GSM8522010: 6857 WT Treg, ATAC, rep1; Mus musculus; ATAC-seq
50,000 Treg were used for ATAC-seq. After being washed by PBS, the cells or epiblasts were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) igepal CA-630 and 1×PIC). After 10 min incubation on ice, cells were spinned down by centrifugation at 500g, 4℃. Then the pellets were resuspended in transposome mix (35 ml ddH2O, 10 ml 5×TTBL, 5 ml TTE mixV50 (TD501, Vazyme) and gently flicked. The reaction was incubated at 37℃ for 0.5 h in a PCR cycler, before 1 ml 0.5M EDTA, 1 ml proteinase K (20 mg/ml) and 5 ml 10% SDS were added to stop the reaction. After another incubation at 50℃ for 1 h, DNA was purified with Phenol-Chloroform-Isoamylol (ThermoFisher) and resuspended with 30 ml ddH2O. 15 ml DNA were amplified with i5 and i7 primers (Vazyme, TD202) and KAPA 2×PCR mix (KM2602) following the cycling program with heated lid, 72℃, 5min; 98℃, 30s; 10 cycles for 98℃, 10 s; 60℃, 30 s; 72℃, 30 s; and 72℃ for 5 min, and hold at 4℃. Upon the program completed, 0.5 volume (25 ml) Ampure XP beads were added to remove fragments above 1 kb and another 0.5 volume (25 ml) Ampure XP beads were added to collect the 200-1,000bp fragments for sequencing. And the rest 15 ml DNA was used as input.