GSM5949799: WT_2 H3K9ac; Danio rerio; ChIP-Seq
About 600 36hpf wild type or sinhcaf mutant embryos were collected. Immunoprecipitation was carried out using ChIP Grade anti- acetyl-histone H3 (K9 9649T) antibody.Chromatin immunoprecipitation assays were carried out using a variation of the protocol (https://wiki.zfin.org/display/prot/ZFIN+Protocol+Wiki) The DNA is combined with End Repair Mix, and incubate at 20℃ for 30 min. Purify the end-repaired DNA with QIAquick PCR Purification Kit (Qiagen), then add A-Tailing Mix, incubate at 37℃ for 30 min. Combine the purified Adenylate 3' Ends DNA, Adapter and Ligation Mix, incubate the ligation reaction at 20℃ for 15 min. Purify the Adapter-ligated DNA with the QIAquick PCR Purification Kit. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the Adapter-ligated DNA fragments. Then the PCR products are selected (about 100-300bp, including adaptor sequence) by running a 2% agarose gel to recover the target fragments. Purify the gel with QIAquick Gel Extraction kit (QIAGEN). The final library is quantitated in two ways: Determine the average molecule length and sample integrity as well as purity using the Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents) and quantify the library by real-time quantitative PCR (RT-qPCR).