GSM5931346: D14_High_CD8_d0_R1_001 [ATAC-seq]; Homo sapiens; ATAC-seq
Accessible chromatin mapping was performed using FAST-ATAC-seq (56) with minor modifications. Briefly, 10000 CD4+ or CD8+ CARHigh T or CARLow T cells from six independent donors were sorted in 1X PBS, 0.05% BSA and pelleted by centrifugation at 500 rcf for 5 min at 4 °C with low acceleration and brake settings in a pre-cooled swinging-bucket rotor centrifuge. All the supernatant but 5 µL was removed. Next, 25 µL of transposase mix (15 µL of 2x TD buffer (Illumina); 1 µL of TDE1 (Illumina), 0.25 µL of 5% digitonin (Promega); 8.75 µL of nuclease- free water) were added to the cells and the pellet was disrupted by pipetting. Transposition reactions were incubated at 37 °C for 30 min in an Eppendorf ThermoMixer with shaking at 450 rpm. Reactions were stopped at 4 °C for 5 min. In order to release tagmented DNA, samples were incubated at 40 °C for 30 min with 5 µL of clean up buffer (900 mM NaCl (Sigma), 30 mM EDTA (Millipore), 2 µL of 5% SDS (Millipore) and 2 µL of Proteinase K (NEB)). DNA was purified using a 2X SPRI beads cleanup kit (Agencourt AMPure XP, Beckman Coulter). CRs were performed using KAPA HiFi DNA Polymerase and customized Nextera PCR indexing primers (IDT). The conditions of the first PCR were: 5 min at 72 °C and 2 min at 98 °C followed by 9 cycles of 20 secs at 9 °C, 30 secs at 63 °C, and 1 min at 72 °C. Depending on the library concentration in this first PCR, a second PCR (2 min at 98 °C followed by 4 to 6 cycles of 20 secs at 98 °C, 30 secs at 63 °C, and 1 min at 72 °C) was performed aiming for a library concentration in the range of 2 to 10 ng/µL. PCR products were purified using a 2X SPRI beads cleanup. Libraries were quantified and their size profiles examined as described above. Sequencing was carried out in an Illumina NextSeq500 using paired-end, dual-index sequencing (Read1: 38 cycles; Read2: 38 cycles; i7 index: 8 cycles; i5 index: 8 cycles).