CUT&RUN libraries were performed essentially as described previously with the modifications described below. After antibody staining, cells were washed twice with Buffer 1 (1x Permeabilization buffer from eBioscience Foxp3/Transcription Factor Staining Buffer Set, 1x EDTA-free protease inhibitors, 0.5 mM Spermidine and 2mM EDTA), and incubated with pA/G-MNase (CST; 40366S) at 1.5 ul per 50 ul Buffer 1 for 1 hour on ice. After washing twice with Buffer 2 (0.05% (w:v:) saponin, 1x EDTA-free protease inhibitors, 0.5 mM spermidine in PBS), cells were resuspended in 100 ul calcium buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, 0.05% (w/v) saponin, 1x EDTA-free protease inhibitors, 2 mM CaCl2) to activate the micrococcal nuclease. After a 30 minute incubation period on ice, 100 ul of 2x stop solution (20 mM EDTA, 4 mM EGTA in Buffer 2) was added and the reaction was incubated for 10 minutes in a 37C incubator to release the cleaved chromatin fragments. 2.5 ul of Sample Normalization Spike-In DNA was added per reaction and supernatants were collected by centrifugation. DNA was purified using a Zymo DNA Clean & Concentrator-5 column (Zymo Research; D4014) and the eluted material was subjected to library preparation using the KAPA HyperPrep kit (Roche; KK8504) as previously described. Libraries were sequenced on the NovaSeq platform (Illumina).