NextSeq 500 paired end sequencing; ChIP-seq of RNA polymerase II from WI-38hTERT/sh3-H2AFJ and WI-38hTERT/sh-NT fibroblasts in proliferation and etoposide-induced senescence.
ChIP-seq of RNA polymerase II from WI-38hTERT/sh3-H2AFJ and WI-38hTERT/sh-NT fibroblasts in proliferation and etoposide-induced senescence.
Cells were crosslinked with 1% formaldehyde for 10 minutes and then quenched with glycine at 200 mM final concentration. Cells were washed with cold PBS, scraped and pelleted by centrifugation at 1,500 x g for 10 minutes at 4°C. Cells were washed again twice with cold PBS and stored at -80°C in 50 µl of PBS.  WI38 human embryonic fibroblasts from the ATCC were immortalized with hTERT and grown in MEM (Invitrogen #21090) + 10% Fetal Bovine Serum + 1 mM Sodium Pyruvate + 2 mM L-glutamine + 0.1 mM non-essential amino acids + pen/strep in an incubator with 5% C02 and 5%02. WI38hTERT fibroblasts cells were grown in the presence of 1 µg/ml doxycycline for 3 days to induce the expression of sh-RNAs before senescence induction by incubation with 100 µM etoposide for 48 hours. Cells were then washed and incubated in fresh medium without etoposide for 7 days before harvesting cells for analysis. Doxycycline was maintained throughout the experiment to ensure continual expression of the sh-RNAs. After MNase, Bioruptor, and IP, beads were treated with RNase and then proteinase K at 65°C O/N. DNA was then then further purified by phenol/chloroform extraction and ethanol precipitation.  NEBNext Ultra II DNA Library Preparation Kit (#E7645S) according to the manufacturer's protocols.