title: GSM4261054: CLIP_1; Homo sapiens; OTHER
library_construction_protocol: Flag-tagged cells were collected and crosslinked with 254 nm UV-crosslinking at 400 mJ/cm2 and 200mJ/cm2 on ice in a Stratalinker 1800 (Stratagene). Lysates were treated with Rnase T1 and protein-RNA complexes were isolated with antibody. Antibody-bound proteins and RNA-protein complex were isolated by magnetic stand and wash with wash buffer (1xPBS, 0.1% SDC, 0.5% deoxycholate, 0.5% NP-40) and high-salt-wash buffer (5xPBS, 0.1% SDC, 0.5% deoxycholate, 0.5% NP-40). Purified RNA fragments were treated with CIP (Takara) and ligated with 3' linker adaptor by using T4 RNA ligase (thermofisher) at 16°C for overnight. Treated RNA fragments were labelled with 32p-γATP, followed by electrophoresis with SDS-PAGE (NuPAGE, thermofisher) and transferred to nitrocellurose membrane. RI-labelled RNAs were detected by autoradiography. Appropriate band or smear-band were picked up from membrane and treated with ProK (Roche).  RNA fragments were purified by acid phenol - chloroform extraction (thermofisher scientific). Purified TruB1-bound RNA fragments were ligated with 5′ linker RNA, followed by the reverse-transcriptase reaction using RT primers and SuperScriptIII (thermofisher scientific). cDNA libraries were amplified by means of Phusion High-Fidelity DNA Polymerase (New England Biolabs).
attributes.tag: GEO Accession
attributes.value: GSM4261054