title: GSM4998032: ChIP-Seq_H3K27ac_Arid1a_Homo_Deletion_1; Mus musculus; ChIP-Seq
library_construction_protocol: Fresh epithelial pellets were fixed in 1% PFA for 10 minutes at room temperature, quenched with 1/20th volume of 2.5 M glycine for 5 minutes, washed twice with cold 1x PBS and flash frozen. When enough sample was acquired (approximately 0.1g of tissue per region), fixed pellets were pooled, dounced in cold PBS with protease inhibitor, and filtered through a 70 µm mesh. Samples were then incubated in cold cell membrane and nuclear lysis buffers containing non-denaturating detergents for 30 mins each on a shaker at 4˚C. Washed samples were pelleted and resuspend in 300µl of sonication buffer containing 0.1% SDS. Sonication was conducted using the Diagenode bioruptor (Diagenode, B01020001) at high power for 30 cycles, 30 seconds ON/OFF. Sonicated samples were cleared with 1:10 dilution of 30% Triton-X and added to antibody bound beads (rabbit anti-H3K27ac: abcam, ab4729). The next day, beads were washed with high and low salt buffers and treated with Proteinase K overnight at 65˚C. DNA was isolated using phenol/chloroform extraction and 10ng of immunoprecipitated DNA was used for cDNA library construction using the Rubicon DNA-seq Thruplex 48S kit. Libraries were size selected and submitted to be sequenced at 50 base pair read length, single end reads with 15-20 million reads per library. ThruPLEX DNA-Seq kit