GSM7349957: HDPC, anti-H3K9ac, abcam, bio rep2 K9acD_2; Homo sapiens; ChIP-Seq
HDPCs were fixed with 1% formaldehyde for 1min at room temperature to crosslink chromatin. 125mM glycine was added to the plates to stop crosslinking. Cells were then washed then lysed with lysis buffer supplemented with phosphatase and protease inhibitor cocktails. Purified nuclei were collected by centrifuge and then incubated with nuclease to get mononucleosomes. Soluble chromatin was then subjected to immunoprecipitation as described in manufacture's protocol(MM_NF-17-375, Millipore). 1.DNA quantification and qualification: ChIP DNA degradation and contamination was monitored on agarose gels. DNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured using Qubit® DNA Assay Kit in Qubit® 3.0Flurometer(LifeTechnologies,CA,USA); 2.Library preparation and quantification: The purified DNA was used for ChIP-seq library preparation. The library was constructed by Novogene Corporation (Beijing, China). Library quality was assessed on the Agilent Bioanalyzer 2100 system. Briefly, In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.