Journal: Nature biotechnology

Article Title: Scalable, multimodal profiling of chromatin accessibility, gene expression, and protein levels in single cells

doi: 10.1038/s41587-021-00927-2

Figure Lengend Snippet: a. Schematic of the cell-processing steps that allow retention and profiling of cell-surface markers jointly with chromatin accessibility. Cells are stained with oligo-conjugated antibodies before fixation, permeabilization and transposition with Tn5. b. In droplets, bridge oligos spiked into the barcoding mix promote templated extension of the antibody tags during the first cycle of amplification rendering them complementary to bead-derived barcoding oligos. Extended antibody tags are subsequently barcoded together with the transposed chromatin fragments. c. Species mixing experiment using the Pre-SPRI approach; number of unique nuclear fragments (left) and protein-tag counts (right) associated with each cell barcode. Points are colored based on species classification using ATAC-derived fragments (97.4% agreement by assignment; all but 1 discrepancy was an errant doublet versus singlet classification) d. TSS enrichment scores of mtscATAC-seq without (left) or with concomitant protein tag capture (right). n indicates the number of cells profiled. e. UMAP showing chromatin accessibility-based clustering of PBMCs stained with a 9-antibody panel, with selected markers highlighted. Color bar: protein tag centered log-ratio (CLR) values. f. Cellular distribution of two most commonly detected mtDNA mutations in the population. Thresholds for + were 5% heteroplasmy based on empirical density.

Article Snippet: Transposition and barcoding for ASAP-seq Cell were subsequently processed according to the Chromium Single Cell ATAC Solution user guide (Versions {"type":"entrez-nucleotide","attrs":{"text":"CG000168","term_id":"33868816"}} CG000168 Rev D for v1 and {"type":"entrez-nucleotide","attrs":{"text":"CG000209","term_id":"33868857"}} CG000209 Rev D for v1.1, 10x Genomics) with the following modifications: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 During the barcoding reaction (step 2.1), 0.5 μl of 1 μM bridge oligo was added to the barcoding mix.

Techniques: Staining, Amplification, Derivative Assay

Journal: Nature biotechnology

Article Title: Scalable, multimodal profiling of chromatin accessibility, gene expression, and protein levels in single cells

doi: 10.1038/s41587-021-00927-2

Figure Lengend Snippet: a. PBMCs and compensation beads were stained with fluorophore-conjugated antibodies and subjected to the ASAP-seq workflow with samples withdrawn at the indicated steps and assessed for fluorophore intensity by flow cytometry. CD19 (staining B cells), CD11c (dendritic cells) and CD4 (lymphocytes and monocytes) signal on fixed cells is hardly affected by permeabilization alone, but after the 37°C incubation for 1h to mimic the Tn5 transposition reaction, some signal reduction is observed. b. Barcoding scheme of TSA tags using the bridge oligo for TotalSeq™-A (BOA). TSA tags do not contain UMIs, so to allow molecule counting, UBIs (N9V) are incorporated via the bridge oligo. c. Species mixing experiment as in Fig. 1c, using the Post-SPRI approach for tag recovery. Points are colored based on species classification using ATAC fragments. d. ATAC library complexity and TSS enrichment for fragments from each species under the two protein-tag library approaches. e. Comparison of protein tag complexity between libraries prepared using the pre- and post-SPRI approach. f. Comparison of ATAC library complexity between mtscATAC-seq and ASAP-seq. g. Two-dimensional embedding of the PBMC hashing data using t-SNE. The four major clusters (black) correspond to the four hashing antibodies used to stain the PBMCs. 13,772 cells were recovered and1,396 doublets (red) were detected. h. UMAP embedding resolving PBMC cell types based on chromatin accessibility for cells processed by mtscATAC-seq and ASAP-seq. Data for the two different samples were processed together using cell ranger-atac aggr before dimensionality reduction. i,j. Selected protein markers (i) and corresponding gene score activities (j) superimposed on the ATAC-clustered PBMCs (for the ASAP-seq sample) as in (h).

Article Snippet: Transposition and barcoding for ASAP-seq Cell were subsequently processed according to the Chromium Single Cell ATAC Solution user guide (Versions {"type":"entrez-nucleotide","attrs":{"text":"CG000168","term_id":"33868816"}} CG000168 Rev D for v1 and {"type":"entrez-nucleotide","attrs":{"text":"CG000209","term_id":"33868857"}} CG000209 Rev D for v1.1, 10x Genomics) with the following modifications: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 During the barcoding reaction (step 2.1), 0.5 μl of 1 μM bridge oligo was added to the barcoding mix.

