GSM8336328: surface epithelium_H3K27me3_rep1; Homo sapiens; ChIP-Seq
Cells were cross-linked with 1% formaldehyde (Thermo Fisher Sci_x0002_entific, 28906) in Dulbecco's Modified Eagle Medium for 10min at room temperature. Formaldehyde was then quenched with 125 mM glycine (Sigma-Aldrich, 50046) for 5min at room temperature. Cells were lysed in sonication buffer [50 mM Hepes-NaOH (pH 7.5), 500 mM NaCl, 1 mM EDTA, 0.1% Na-deoxycholate, 1% Triton X-100, and 0.1% SDS]. Chromatin was sheared with Covaris M220 focused-ultrasonicator to an average DNA fragment length of 200 to 500base pairs (bp). Chromatin was then diluted in lysis buffer without SDS and immunoprecipitated with primary antibodies overnight at 4°C. The immunocomplexes were captured with Protein A and Protein G magnetic beads (1:1, Invitrogen). After three washes with sonication buffer, two washes with low-salt wash buffer [10 mM tris-HCl (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, and 0.5% Na-deoxycholate], and one wash with TE buffer [10 mM tris-HCl (pH 8.0) and 1 mM EDTA], chromatin was then eluted from beads and de–cross-linked with 1% SDS in TE for 4 hours. After 1-hour digestion at 37°C with proteinase K (Invitrogen) and ribonuclease A (Invitrogen), DNA was purified using the MinElute PCR Purification Kit (QIAGEN) Purified DNA was used to prepare ChIP-seq DNA libraries for Illumina sequencing by KAPA Hyper Prep Kit (KAPABIOSYSTEMS).