GSM3855388: OTX2low; Homo sapiens; ChIP-Seq
The reaction was then quenched with 0.125 M Glycine for 10 minutes. Cells were washed twice with 1X PBS. Chromatin was mechanically sheered in sonication buffer (HEPES pH 7.9 (50 mM), NaCl (140 mM, EDTA (1 mM), Triton (1%), Na-deoxycholate (0.1%), SDS (1%), PIC (1X)). Immunoprecipitation was carried out with H3K27me3 (07-449, Millipore) and H3K4me3 (07-473, Millipore) antibodies overnight at 4°C. Antibody complexes were captured on Dynabeads (Invitrogen) for 4 hours at 4°C then washed with Low Salt buffer (SDS (0.1%), Triton (1%), EDTA (2mM), Tris-HCl pH 8.1 (20 mM), NaCl (150 mM)), High Salt buffer (SDS (0.1%), Triton (1%), EDTA (2mM), Tris-HCl pH 8.1 (20 mM), NaCl (500 mM)), LiCl buffer (LiCl (0.25M), Na-deoxycholate (1%), EDTA (1mM), Tris-HCl pH 8.1 (10mM), NP-40 (1%)) and TE. Antibody complexes were eluted from the beads and reverse cross-linked for 12-16 hours at 65°C. Chromatin was then incubated in RNase A (0.2mg/mL) and NaCl (0.2 M/mL) for 2 hours. Proteinase K (0.2 mg/mL) was then added for an additional 2 hours. Chromatin was then cleaned using the Qiagen PCR Purification Kit according to manufacturer's instructions. NextFlex Illumina Chip-seq kit