GSM6912005: P42_10wks_M_Input; Homo sapiens; ChIP-Seq
Frozen tissue was pulverized using a liquid nitrogen-chilled hammer mill, crosslinked with 1% formaldehyde in a volume of 10mL, quenched by addition of 5mL 2.5 M   glycine for 5 min at 4°C, washed generously in ice-cold PBS, and homogenized in Farnham Lysis Buffer [5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40, 1 mM DTT, 1x cOmplete protease inhibitor cocktail (Roche, 11697498001)], as described (Murakami et al., 2017).  The nuclei were collected by brief centrifugation and resuspended in SDS Lysis Buffer (50 mM Tris-HCl pH 7.9, 1% SDS, 10 mM EDTA, 1 mM DTT, 1x cOmplete protease inhibitor cocktail) by pipetting and incubating on ice for 10 min. The chromatin was sheared to ~200 bp DNA fragments by sonication using a Bioruptor sonicator (Diagenode) for 24-28 cycles of 30 sec on and 30 sec off.  Fragment size was verified by agarose gel electrophoresis before quantification of protein concentrations using a BCA protein assay kit (Pierce, 23225). 100 µg of soluble chromatin was precleared with Protein A Dynabeads (Invitrogen, 10001D) before incubation overnight with 2.5 g of antibody: H3K4me1 (Abcam, ab8895) or H3K27ac (Abcam, ab4729).  ChIP-seq libraries were constructed as described previously (Franco et al., 2015)