Techniques: Staining, Flow Cytometry, Incubation, Comparison

Journal: Nature biotechnology

Article Title: Scalable, multimodal profiling of chromatin accessibility, gene expression, and protein levels in single cells

doi: 10.1038/s41587-021-00927-2

Figure Lengend Snippet: a. Barcoding scheme of TSB tags using the bridge oligo for TotalSeqB (BOB). TSB tags contain UMIs (encompassing the antibody barcode), negating the requirement for a UBI on the bridge oligo. b. Pairwise comparison of centered log-ratio (CLR) normalized TSA and TSB counts under OMNI lysis conditions (n=5,236 cells). Counts were collapsed for unique molecules using UBIs (TSA panel) or UMIs (TSB panel). c. Comparison of CLR normalised TSB counts under the two lysis conditions. Statistical comparisons are Wilcoxon rank sum test with Bonferroni adjusted p-values (ns = not significant; *padj < 0.05; ** padj < 0.01; *** padj < 0.001). d. UMAP embedding and cluster annotation of the LLL (n=5,236) and OMNI (n=4,748) processed cells. Data for the two different samples were processed together using cell ranger-atac aggr before dimensionality reduction. e. TSA and TSB CLR counts projected on the LLL embeddings. f. TSA and TSB CLR counts projected on the OMNI embeddings.

Article Snippet: Transposition and barcoding for ASAP-seq Cell were subsequently processed according to the Chromium Single Cell ATAC Solution user guide (Versions {"type":"entrez-nucleotide","attrs":{"text":"CG000168","term_id":"33868816"}} CG000168 Rev D for v1 and {"type":"entrez-nucleotide","attrs":{"text":"CG000209","term_id":"33868857"}} CG000209 Rev D for v1.1, 10x Genomics) with the following modifications: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 During the barcoding reaction (step 2.1), 0.5 μl of 1 μM bridge oligo was added to the barcoding mix.

Techniques: Biomarker Discovery, Comparison, Lysis

Journal: Nature biotechnology

Article Title: Scalable, multimodal profiling of chromatin accessibility, gene expression, and protein levels in single cells

doi: 10.1038/s41587-021-00927-2

Figure Lengend Snippet: a. Schematic workflow for combinatorial multiplexing with ASAP-seq. CRISPR-edited cells are first stained with oligo-conjugated hashtag antibodies and then pooled for downstream processing by ASAP-seq. gRNA identities are demultiplexed using hashing antibody counts. b. UMAP embedding of n = 5,825 single cells and their associated gRNAs. c. Heatmap showing mean expression for 27 selected surface protein markers across gRNA perturbations in stimulated cells. d. Heatmap representation of chromVAR bias-corrected transcription factor motif deviation scores for the top 100 most variable transcription factors across perturbation conditions. Associated gRNA and donor information are color-coded and indicated at the top of the plot. e. Overlay on ASAP-Seq UMAP of chromVAR transcription factor motif deviations. The motif for the given transcription factor is indicated at the top of the plot. f. Volcano plots showing transcription factor motifs with significantly changed chromatin accessibility profiles between NTC cells and guides targeting CD3E and ZAP70 (FDR <= 0.05, chromVAR accessibility change >= 0.25). g. Scatterplot of mean gene activity scores for 22 individual gene loci plotted against CLR-normalized mean protein tag counts associated with each gRNA. Values are normalized against NTC cells. h,i. Genomic tracks of (h) PDCD1 (gene encoding PD-1) and (i) IL2RA (gene encoding CD25), indicating pseudo-bulk ATAC signal tracks across gRNAs with corresponding CLR-normalized protein abundance ridge plots. Differentially accessible regions are highlighted in red. Differentially accessible regions not overlapping CARE enhancers are highlighted in blue (i) and the TSS is highlighted in green (i). NFKB2 sequence motif matches are indicated by *.

Article Snippet: Transposition and barcoding for ASAP-seq Cell were subsequently processed according to the Chromium Single Cell ATAC Solution user guide (Versions {"type":"entrez-nucleotide","attrs":{"text":"CG000168","term_id":"33868816"}} CG000168 Rev D for v1 and {"type":"entrez-nucleotide","attrs":{"text":"CG000209","term_id":"33868857"}} CG000209 Rev D for v1.1, 10x Genomics) with the following modifications: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 During the barcoding reaction (step 2.1), 0.5 μl of 1 μM bridge oligo was added to the barcoding mix.

Techniques: Multiplexing, CRISPR, Staining, Expressing, Activity Assay, Quantitative Proteomics, Sequencing