GSM5452342: MCF-7 H3K27ac; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 10 min at room temperature, and quenched by the addition of glycine to a final concentration of 125 mM for 5 min. The fixed cells were re-suspended in SDS lysis buffer in the presence of protease inhibitor Cocktail II, then subjected to 12 cycles (turn on for 20 s and turn off for 10 s) of sonication (Biorupter, Ningbo, China) to generate chromatin fragments of 200-1000 bp in length. Lysates were centrifuged at 14000 g for 10 min at 4°C to remove insoluble material. Supernatant was diluted in ChIP Dilution Buffer. For immunoprecipitation, the diluted chromatin was incubated with specific antibodies overnight at 4 °C with constant rotation, followed by incubation with protein G agarose for 1 h. Beads were washed continuously with buffers. The pulled-down chromatin complex was de-crosslinked at 65 °C for 5 h in elution buffer. Eluted DNA was digested with RNase at 37 °C for 30 min, and with proteinase K at 45 °C for 2 h to further purify. Purified DNA was enriched by spin columns. To construct the ChIP-Seq library, DNA was combined with end repair mix, and incubated at 20 °C for 30 min. The end-repaired DNA was purified with QIAquick PCR-purification kit (Qiagen, Beijing local agent, China), then added with A-Tailing mix, incubated at 37 °C for 30 min. the purified Adenylate 3' ends DNA, adapter and ligation mix were combined, and incubated the ligation reaction at 20 °C for 15 min. the Adapter-ligated DNA was purified with QIAquick PCR Purification Kit. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the Adapter-ligated DNA fragments. Then the PCR products were selected (about 100-300 bp, including adaptor sequence) by running a 2% agarose gel to recover the target fragments. The gel was purified with QIAquick Gel Extraction Kit (Qiagen, Beijing local agent, China). The final library was quantified in two ways: the average molecule length and sample integrity as well as purity were quantified using Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents), and quantified using real-time quantitative PCR.   The double-stranded PCR products were heat-denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formulated as the final library. The library was qualified by Qubit ssDNA kit. The sequencing was performed with the BGISEQ-500 sequencing system, featuring combinatorial probe-anchor synthesis (cPAS) and DNA Nanoballs (DNB) technology for superior data quality (BGI-Shenzhen, China). For Illumina, the qualified libraries were amplified on cBot to generate the cluster on the flowcell (TruSeq PE Cluster Kit V3-cBot-HS, Illumina). And the amplified flowcell was sequenced pair end on the HiSeq 2000 System (TruSeq SBS Kit-HS V3, Illumina).