GSM8691752: RNA-seq_HIGM1_patient_2; Homo sapiens; RNA-Seq
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 manufacture'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 100000000 isolated B cells were irradiated at 400mJ/cm2 with 254nm UV light. The crosslinked cells were lysed and 15ug of BCAS2 (Abmart, Catalog # PA2466S) 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 BCAS2 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. RNA-seq was performed as follows. Total RNA of B cells was extracted by Trizol while RNA from human peripheral blood mononuclear cells was extracted by using RNA extraction kit (Tiangen, Catalog # A0801A). mRNA from B cells and peripheral blood mononuclear cells was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, a transcriptome sequencing library was established. Utilizing a TruSeq PE Cluster kit v3-cBot-HS (Illumina) on a cBot Cluster Generation System, index-coded samples were clustered in strict accordance with the manufacturer's guidelines. Upon completion of the clustering process, the library preparations were subjected to sequencing on the Illumina NovaSeq platform, generating 150 bp paired-end reads. Rigorous quality control measures were implemented post-sequencing to ensure that downstream analyses were performed exclusively on pristine, high-quality data.