GSM9066009: Chd2KD_rep2_noab; Mus musculus; OTHER
Nuclei were incubated for 1 hour at room temperature with rotation with rabbit anti-H3K4me3 (Millipore Sigma, Cat# 05-745R, 3697296). Control samples were treated in the same conditions without antibody. Samples were washed twice with wash buffer and incubated for 30 min at room temperature with rotation in wash buffer containing recombinant Protein A-MNase (pA-MN) (1:250). Samples were washed twice with wash buffer and equilibrated at 0°C in an ice bath. Three mM CaCl2 was added to each sample to activate MNase cleavage and digested in an ice bath for 30 min. Digestion for H3K4me3 samples was stopped after 30 min using a stop buffer (20 mM EDTA. 4 mM EGTA, 200 mM NaCl, 50 ug/mL RNase A). Samples were incubated at 37°C for 20 min to release genomic fragments and fragments were then separated through centrifugation. Samples were incubated at 4°C for 1 hr to release genomic fragments. The salt concentration was increased to 500 mM for Chd1 samples, RNase A was added, and samples were incubated at 37°C for 20 min to release genomic fragments. Fragments for all samples were isolated and extracted to be used as input for library build. Libraries were built using NEB Next II Ultra DNA library kit (NEB E7645L) and AMPure XP beads (Beckman Coulter A63881) purifications. Fragments were PCR amplified with 15 cycles. Samples were then pooled and paired-end sequenced on an Illumina NextSeq2000 to a depth of ~10 million uniquely mapped reads per sample.
GEO
GSM9066009_r1