GSM6701646: Foxp3_CnT_DSM2; Mus musculus; OTHER
Briefly, a single cell suspension from the thymus or the spleen was prepared, then pre-enriched using EasySep Mouse CD4+ T cell Isolation Kit (STEMCELL Technologies, Cat# 19852), and stained with fluorescent-conjugated primary antibodies on ice for 30 minutes. The individual cell population was sorted by FACS. CUT&RUN experiments were performed as previously described with minor modifications.  To avoid the potential activation of T cells by Concanavalin-A (ConA) beads in the standard protocol, we instead used a spin-down method to collect cells as previously reported. Briefly, 0.5×106 cells per biological replicate were collected in V-bottom 96-well plate. Cells were first washed twice in Antibody Buffer (2mM EDTA, 1X EDTA-free protease inhibitors, 0.5mM spermidine, 1x permeabilization buffer from eBioscience Foxp3/Transcription Factor Staining Buffer Set) by centrifugation at 1900rpm for 6min, at 4°C, and then incubated with IgG and Foxp3 antibodies on ice for 1 hour. After two washes with buffer 1 (1X EDTA-free protease inhibitors, 0.5mM spermidine, 1x permeabilization buffer), cell pellets were incubated with pA/G-MNase (20X) enzyme in 50µl buffer 1 and incubated at 4°C for 1 hour. Cells were washed twice in saponin buffer (0.05% (w/v) saponin, 1X EDTA-free protease inhibitors, 0.5mM spermidine in PBS) and resuspended in 100µl calcium buffer (2mM CaCl2 in buffer 2) on ice for 30 min. 100µl 2x stop buffer (20mM EDTA, 4mM EGTA in saponin buffer) was then added and incubated in a 37°C incubator for 10-20 min to release cleaved chromatin fragments. Last, the supernatant containing chromatin fragments was collected by centrifugation and DNA was extracted using a QIAGEN MinElute kit according to manufacturer's protocol. CUT&RUN libraries were prepared using the NEBNext UltraTM II DNA Library Prep Kit for Illumina® (E7645) according to manufacturer's instructions.