title: GSM2906319: GRO-seq WT rep2; Mus musculus; RNA-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 100000000 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. GRO-seq was performed as previously described (Core et al., 2008). Briefly, Splenic B cells from 8-week-old ROD1 WT and KO mice were ex vivo cultured and stimulated by LPS for 3 days. Nuclei was isolated with ice cold swelling buffer (10 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 3 mM CaCl2) and stored in freezing Buffer (40% Glycerol, 5 mM MgCl2, 0.1 mM EDTA, 50 mM Tris-HCl, pH8.3, 2U/ml RNaseOut). 5 x 10^7 nuclei were mixed with an equal volume of reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 200 U/ml RNaseOut, 1% sarkosyl, 500 uM ATP, GTP, and Br-UTP and 2 µM CTP) and incubated at 30°C for 5 min. The RNA was extracted by Phenol:chloroform and precipitated by ethanol. The pellet resuspended in 20 ul of DEPC-treated water. RNA was then hydrolysed and immunopurified with anti-BrdU beads (Santa Cruz Biotech). The BrdU enriched RNA was polyA-tailed by using a E.coli Poly (A) polymerase and then reverse transcribed, after circulation and relinerization by APE1 (15 U; M0282S, NEB), the cDNA was PCR amplified and gel purified for Illumina HiSeq 2500.
identifiers.namespace: GEO
identifiers.id: GSM2906319