GSM6505986: Spleen, Cdca7G305V/G305V, H3K9me3, rep2; Mus musculus; ChIP-Seq
For chromatin preparation, frozen tissue was smashed using pestle and mortar (done in liquid nitrogen), and cross linked with 1% formaldehyde (344198; Calbiochem) in 9 ml of 1% PBS for 15 min at room temperature. Glycine (125 mM) was used to quench cross-linking for 5 min, and tissue was washed twice with cold PBS containing proteinase inhibitors (cOmplete™, EDTA-free Protease Inhibitor Cocktail, 5056489001, Roche). For lysis 1 ml of Cytoplasmic lysis buffer (10 mM Tris (pH 8), 10 mM NaCl, 0,2 % Igepal, and freshly added Protease Inhibitor Cocktail (05056489001; Roche)) was used. Tissue was homogenized 2 times for 10 sec (IKA model - T10 basic, at max speed – 6). Samples were spun down at 4000 rpm for 8-10 min at 4°C, and the pellets were resuspended in 1 ml SDS lysis buffer (1% SDS, 50 mM Tris-HCl (pH 8.1), 10 mM EDTA and freshly added Protease Inhibitor Cocktail). Lysed samples were sheared by sonication (Diagenode Biorupter Pico, - 30 sec ON/ 30 sec OFF, 15-25 cycles). Sheared chromatin was centrifuged at 12,000 g for 15 min at 4°C to discard the pellets For library preparation the Kapa Hyper Prep Kit (Roche, KK8504, 07962363001) was used, following the manufacturer's instructions