GSM3963951: T cell Input control; Mus musculus; ChIP-Seq
~60x106 CD8+ T cells from 8 mice were resuspended in PBS and crossed-linked with fresh formaldehyde solution for 30 min at room temperature. Excess formaldehyde was quenched with 125mM Glycine. Cells were incubated with nuclear extraction buffer with protease inhibitors on ice for 5 min. Nuclear extracts were resuspended in sonication buffer and sonicated using the Covaris S220 Focused Ultrasonicator.  Protein A and Protein G Dynabeads were washed and resuspended in ChIP IP buffer then  added to diluted lysates with 20µg of a previously validated polyclonal anti-EGR2 ChIP antibody (ab43020, Abcam). IP samples were incubated overnight at 4oC. Protein A/G beads were washed then incubated with Reverse Crosslinking Buffer containing 45mg Proteinase K at 55oC for 1h and the supernatant was then isolated and incubated at 65oC overnight. DNA was isolated using the ChIP DNA Clean & Concentrator Kit (Zymo Research). Indexed libraries were prepared using KAPA Hyper Prep Kit for Illumina platforms  and the SeqCap Adapter Kit (Roche) following vendor's instructions. Library QC and quantiﬁcation was performed using D1000 high-sensitivity screen tape with a 4200 TapeStation Instrument (Agilent Technologies) and size selected for between 200 bp and 500 bp using a Pippin Prep system.