Input_ChIP_R1_001.fastq.gz
Approximately 3-6  106 splenic Treg cells were sorted and cross-linked with fresh 1% formaldehyde (Fisher Scientific) for 8 min at room temperature (RT) and quenched with 125 mM glycine for 5 min at RT. Nuclei were isolated using the truChIP kit (Covaris Inc.), and chromatin was sheared at 4C, 140 W, and 200 cycles per burst with 10% duty factor for 8 min (Covaris). Input fraction was saved (1%), and the remaining sheared chromatin was used for ChIP with a mouse anti-NFB p65 antibody (F-6) (sc-8008 X, Santa Cruz) or an immunoglobulin G isotype control (Millipore) in immunoprecipitation buffer at 4 C, rotating overnight, followed by incubation with Dynabeads Protein G (10003D, Life Technologies) for 4 hours.