title: GSM2480488: AID CLIP-seq rep1; Mus musculus; RIP-Seq
library_construction_protocol: The spleen was isolated from 8-week-old mice and disrupted in PBS containing 2% fetal bovine serum. Remove aggregates and debris by passing cell suspension through a 70 μm mesh nylon strainer. The untouched and highly purified B cells were isolated from mouse splenocytes by immunomagnetic negative selection following the manufacturer's protocol for EasyStepTM mouse B cell isolate kit (Stem cell, Catalog # 19754). CLIP-seq was performed as previously described (Xue et al., 2009). Briefly, the 10000000 isolated B cells were irradiated at 400mJ/cm2 with 254nm UV light. The crosslinked cells were lysed and 15ug of ROD1/PTBP3 (Abnova, Catalog # H00009991-M01) or AID (Life Technologies, Catalog # 392500) specific antibody was applied to pull-down protein-RNA complexes. After micrococcal nuclease treatment, 3' RNA linker ligation and 5' end 32P labeling, the immunoprecipitated complexes were fractionated on 10% NuPAGE bis-tris gel and transferred to nitrocellulose membrane. The auto-radiographed ROD1/PTBP3 or AID specific smear bands were cut and treated with Proteinase K to extract respective RNA by phenol and chloroform. The isolated RNA was then added a 5' RNA linker and reverse transcribed by superscript reverse transcriptase III (Life Technologies, catalog# 18080-051). The PCR products were sequenced with Illumina Hiseq 2500 platform.
identifiers.namespace: GEO
identifiers.id: GSM2480488