GSM1437472
GSM1437472: mmu_ezh2_H3K27me3_ChIPSeq; Mus musculus; ChIP-Seq
Cross-linked cell pellet was resuspended in SDS-ChIP buffer and chromatin was sonicated to around 200-500 bp. Sonicated chromatin were incubated with 5-10 µg antibody overnight at 4oC. After overnight incubation, protein A or G agarose beads were added to the ChIP reactions and incubated for 2-3 hours at 4oC to IP chromatin. Subsequently, beads were washed twice with 1 ml of low salt wash buffer, once with 1 ml of high salt wash buffer, once with 1 ml of LiCl wash buffer, and twice with 1 ml of TE buffer. The chromatin was eluted and reverse-crosslinked at 65oC overnight in SDS elution buffer (300 µl). The next day, an equal volume of TE was added (300µl). ChIP DNA was treated with 1 µl of RNaseA (10 mg/ml) for 1 hour, and with 3 µl of proteinase K (20 mg/ml) for 3 hours at 37ºC, and purified using phenol-chloroform extraction, followed by QIAquick PCR purification spin columns (Qiagen). Finally, ChIP-DNA was eluted from the column with 40 µl of water. Input ChIP samples were reserved after the sonication and continued from reverse cross-linking step until the end, same as other ChIP samples. Purified ChIP DNA was measured in Qubit (Invitrogen). 2-10 ng of purified ChIP DNA was used to prepare sequencing libraries, using NEB next generation ChIP sequencing Kit (NEB) and Illumina ChIP seq kit (illumina) according to the manufacture’s instructions. All libraries were checked through a Bio-analyzer for quality control purposes. ChIP sequencing was performed using illumina Hiseq 2000 sequencing system. Raw data were processed through the illumina software pipeline.
GEO
GSM1437472
GEO Accession
GSM1437472