GSM8779912: bulkATAC_D3_CD4_Naive; Homo sapiens; ATAC-seq
Cells sorted in collection media (1:1 FBS:PBS) were counted, and 55,000 cells were taken in LoBind 0.6 mL microcentrifuge tubes (Eppendorf) and washed with 0.5 mL chilled PBS. Cell pellet was resuspended in 50 uL chilled freshly-made lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL-CA630, 0.1% Tween-20, 0.01% Digitonin, 1X Protease Inhibtor, 20 mM NaBu) supplemented with 1 U/uL recombinant RNase Inhibtor. After 5 minutes of lysis on ice, 0.2 mL of wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) supplemented with 1 U/uL recombinant RNase Inhibitor, was added. Pelleted nuclei were transposed using Tn5 transposase from Illumina, in a 50 uL reaction mix supplemented with 1X PBS, 0.1% Tween-20 and 0.01% Digitonin, at 37°C for 30 minutes. Tagmented DNA was purified using Zymo DNA Clean and Concentrator-5 kit followed by amplification using barcoded Nextera XT primers with KAPA Hi-Fi HotStart Ready Mix. After the initial 5 cycles of amplification, cycle threshold determination (CTD) was performed using SYBR and ROX. Based on the CTD the additional cycles (CTD-2 cycle) were performed on the amplified DNA. Final libraries were purified and size-selected using double sided purification by Ampure XP beads (0.5X / 1.8X). Pooled libraries were sequenced using NovaSeq 6000 (Illumina) for 150-bp paired-end sequencing.