alias: GSM3227974
title: GSM3227974: CS2: ChIP_seq_RPA_aB_28h_HU_28h_10mM_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: 20 million fixed B-cells were washed twice with cold PBS and pellets were snap frozen in dry ice and stored at −80°C. Fixed cell pellets of 20 million cells were thawed on ice and resuspended in 1 mL of cold RIPA buffer (10 mM TrisHCl pH 7.5, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1 × Complete Mini EDTA free proteinase inhibitor (Roche)). Sonication was performed using the Covaris S220 sonicator at duty cycle 20%, peak incident power 175, cycle/burst 200 for 30 min. Chromatin were clarified by centrifugation at 21,000 g at 4°C for 10 min and precleared with 80 μL prewashed Dynabeads protein A (ThermoFisher) for 30 min at 4°C. 40 μL prewashed Dynabeads protein A were incubated with 10 μg of Anti-RPA32/RPA2 antibody (Abcam, ab10359) in 100 μL of PBS for 20 min at room temperature in continuous mixing, washed twice in PBS for 5 min and added to 1 mL of chromatin followed by overnight incubation at 4°C on a rotator. Beads were then collected in a magnetic separator (DynaMag-2 Invitrogen), washed twice with cold RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (10 mM Tris pH 8.0, 1mM EDTA) plus 0.2% Triton X-100, and once with TE. Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1mg/ml of Proteinase K (Ambion). DNA was purified by standard phenol-chloroform method and eluted in 20 μl. 100ng ChIP DNA was used to prepare Illumina sequencing libraries. libraries were prepared and sequenced following standard Illumina protocols
identifiers.namespace: GEO
identifiers.id: GSM3227974