An RFX/miR-150 axis regulates proliferation of primary human T lymphocytes

ATAC-seq was performed to identify the cis-regulatory genomic elements in in primary human naïve and memory T cells untreated or activated with anti-CD3 and anti-CD28 antibodies for one or three days. Overall design: Primary Naïve and memory T cells from two independent donors were sorted based on the expression of the following surface markers: Naive: CD4+CD25–CD45RA+CCR7+; Memory: CD4+CD25–CD45RA–CCR7+/-and activated for 48hrs on anti-CD3 and anti-CD28 antibodies. From each subset 10^5 cells were taken at day 0 (ex vivo, immediately after sorting), at day1 (24h after activation) and at day3 and lysed. Nuclei were than isolated by centrifugation and treated with Tn5 transposase (made in-house) for 1hr at 37°C. Chromatin tagmentation was inactivated by incubating the samples for 30min at 40°C with a cleanup buffer containing EDTA. Tagmented DNA was isolated using 2× AMPure XP beads and amplified by PCR with barcoded primers using 14 cycles of PCR. Finally, fragments smaller than 500 bp were purified with 0,65x AMPure XP beads and primers removed by purification with 1,8x AMPure XP beads. Libraries were sequenced paired-end on an Illumina NOVAseq.