alias: GSM3681103
title: GSM3681103: PD-L1 repeat 1; Homo sapiens; RIP-Seq
library_construction_protocol: Crosslinked RIP was performed as previously described with minor modification(Gilbert and Svejstrup, 2006). Briefly, cells were first fixed with 0.3% formaldehyde for 10min followed by stopping crosslinking reaction with 0.2M glycine. Then the cells were lysed with FA lysis buffer (50 mM This.HCl, pH=7.5, 140 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate) followed by sonication (10% AMP, 5s sonication, 5s pause, 10 cycles). The supernatant of the cell lysate was then mixed with antibody and protein A/G magnetic beads followed by incubation overnight at 4℃. Then the beads were washed two times of FA lysis buffer, FA500 buffer (50 mM This.HCl, pH=7.5, 500 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate), LiCl buffer (10 mM Tris HCl, pH 8, 250 mM LiCl, 0.5% (v/v) NP-40, 0.1% (w/v) sodium deoxycholate, 1 mM EDTA), and TE/100 mM NaCl buffer (10 mM Tris HCl, pH 8, 1 mM EDTA, 100 mM NaCl). The RNA was then eluted by elution buffer (100 mM Tris⋅Cl, pH 8, 10 mM EDTA, 1% (w/v) SDS) and extracted using Trizol (invitrogen). Paired-end sequencing libraries were prepared using the TruSeq Stranded Total Sample Preparation kit (Illumina) by the Mayo Clinic sequencing core facilities
identifiers.namespace: GEO
identifiers.id: GSM3681103