beads were incubated with calf intestinal phosphatase (NEB) and RNA fragments were radioactively end-labeled using T4 polynucleotide kinase (Fermentas) and γ-(32P)-ATP. The crosslinked protein-RNA complexes were resolved on a 4-12% NuPAGE gel (Invitrogen). The SDS-PAGE gel was transferred to a nitrocellulose membrane (Whatman) and the protein-RNA complex migrating at an expected molecular weight was excised. RNA was isolated by Proteinase K (Roche) treatment and phenol-chloroform extraction. In subsequent steps barcoded 3’ adapters (NBC6 AppTCTCCATTCGTATGCCGTCTTCTGCTTG-InvdT for LARP4B_4SU_1, NBC8 AppTCTCTGCTCGTATGCCGTCTTCTGCTTG-InvdT for LARP4B_4SU_2) and 5’ adapters (OR5 GUUCAGAGUUCUACAGUCCGACGAUC for LARP4B_4SU_1, OR5-NN GUUCAGAGUUCUACAGUCCGACGAUCNN for LARP4B_4SU_2)  were ligated to the isolated RNA. Adapter-ligated RNA was reverse transcribed and PCR-amplified. The amplified cDNA was sequenced on a HighSeq2000 (Illumina) with a 1x51 nt cycle. PAR-CLIP
GEO Accession
GSM1694273