GSM8173916: FlcnKO_Treg_rep2; Mus musculus; ATAC-seq
5x104 total Treg cells were sorted from the indicated mice and lysed in 50 uL ATAC-seq lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) on ice for 10 min, followed by centrifugation at 500g for 10 min at 4 ºC to pellet nuclei. After careful removal of supernatant, nuclei were resuspended in 50 uL transposase reaction mix (Illumina) and incubated at 37 ºC for 30 min. Following tagmentation, DNA was purified using MinElute kit (Qiagen). Purified DNA was then indexed using Nextera barcoding and amplified for 5 cycles using NEBNext HiFi kit (NEB). A 5 uL aliquot from the previous PCR reaction was used for real-time PCR (KAPA SYBR Fast; Kapa Biosystems) to determine the optimal remaining cycles. The final PCR products were cleaned using AMPure XP beads (Beckman Coulter).