GSM4217410: CDK9-siKDM5C-CTL-MCF-7; Homo sapiens; ChIP-Seq
For ChIP assays, cells were fixed with disuccinimidyl glutarate (DSG) (2 mM) (Proteochem) for 45 mins at RT, washed twice with PBS and then double-fixed with 1% formaldehyde for another 10 mins at RT (for KDM5C (Bethyl Laboratory, A301-034A), ZMYND8 (Bethyl Laboratory, A302-090A)) or fixed with 1% glutaraldehyde for 10mins at RT(for CDK9 (CST, 2316S) ChIP). Fixation was stopped by adding glycine (0.125 M) and incubating for 5 mins at RT, followed by washing with PBS twice. Chromatin DNA was sheared to 200-500 bp average in size through sonication. Resultant was immunoprecipitated with control IgG or specific antibodies overnight at 4 ℃, followed by incubation with protein G magnetic beads (Biorad) for an additional 3 hrs. After washing and elution, formaldehyde or DSG and formaldehyde fixed chromatin was treated at 65 ℃ overnight whereas glutaraldehyde fixed chromatin was treated with 0.2mg/mL proteinase K at 55℃ for two hours then 65℃ overnight with interval shaking. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen) and subjected to either qPCR analysis or high throughput sequencing. The libraries were constructed following Illumina ChIP-seq Sample prep kit. Briefly, ChIP DNA was end-blunted and added with an 'A' base so the adaptors from Illumina with a 'T' can ligate on the ends. Then 200-400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.
GEO Accession
GSM4217410