GSM3956439
GSM3956439: H3K27ac ChIP-Seq in Mouse E16 Forestomach; Mus musculus; ChIP-Seq
After isolation of embryonic epithelium using dispase for forestomach and EDTA for the hindstomach, intestine and colon, epithelial pellets were fixed in 1% PFA for 10 minutes at room temperature, quenched with 1/20th volume of 2.5M 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 70µm 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 then 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 40 cycles, 30s ON/OFF. Sonicated samples were cleared with 1:10 dilution of 30% Triton-X and added to antibody bound beads. 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
GEO
GSM3956439
GEO Accession
GSM3956439