GSM8835008: MCF-7, 5a_DTT, ChIP; Homo sapiens; ChIP-Seq
Cells were cultured, treated as required, and cross-linked with 1% formaldehyde for 10 minutes at room temperature. To quench the cross-linking, 1X glycine was added for 5 minutes. The cells were then washed with PBS, scraped in PBS containing Halt cocktail, and pelleted by centrifugation at 3,000 × g for 5 minutes. The pellet was resuspended in membrane extraction buffer with inhibitors, vortexed briefly, and incubated on ice for 10 minutes. Nuclei were isolated by centrifugation at 9,000 × g for 3 minutes, resuspended in MNase digestion buffer, and digested with 2 μL of diluted MNase (1:10) per 4 × 10⁶ cells at 37°C for 15 minutes. The reaction was stopped, and the nuclei were pelleted, resuspended in IP dilution buffer, and sonicated (three 20-second pulses). The lysate was centrifuged at 9,000 × g for 5 minutes, and the supernatant containing chromatin was collected. Chromatin was then diluted in IP dilution buffer and incubated overnight at 4°C with antibodies (e.g., anti-RNA Pol II or target-specific antibodies). The following day, protein A/G magnetic beads were added and incubated for 2 hours. The beads were washed three times with wash buffer 1 and once with wash buffer 2. Chromatin was eluted with IP elution buffer for 30 minutes at 65°C and treated with NaCl and proteinase K for 1.5 hours at 65°C to reverse cross-linking. Finally, DNA was purified using a DNA cleanup column and eluted in elution buffer for use in downstream applications. ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina following manufacturer's protocols. Post ampilfication libraries were cleanup using AMPure XP Beads from Beckman Coulter. Libraries were validated using the Agilent Tapestation 4200 instrument.
GEO
GSM8835008_r1