title: GSM2113569: Rag-/- pro-B nuclear RNA-seq Rep1; Mus musculus; RNA-Seq
library_construction_protocol: ChIP-seq: ChIP was performed as described (Schoenfelder et al., 2010), using an antibody against histone H3K4me3 (Ab8580, Abcam). Cross links were reversed with 200 mM NaCl, 12.5 mM EDTA, 50 mM NaHCO3, 0.5% SDS, 100 _g/ml proteinase K overnight at 65°C, and ChIP DNA was purified by PCI extraction and isopropanol precipitation. RNA-seq: Nuclei were obtained from 2-5 x 10(6) flow-sorted Rag1-/- pro-B cells (B220+CD19+CD43+) by incubation in 5 ml 50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.4% NP40, 5 min on ice. RNA was isolated with a RNeasy mini kit (Qiagen) and treated with Turbo DNase (Ambion). VDJ-seq:  Genomic DNA was isolated using a Qiagen Dneasy blood and tissue kit ChIP-seq: Paired end ChIP-seq libraries were prepared according to standard Illumina ChIP-seq library protocols. RNA-seq: Paired-end strand-specific RNA-seq libraries for Illumina sequencing were generated as described (Parkhomchuk et al., 2009) except polyA+ RNA selection was omitted, first strand cDNA synthesis was performed with solely random hexamer primers, and double-stranded cDNA was fragmented with a Diagenode Bioruptor. VDJ-seq: For each sample 10mg of DNA were sonicated to 500bp using a Covaris E220 sonicator using recommended settings then end-repaired and A-tailed using standard protocols and a short asymmetric adaptor ligated to both fragment ends. DNA was cleaned with a PCR cleanup kit (Qiagen) after end-repair, following A-tailing and following adaptor ligation. Biotinylated primers located in JH intergenic regions were then used in primer extension reactions (8 x 50_l) using Vent Exo- polymerase (2 units per tube; NEB) followed by purification with a PCR cleanup kit. Due to the placement of these primers and the fragment size of the sonicated DNA, primer extension of unrecombined JH segments would be favoured over that of DJH or VDJH recombined segments. Primer-extended sequences (enriched for unrecombined JH intergenic sequences) were then removed using streptavidin beads (My-one C1; Invitrogen) following the manufacturers protocol with incubation for 4 hours at room temperature (20 _l beads per sample). Following a further cleanup, a second primer extension (6 x 50 _l reactions, 2 units per tube) was performed using biotinylated reverse primers located immediately downstream of each JH gene. Streptavidin beads (20_l) were again used to isolate primer extended JH-specific products by incubation overnight with rotation. Illumina PE1 primers corresponding to the long strand of the asymmetric adaptor and JH-specific PE2 primers were then used to amplify the library off the beads in 15 cycle PCR reactions (4 x 25_l) using Pwo master mix (Roche). Following 1x size selection (to remove small products) and cleanup using AMPure XP beads (Beckman Coulter), a second round 5 cycle PCR was performed to add the remainder of the Illumina PE1 and PE2 adaptors, incorporating Truseq bar codes at the PE2 end, followed by a second 1x size selection/cleanup with AMpure XP beads. If necessary, a 'double-sided' Ampure XP size selection (0.5x followed by 1x) was used to remove a low quantity of library products >1kb as these are too large for efficient cluster generation in Illumina sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2113569