GSM9006070: MCF7, scramble RNA, Rep2, ERalpha, ChIP; Homo sapiens; ChIP-Seq
Cells were fixed using 1% formaldehyde for 10 mins at room temperature on an orbital shaker. After 10mins, formaldehyde was quenched using 0.125M glycine for 5mins at room temperature on the orbital shaker. The cells were washed with cold 1XPBS and scraped. The cells were pelleted down at 3000rpm for 5mins at 4°C. The pellets were stored at -80°C. 10 million cells were resuspended in 1 ml of nuclear lysis buffer (NLB) (50mM Tris-HCl pH 7.4, 1% SDS, 10mM EDTA pH 8.0, and 1X PIC) and incubated in ice for 10mins. After nuclear lysis, cells were sonicated using Bioruptor pico for 28cycles with 30secs ON and 30secs OFF settings. The lysate was then spun at 12000rpm for 12mins at 4°C to clear off the cell debris. 100μg of chromatin was used for IP, further 3μg of sonicated S2 chromatin was added as spike-in for only H3K27ac ChIP. The IP was diluted 2.5 times with dilution buffer (20mM Tris-HCl pH 7.4, 100mM NaCl, 2mM EDTA pH 8.0, 0.5% Triton X-10, and 1X PIC). IP was set in a volume of 500μl. Additionally, 50μl was taken out and labelled as 10% input. For IP, main as well spike-in antibody was added in their respective amounts (ERα (sc-8002, Santa Cruz Biotech) 1μg, anti-PolII (Diagenode C15200004) 2μg, H3K27ac (ab4729, Abcam) 1μg and spike-in H2av 0.3ug (61686, Active Motif)). IP was incubated with antibodies overnight at 4°C at constant rotation of 12rpm. After incubation, 15μl of Protein G beads pre-blocked in BSA were added to the mix. It was incubated for 4 hours at 4°C on the rotor. After incubation, the beads were washed with wash buffers I, II, III and 1XTE sequentially (Li et al., 2013). The DNA-protein complexes were eluted from the beads by resuspending the beads in (100mM NaHCO3, 1%SDS, 2μg of RNaseA) at 37°C in a thermomixer with 1400rpm for 30mins. The crosslinked DNA-protein complexes were reverse-crosslinked over night at 65°C by the addition of NaCl. After that 2μl of Proteinase K (20mg/ml) was added to the mix and incubated for 2hours at 45°C. The ChIP DNA was then purified using PCI (Phenol:Chloroform:Isoamylalcohol) and ethanol precipitation. The pellet was resuspended in 10μl of NFW. ChIP-seq