title: inChIP_Foxp3posi
library_construction_protocol: For each reaction, 30 ul Dynabeads Protein G and Protein A magnetic beads (1:1 ratio; Invitrogen) were resuspended in 500 ul bead wash buffer (0.5% BSA in PBS) and then pre-incubated with approximately 3 ug of the appropriate antibody at 4C on a rotator for at least 2 hours. Subsequently, the reactions were placed on a magnet and washed three times with 500 ul bead wash buffer to remove excess antibodies and then resuspended to the original bead volume with bead wash buffer. Fixed cells were sorted and resuspended in 100 ul nuclear lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS), supplemented with 1x protease inhibitor cocktail (Roche, 11873580001) and incubated on ice for 10 minutes. Sonication was performed on the Picorupter platform (Diagenode) for 5 cycles of 30s ON/30s OFF. Sheared chromatin was centrifuged at 10,000 g, 10 minutes, 4C and the clarified supernatant was transferred to a new DNA LoBind Eppendorf tube (Eppendorf). Chromatin was diluted 10x using ChIP dilution buffer (16.7 mM Tris pH 7.5, 167 mM NaCl, 1.2 mM EDTA, 1.1% TritonX-100, 0.01% SDS) supplemented with 1x protease inhibitor cocktail and blocked with 5 ul Fab Fragment Goat Anti-Mouse IgG (Jackson ImmunoResearch, 115-007-003) on ice for at least one hour, with occasional mixing by inversion. The reaction was incubated overnight at 4C on a rotator with antibody-conjugated beads. On the next day, beads were washed and reverse-crosslinked with the iDeal ChIP-seq kit for Histones (Diagenode; C01010173) as per manufacturer's recommendations and then column purified with ChIP DNA Clean & Concentrator (Zymo Research; D5205). Library preparation was performed with the KAPA HyperPrep kit (Roche; KK8504) according to manufacturer's instructions and then sequenced on the Illumina NovaSeq platform.