GSM6043961: Y3_bef_liver_H3K27me3; Mus musculus; ChIP-Seq
Crude nuclei preparations were made from ~100 mg frozen tissue by douncing in nuclei preparation buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20, 0.1% NP-40, 0.01% digitonin and 1 mM BSA, supplemented with protease inhibitors and 1 mM sodium butyrate). After centrifugation, the nuclei pellet was crosslinked with 1% formaldehyde in 2 ml PBS by constant rotation at room temperature for 10 min followed by quenching with 125 mM glycine and two PBS washes. The nuclei were lysed with nuclei lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium-deoxycholate, 0.5% N-lauroylsarcosine supplemented with protease inhibitors and 1mM sodium butyrate) and sheared to <500bp using a Covaris S220 Ultrasonicator. An aliquot of the sheared chromatin sample was removed to check sonication success and to quantify the amount of chromatin. The immunoprecipitation, wash and elution steps were performed as reported previously (Sen et al., 2019) except ~4% HeLa chromatin was spiked in to 2 µg mouse chromatin prior to immunoprecipitation (only for liver samples). DNA (~5 ng) was used to prepare libraries with the NEBNext Ultra II library preparation kit with unique dual index primers (New England Biolabs). The library quality and quantity were verified by BioAnalyzer DNA 1000 (Agilent) run and qPCR with NEBNext Library Quant kit (New England Biolabs) respectively.