DOGMA-seq Assay for Transposase Accessible Chromatin (ATAC)  library
25 healthy study participants with ages 18-35 years were enrolled with informed consent in a research protocol compliant with all relevant ethical regulations, which was approved by the University of Pittsburgh Institutional Review Board.  T cells were enriched from peripheral blood samples; cryopreserved; thawed in batches; exposed to anti-CD3/CD28/CD2 activation stimuli, IL-1β and IL-23 alone or in combination with PGE2, TGF-β, or both PGE2 and TGF-β for 12 hours in tissue cultures; and profiled using DOGMA-seq, cell hashing, and cell permeabilization with digitonin (Mimitou et al. Nat Biotechnol. 2021 Oct;39(10):1246-1258. PMID: 34083792; PMCID: PMC8763625) modifications to the Chromium Next GEM Multiome ATAC + Gene Expression protocol (kit 1000285, user guide CG000338 Rev D, 10X Genomics).  DOGMA-seq incorporates simultaneous assays for gene expression (GEX), transposase accessible chromatin (ATAC), and cell surface protein antibody-derived tags (ADT) in the same single cells.  Cell hashing with hashtag oligonucleotides (HTO) mitigated batch effects between samples from the same study participant but stimulated differently.  GEX libraries were indexed using the Single Index Kit T Set A (kit 1000213, 10X Genomics) instead of the Dual Index Kit TT Set A (kit 1000215, 10X Genomics).  ADT and HTO libraries were generated using TotalSeq™-A antibodies (BioLegend) and amplified with custom index primers.  Final libraries were quantified, pooled, and sequenced on Illumina NovaSeq 6000 instruments to generate paired-end FASTQ files for downstream analysis.
NIDDK-phs004468
Duerr_20220524_DOGMAseq-2_ATAC