title: GSM2810037: Murine_Bcells_Activated_DMSO_24h_2449; Mus musculus; RNA-Seq
library_construction_protocol: ChIP assays were performed with activated B cells by  fixing for 10 min at 25 °C with 11% formaldehyde followed by chromatin isolation and shearing  by sonication for 7 min (10% duty cycle, 175 pulses and 200 bursts; Covaris). 5 or 10 μg  chromatin was subjected to immunoprecipitation with a-H3K27Ac (1 μg; Diagenode), a-  NFATc1 (1 μg, 7A6; Santa Cruz Biotechnology), a-NFATc2 (1 μg, 4G6-G5; Santa Cruz  Biotechnology), a-IRF4 (1 μg, M-17; Santa Cruz Biotechnology) or a-IRF8 (2 μg, C-19; Santa  Cruz Biotechnology).   After washing, elution, and reversal of cross-links, DNA was isolated by  phenol/chloroform extraction and used in PCR reactions that were performed with the primers  provided in the Extended Data For ChIP-seq analyses, activated B cells (24 h) were  transduced with retroviral vectors as described above. Cells (48 h after transduction) were  crosslinked with formaldehyde as described above and chromatin was subjected to  immunoprecipitation with a-HA (1 μg, 12CA5; Roche). For ChIP-seq analysis with hCD4 T  cells a-CD3 and a-CD28 beads were used for activation. Cross-linked chromatin was  immunoprecipitated with a-NFATc1, a-NFATc2 (1 μg, 25A10.D6.D2, ThermoFIscher  Scientific) or a-IRF4 using IP-STAR robot (Diagenode). DNA libraries were prepared with a ThruPLEX-FD Prep Kit (Rubicon  Genomics) or using chipmentation aproach. The amplified products were purified with AMPure XP beads (Beckman Coulter)  and subjected to single or paired end-sequencing on an Illumina HiSeq2500 machine. Each sample was  used to generate ~1 X 10^7 reads. RNA-seq libraries were generated from 300 ng total RNA with an  RNA Sample Prep Kit (Illumina) according to the manufacturer's protocol. The libraries were  subjected to paired-end sequencingat CCHMC DNA sequencing core using . Each sample was used to generate ~2 X 10^7 reads. RNA  libraries for single read sequencing were prepared by using standard Illumina protocols. ChIP libraries were prepared by using ThruPLEX DNA kit (Rubicon) or by chipmentation.
attributes.tag: GEO Accession
attributes.value: GSM2810037