H3-K27Ac Cut & Tag of primary human fibroblasts
H3-K27Ac Cut & Tag of primary human fibroblasts
Trypsinized cells and used 50,000 for Cut & Tag with the nuclear permeabilization protocol described by the Henikoff lab. DMEM (4.5g/l glucose) + 10% FBS + stabilized glutamate + pyruvate + pen/strep in an incubator with 5% CO2 + 5% O2 For irradiation, 40 Gy of 120 kV X-rays, then additional incubation for 10 days. For irradation + IL1-alpha, 40 Gy of 120 kV X-rays, then additional incubation for 9 days followed by incubation with 20 pg/ml IL1-apha for 1 day. For etoposide-induced senescence, treatment with 100 microM etoposide for 2 days followed by incubation for 8 days without etoposide. Phenol-chloroform extraction after the Cut & Tag reaction followed by ethanol precipitation. PCR amplification of Cut & Tag fragmented DNA using Illmina multiplex oligonucleotide primers.