GSM8256963: Dnmt1tet/tet mESCs, Dnmt1 control (CTL), replicate 2, H3K4me3 ChIP-seq; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation (ChIP) sequencing was performed in duplicates for each histone modification per experimental condition and one aliquot of non-immunoprecipitated chromatin (i.e. input) was sequenced per experimental condition. First, cells were fixed with a solution consisting of 13% formaldehyde (Sigma-Aldrich, 47608) in DPBS (Corning, 21-031-CV) for 10 minutes at room temperature with gentle rocking. Then, cell and nuclei lysis was performed, and chromatin was purified and sonicated using buffers and protocols from the IDeal ChIP-Seq Histone Kit (Diagenode, C01010051). Sonication was conducted with the Bioruptor® Pico sonication device (Diagenode, B01060010) for 15 cycles (30 seconds ON/ 30 seconds OFF) to obtain DNA fragments between 150-400bp. Next, chromatin was pre-cleared via incubation with protein G Dynabeads™ (Thermo Fisher, 10009D) for 1h at 4°C on a rotator, after which samples were placed on a magnetic rack and input aliquots were taken from the supernatant. Chromatin immunoprecipitation was then conducted as follows. Pre-cleared chromatin samples were incubated overnight at 4°C on a rotator with histone modification antibodies–either anti-H3K4me3 (Active Motif, 39159), anti-H3K27ac (Abcam, ab4729), anti-H3K4me1 (Cell signaling technology, 5326), anti-H3K27me3 (Sigma-Aldrich, 07-449) or anti-H3K9me3 (Abcam, ab8898)–in quantities recommended by the antibody manufacturer. Dynabeads™ were added to the chromatin-antibody mixtures and were incubated for an additional 2h at 4°C on a rotator. Protein A Dynabeads™ (Thermo Fisher, 10001D) or protein G Dynabeads™ (Thermo Fisher, 10009D) were used depending on the antibody source. Washing, elution, decrosslinking and DNA purification were then conducted using the IDeal ChIP-Seq Histone Kit (Diagenode, C01010051). Libraries were prepared with the KAPA HyperPrep Kit (Roche, KK8502).