Tn5 transposase

ATAC-seq was performed as previously described with minor modifications ^{27 (link),70 (link)}. Fifty thousand cells were pelleted at 550 × g and washed with 50 μL ice-cold 1× PBS, followed by treatment with 50 μL lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Nuclei pellets were resuspended in 50 μL transposition reaction containing 2.5 μL Tn5 transposase (FC-121-1030; Illumina). The reaction was incubated in a 37°C heat block for 45 min. Tagmented DNA was purified using a MinElute Reaction Cleanup Kit (QIAGEN) and amplified with varying cycles, depending on the side reaction results. Libraries were purified using a QIAQuick PCR Purification Kit (QIAGEN). Libraries were validated for quality and size distribution using a TapeStation 2200 (Agilent). Libraries were paired-end sequenced (38bp+38bp) on a NextSeq 550 (Illumina).

Human M(IFN-γ) macrophages were left unstimulated [M(IFN-γ)] or stimulated with LPS (100 ng/ml for 4 hours) [M(IFN-γ+LPS 4h)] and subjected to ATAC-seq as previously described (40 (link)). Briefly, 50,000 cells were pelleted, resuspended in 50 μl of lysis buffer [10 mM tris-HCl (pH 7.4), 3 mM MgCl₂, 10 mM NaCl, and 0.1% NP-40 (CA-630, IGEPAL)], and immediately centrifuged at 500g for 10 min at 4°C. The nuclei pellets were resuspended in 50 μl of transposition buffer (25 μl of 2× 13 tagmentation DNA buffer, 22.5 μl of dH₂O, and 2.5 μl of Illumina Tn5 transposase) and incubated for 30 min at 37°C. Transposed DNA was purified with the MinElute PCR Purification Kit (Qiagen) and eluted in 10 μl of elution buffer (Qiagen). Barcoded ATAC-seq libraries were prepared as previously described and sequenced on an Illumina HiSeq 2500.

Omni ATAC-seq libraries were made as previously described (Corces et al., 2017 (link)). Briefly, nuclei were isolated from 30,000 sorted CD8+ surface CAR+ T cells, followed by the transposition reaction using Tn5 transposase (Illumina) for 30 min at 37° C with 1000 rpm mixing. Purification of transposed DNA was completed with DNA Clean and Concentrator (Zymo) and fragments were barcoded with ATAC-seq indices (Buenrostro et al., 2013 (link)). Final libraries were double size selected using AMPure beads prior to sequencing. Paired-end sequencing (2 × 75 bp reads) was carried out on an Illumina NextSeq 500 platform.

Following culture, CD8+ CAR+ TCRVβ5.1+ and CD8+ CAR− TCRVβ5.1-T-cells were sorted on a FACSAria (BD). ATAC-seq was carried out as previously described^{37 (link),38 (link)}. Two replicates were performed for each ex vivo expanded CD8+ CAR+ TCRVβ5.1+ and CD8+ CAR− TCRVβ5.1- T-cell culture. Briefly, nuclei were isolated from 200,000 sorted CD8+ T-cells for each replicate, followed by the transposition reaction in the presence of Tn5 transposase (Illumina, Inc.) for 45 minutes at 37°C. Purification of transposed DNA was subsequently completed with the MinElute Kit (Qiagen) and fragments were barcoded with dual indexes (Illumina Nextra). Paired-end sequencing (2 × 75 bp reads) was carried out using the Illumina NextSeq 500. Raw sequencing data were processed and aligned to the GRC37h/hg19 reference genome using Bowtie2 and regions of significant enrichment were identified using MACS v1.4.2. Merged peak lists were created using BedTools. ATAC sequencing tag enrichment and DNA motif analysis across the merged peak list were carried out using HOMER (homer.salk.edu). Gene Ontology (GO)/Pathway analysis was performed with Metascape (http://metascape.org). Only high-confidence peaks were used for gene ontology and DNA motif analyses. For these evaluations, peaks with an enrichment score less than 5 were filtered out as previously established^{39 (link)}.