title: GSM7025971: 4CBS_CUT&Tag_ab192985; Mus musculus; OTHER
library_construction_protocol: To prepare nuclei, mESC were dissociated with Accutase (Innovative Cell Technologies, AT104), collected by centrifugation, washed twice with PBS (Gibco, 10010023) then resuspended in cold nuclei permeabilization buffer (10 mM Tris-HCl pH 7.4 (Sigma, T4661), 10 mM NaCl (Sigma, S7653), 3 mM MgCl2 (Sigma, 63069), 1×Protease Inhibitor (Roche, 05056489001), 0.5 U µl−1 RNase OUT (Invitrogen, 10777-019), 0.5 U µl−1 SUPERase Inhibitor (Invitrogen, AM2694), 0.1% IGEPAL CA630 (Sigma, I8896) and 0.02% Digitonin (Sigma, Cat#D141)) for 1 minute. Nuclei were counted using a BioRad TC20 cell counter with 0.4% Trypan Blue (Gibco, 15250061) staining. For each experiment, around 0.5 million nuclei were aliquoted, and centrifuged for 10 min at 1,000g, 4 °C before proceeding to Bulk CUT&Tag. Tagmentated products are extracted using QIAGEN MiniElute after CUT&Tag, and directly amplified with indexed primers. Size selection using SPRI Beads is performed with amplified products to obtain the final sequencing library (150-800 bp)