Cord blood CD4 T cells - ATAC Seq
Chromatin accessibility was profiled using bulk ATAC-Seq as originally described (15). Briefly, 1x105 purified CD4+ T cells (n=2/group) were lysed in lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and NP-40 for 10 min on ice to prepare the nuclei. Immediately after lysis, nuclei were spun at 500g for 5 min to remove the supernatant. Nuclei were then incubated with transposition mixture (100 nM Tn5 transposase, 0.1% Tween-20, 0.01% Digitonin and TD buffer) at 37C for 30 min. PCR was performed to amplify the library for 6-10 cycles using the following PCR conditions: -72C for 3 min; 98C for 30s and thermocycling at 98C for 10s, 63C for 30s and 72C for 1 min. After PCR reaction, libraries were purified with the 1.2X AMPure beads. Libraries were quality checked on the Bioanalyzer, multiplexed and sequenced on 75 bp paired ended on NextSeq500.