GSM9154203: 688M PDA cells, H3K36me2, shKdm8-2, normoxia, replicate 1; Mus musculus; ChIP-Seq
Briefly, 2.5x10^7-1x10^8 688M cells were cultured for each ChIP. To crosslink, formaldehyde solution was directly added to cell culture media to a final concentration of 1% for 10 min, followed by quenching with 125 mM glycine solution. Fixed cells were rinsed twice in ice-cold PBS and lysed in ice-cold lysis buffer (50mM HEPES-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, with protease inhibitor cocktail). Lysates were transferred to a 1.5 ml TPX tube (Diagenode) and sonicated using Bioruptor (Diagenode) three times of 10 min (output level: high, interval: 0.5). An aliquot (50 μl) of the lysates was reserved for QC and input library. For immunoprecipitation, 4mg of ChIP-grade antibodies against H3K36me2 (ab9049, abcam) or H3K27me3 (ab192985, abcam) were added to the lysate (containing ~50μg of sheared gDNA) and rotated overnight at 4°C, followed by an incubation with 25 μl of Protein A Dynabeads (Life Technology) for 2 hours at 4°C. The beads were washed three times with 800 μl ice-cold LiCl washing buffer (100 mM Tris-HCl, pH7.5, 500 mM LiCl, 1% NP-40 and 1% sodim deoxycholate) prior to elution and reverse crosslink through an incubation with 400 μl of digestion buffer (0.1 M Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, and 200 μg/ml of proteinase K) at 65°C for 2 hours with agitation every 30 min. Eluted DNA samples were purified by organic extraction and ethanol precipitation. DNA libraries were prepared using KAPA Hyperprep Kit (Roche) and sequenced on the Illumina Nova-seq X platform.