GSM5051697: noCadmium_231_k27me3; Homo sapiens; ChIP-Seq
Cells were crosslinked by adding 1% formaldehyde to media for 10 minutes at 37°C. Crosslinking was stopped by addition of Glycine to 0.125M and incubating for 5 minutes at room temperature. Cells were washed with PBS and collected and centrifuged. Cells were resuspended in cellular lysis buffer (5mM PIPES pH 8, 85 mM KCl, 0.5% NP40), incubated on ice for 5 minutes and centrifuged. Samples were resuspended in nuclear lysis buffer (50 mM Tris pH 8, 10 mM EDTA pH 8, 1% SDS) and sonicated for 45 minutes using 30 second on/off cycle repeats at 70% intensity (using a Qsonica Q800R). 10 ug crosslinked DNA and 2 ug crosslinked Drosophila control spike-in chromatin was used per immunoprecipitation. Chromatin was pulled down overnight using Invitrogen Protein A Dynabeads with antibodies for H3 (Cell Signaling Technology), H3K27m33 (Abcam) or IgG (Cell Signaling Technology). Beads were washed twice with IP buffer (16.7 mM Tris pH 8, 1.2 mM EDTA pH 8, 167 mM NaCl, 0.1% SDS, 1.84% Triton X100), once with TSE buffer (20 mM Tris pH 8, 2 mM EDTA pH 8, 500 mM NaCl, 1% Triton X100, 0.1% SDS) and LiCl buffer (100mM Tris pH 8, 500 mM LiCl, 1% deoxycholic acid, 1% NP40), and twice with TE (10mM Tris pH 8, 1 mM EDTA pH 8). DNA was eluted from beads with proteinase K and purified with Promega Wizard SV PCR clean up kit. DNA was analyzed with qPCR and compared to 10% input collected prior to pulldown. ChIP-seq libraries were prepared using the Illumina NEBNext Ultra II ChIP-Seq sample kit, according to the manufacturer's protocol. Libraries were validated using the Agilent Technologies 2100 Bioanalyzer. Samples were sequenced on a llumina NovaSEQ6000 paired end