GSM8067770
Briefly, cells were cultured to ~80% confluency in a petri dish containing 10 mL of growth media, and then fixed in 1% formaldehyde by adding 275 μl of 37% formaldehyde for 10 minutes to cross-link protein–DNA complexes at room temperature. The unreacted formaldehyde was quenched by adding Glycine to a final concentration 0.125 M. Gently swirl dish to mix. The nuclear pellet was isolated with Cell Lysis Buffer. The pellet was resuspended with 500 μl SDS Lysis Buffer containing 1X Protease Inhibitor Cocktail II before sonication for 4 min (10 s on, 30 s off, 10% strength in a Bioruptor to yield DNA fragments of 0.2-1.0 kb in length. The lysates were cleared by centrifugation (12,000g for 10 min at 4 °C) and diluted tenfold in ChIP dilution buffer to decrease the concentration of SDS. After keeping 10% of the sample as input, 500 μl supernatant was incubated overnight at 4 °C with antibody and 20 μL of fully resuspended protein A/G magnetic beads. The washing, elution, reverse cross-linking, and purification steps were performed according to manufacturer's description. ChIPed Eluted DNA was quantified by Qubit dsDNA HS Assay Kit.Total RNA was extracted from the cell pellets using AllPrep DNA/RNA kit (Qiagen) ChIP-seq libraries were prepared with Kapa DNA HyperPrep Kit (Kapa, Cat KK 8700) according to the manufacturer's protocol. Briefly, 5-10 ng of immunoprecipitated DNA was underwent end-repaired, A tailing and adaptor ligation. A 10 cycles of PCR was performed to produce the final sequencing library. As for ATAC, the Tn5 treated DNA was amplified with a 5-cycle PCR in 50µl reaction volume. The tubes were removed from thermocycler and used 5 µl of partially amplified library to perform qPCR to determine how many additional PCR cycles were needed. For the samples in this study, an additional 4-5 cycles of PCR was performed on the remaining 45ul of each partially amplified product. 1.8X AmpurXP beads purification was used for the final PCR cleanup. The libraries were validated with the Agilent Bioanalyzer DNA High Sensitivity Kit, and quantified with qPCR. For RNA-seq, sequencing libraries were prepared with TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, USA) with minor modifications.mRNA library was constructed with TruSeq RNA Sample Preparation Kit and sequenced on the Illumina Hiseq 2500 with pair-end 150bp reads following the manufacturer's recommendations by Novogene.