GSM9708907: CN,RAD21_ICRF_ChIP_2; Mus musculus; ChIP-Seq
ChIP was performed for two biological replicates using a ChIP assay kit (MilliporeSigma) as described by the manufacturer. Briefly, DIV13 primary cortical neurons (~15M cells per replicate) were crosslinked with 1% formaldehyde for 10 minutes at 37°C, washed twice with ice-cold PBS, scraped, and resuspended in SDS lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS, protease and phosphatase inhibitor cocktail). Cells were sonicated to shear DNA to 200-500 bp using a bioruptor (Diagenode; high power, 30 seconds on/30 seconds off for 40 cycles). Samples were then centrifuged at 13,000 RPM for 10 minutes at 4°C to remove cellular debris, and the supernatant was diluted up to 2 mL in ChIP dilution buffer (16.7 mM Tris-HCl pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS). Prior to immunoprecipitation, 1% of each sample transferred to a clean tube to be used for input normalization. Samples were then incubated with appropriate antibody overnight at 4°C followed by incubation with ChIP-grade protein A/G magnetic beads (Thermo) for 3 hours at 4°C. Antibody-conjugated beads were washed with low salt buffer (20 mM Tris-HCl pH 8, 1% Triton-X-100, 2 mM EDTA, 0.1% SDS, 150 mM NaCl), high salt buffer (20 mM Tris-HCl pH 8, 1% Triton-X-100, 2 mM EDTA, 0.1% SDS, 500 mM NaCl), LiCl buffer (10 mM Tris-HCl pH 8, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 0.25 M LiCl), and twice with TE buffer (50 mM Tris-HCl pH 8, 1 mM EDTA) for 5 minutes each with agitation at room temperature. Immunoprecipitated protein-DNA complexes were eluted from the magnetic beads in 500 µl ChIP elution buffer (1% SDS, 0.1 M NaHCO3). DNA-protein crosslinks for immunoprecipitated samples and input DNA were reversed via incubation with an additional 20 mM NaCl for 4 hours at 37°C and residual protein was digested with Proteinase K (NEB) for 1 hour at 45°C. DNA was precipitated using phenol/chloroform isolation and ethanol precipitation, washed with 70% ethanol, and resuspended in 50 µl TE buffer. Samples were prepared for sequencing using a paired-end library preparation kit compatible with Illumina sequencing instruments (KAPA biosciences) according to the manufacturer's instructions. Following library amplification, DNA fragment size was assessed via Tapestation (Agilent) to ensure a final library size of 350-450 bp. DNA concentration was quantified by fluorometry (dsDNA high sensitivity Qubit assay, Thermo) to ensure equal DNA concentration for each replicate before replicates were pooled and sequenced on an Illumina NextSeq 500 instrument using V2.5 reagents to an average depth of 25-35 million reads per replicate.