Fig. 1

Comparison between DIG and LLL conditions. A Overview of study design for comparison between DIG and LLL conditions. Each of four aliquots of T cells from a human donor were activated and stimulated under a different stimulation condition (total of four stimulation conditions in four tissue culture wells) in a 12-h tissue culture. Each of two aliquots of cells from each of the four tissue culture wells were then labeled with a unique hashtag (total of eight unique hashtags). Approximately equal numbers of the eight uniquely hashtagged cell populations were then pooled and labeled with antibody cocktail. An aliquot of the hashtag- and antibody cocktail-labeled pool of cells was permeabilized with DIG, and another aliquot of the hashtag- and antibody cocktail-labeled pool of cells was permeabilized with LLL prior to the subsequent DOGMA-seq library preparation steps. B–H Boxplot showing quality control metric comparisons between DIG and LLL conditions. Median values are indicated with corresponding colors. B Protein tag complexity per cell. Median ratio = 2.30. C ATAC fragment complexity per cell. Median ratio = 5.67. D Percentage of ATAC fragments mapped to mtDNA per cell. Median ratio = 0.01. E Number of genes per cell. Median ratio = 0.96. F Percentage of UMIs mapped to mtRNA per cell. Median ratio = 1.88. G Percentage of UMIs mapped to exons per cell. Median ratio = 0.71. H Percentage of UMIs mapped to exons per cell, excluding those mapped to mtRNA. Median ratio = 0.65. I TSS enrichment scores over distance from TSS. Maximum values are indicated with corresponding colors. J Pairwise comparison of protein tag detection frequencies under LLL (x-axis) and DIG (y-axis) conditions. Each point represents a single protein tag. Blue points highlight isotype control protein tags. Grey points are all other protein tags. K Correlation of protein tag fold change (log₂) under DIG and LLL conditions in two groups of T cells that were both activated and cultured with IL-1β and IL-23, and one of the two groups was also cultured with PGE2. Top upregulated protein tags are highlighted in red; downregulated protein tags are highlighted in blue. L Pairwise comparison of gene detection frequencies under LLL (x-axis) and DIG (y-axis) conditions. Each point represents a single gene. Blue points highlight ribosomal protein genes (RPL/S); red points highlight mitochondrial genes (MT-). Grey points are all other genes. M Correlation of gene fold change (log₂) under DIG and LLL conditions in two groups of T cells that were both activated and cultured with IL-1β and IL-23, and one of the two groups was also cultured with PGE2. Top upregulated genes are highlighted in red; downregulated genes are highlighted in blue. N (i) “Harmonized” 3WNN UMAP plot showing clusters identified in 3WNN clustering of DOGMA-seq data under DIG condition. (ii) “Harmonized” 3WNN UMAP plot showing cell types predicted after projection into the Azimuth PBMC reference. O (i) “Harmonized” 3WNN UMAP plot showing clusters identified in 3WNN clustering of DOGMA-seq data under LLL condition. (ii) “Harmonized” 3WNN UMAP plot showing cell types predicted after projection into the Azimuth PBMC reference. P Line plots showing average ROGUE (purity of identified clusters) for clustering based on 3WNN, RNA, ADT, and ATAC spaces, with resolution ranging from 0.02 to 0.3. Number of clusters identified in each clustering was labeled. Q Bar plot show that proportions of predicted cell types in DOGMA-seq data under DIG and LLL conditions are quite similar. R, S “Harmonized” 3WNN UMAP plots highlight canonical markers for Th17 cells in DOGMA-seq data. ATAC marker is motif activity (the deviations in chromatin accessibility across the set of regions related to the motif) calculated from ATAC-seq peaks. R Under DIG condition. Cluster 1 in Fig. 1Ni is enriched for Th17 cells based on CCR4 ADT, CCR6 ADT, and RORC ATAC signals. S Under LLL condition. Cluster 1 in Fig. 1Oi is enriched for Th17 cells based on CCR4 ADT, CCR6 ADT, and RORC ATAC signals.