GSM7898077: KO_NPC_H3K9me3_Rep2; Mus musculus; ChIP-Seq
Three million NPCs were harvested, washed in PBS and lysed with NP40 lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM EDTA, 10 mM Tris pH8, 0.1% NP40). For native ChIP (nChIP, for H3K9me3), nuclei were collected through centrifugation, resuspended in douncing buffer (10 mM Tris pH8, 4 mM MgCl2, 1 mM CaCl2) and chromatin was fragmentized with micrococcal nuclease (Sigma N3755). Hypotonic solution (0.2 mM EDTA, cOmplete inhibitor (Roche 11697498001), 0.1% NP40) was added for nuclei lysis and chromatin release. For fixed ChIP (xChIP, for CTCF), nuclei were collected through centrifugation, fixed in 1% formaldehyde (Sigma 252549) at room temperature for 5 minutes with rotation, and quenched in 0.125 M glycine at RT for 10 minutes with rotation. After collected with centrifugation, the fixed nuclei were resuspended in FSB solution (5 mM EDTA, 20 mM Tris pH8, 500 mM NaCl) supplemented with 0.1% SDS and cOmplete inhibitor, and sonicated with Covaris E220 ultrasonicator system to 300~500 bp. For both nChIP and xChIP, protein-DNA complexes were isolated with antibody. ChIP DNA was blunted with End-it DNA Repair Kit (Epicentre ER0720), A-tailed with Klenow Exo-minus (Epicentre KL06041K), ligated with adapter (Vazyme N802-01) using Fast-Link kit (Epicentre LK11025) and amplified with VAHTS HiFi Universal Amplification Mix for Illumina (Vazyme N618-02). The library was size-selected with SPRIselect beads (Beckman B23318) and sent for sequencing on Illumina NovaSeq 6000 (paired-end, 150 bp).