title: GSM1620002: Th2 H3K27ac ChIP-Seq; Mus musculus; ChIP-Seq
library_construction_protocol: For generating ChIP-Seq libraries, chromatin extracts were prepared by sonication of cells following crosslinking with 1% formaldehyde for 10min at room temperature. The ChIP was performed with 1 ug specific antibodies as described (Barski et al., 2007) and ChIP-Seq libraries were prepared using indexing primers as described (Hu et al., 2013a). RNA-Seq libraries using polyA RNA isolated from both the WT and KO cells were prepared as described (Chepelev et al., 2009). Murine R1 ES cells and in vitro inducted Th2 cells were cross-linked with 1% formaldehyde for 10min at room temperature. 10 X106 cells were lysed by resuspending in 10ml lysis buffer (10mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% NP40; 10μl protease inhibitors (Sigma)) and incubation at 4°C with rotation for one hour. The cells were then treated with 400 μl 1xNEB cutsmart buffer + 0.1% SDS at 65°C for 10 minutes, followed by addition of 44μl 10% Triton X-100 to quench SDS. Chromatin was subsequently digested with 20 Units CviQ I (NEB), and 20 Units CviA II (NEB) at 25°C for 20 minutes, then 20 Units Bfa I (NEB) at 37°C for 20 minutes. The reaction was stopped by washing the cells twice with 600 ul wash buffer (10mM NaCl, 1mM EDTA, 0.1% triton-100). Then the samples were processed as the Hi-C protocol (Lieberman-Aiden et al., 2009) with modifications briefly described below. The DNA ends were blunted and labeled with biotin by Klenow enzyme in the presence of dCTP, dGTP, dTTP, biotin-14-dATP, followed by 3C ligation using T4 DNA ligase. After reverse crosslinking, the DNA was fragmented to 300-500 bp by sonication with a Bioruptor sonicator (Diagenode UCD-200). The samples were treated with T4 DNA polymerase to remove biotin labels at the DNA ends. The remaining biotinylated DNA fragments were then captured using Dynabeads MyOne Streptavin C1 Beads (Invitrogen) by incubating for 30 minutes at RT with rotation. The DNA on beads was end-repaired, followed by A-addition and ligation to the Illumina Paired End Adaptors as described previously. Following PCR-amplification of the libraries, DNA fragments from 300 to 600 bp were purified from 2% agarose gel and sequenced on HiSeq2000.
attributes.tag: GEO Accession
attributes.value: GSM1620002