Journal: bioRxiv

Article Title: An interwoven network of transcription factors, with divergent influences from FoxP3, underlies Treg diversity

doi: 10.1101/2023.05.18.541358

Figure Lengend Snippet: A) Sort strategy used for cell hashtagging in WT and KO FoxP3 heterozygote scATAC-seq experiment in . B) Combined UMAP of Tconv, KO Treg, and WT Treg as in , colored by Leiden clusters. C) scATAC-seq UMAP from , colored by number of unique reads per cell. D) UMAP from separated by hashtag assignment, with colored boxes corresponding to sort gates from S8A. E) Overlap between FoxP3 KO and WT populations (left) vs between Gata3 KO and WT populations (right), as measured by the Local Inverse Simpson’s Index (LISI). LISI values range from 1 for complete separation to 2 for complete overlap (p value from Wilcoxon Rank Sum Test). F) Quantification of CD44+ Tregs from across biological replicates.

Article Snippet: Before sorting, cells were hashtagged with mouse TotalSeqA DNA-barcoded hashtag antibodies at the same time as staining with fluorophore-conjugated antibodies (BioLegend).

Techniques:

Journal: BMC Biology

Article Title: No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells

doi: 10.1186/s12915-020-00941-x

Figure Lengend Snippet: Schematic overview of experimental design. PBMCs from 8 healthy HLA-mismatched donors (tubes on left) were barcoded with MULTI-seq LMOs (black double-helix hybridized to red DNA barcode) and BD single-cell multiplexing kit (SCMK) antibodies (black antibody conjugated to teal DNA barcode). Cells were then strategically pooled to directly assess whether mixing HLA-mismatched PBMCs during scRNA-seq causes alloreactivity

Article Snippet: PBMCs from 8 healthy HLA-mismatched donors (tubes on left) were barcoded with MULTI-seq LMOs (black double-helix hybridized to red DNA barcode) and BD single-cell multiplexing kit (SCMK) antibodies (black antibody conjugated to teal DNA barcode).

Techniques: Multiplexing

Journal: BMC Biology

Article Title: No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells

doi: 10.1186/s12915-020-00941-x

Figure Lengend Snippet: MULTI-seq and SCMK classifications largely match in silico genotyping, with lower SCMK classification efficiency and bias against activated CD4+ T cells. a Sample classification results from three demultiplexing pipelines (e.g., deMULTIplex, souporcell, and demuxEM) projected onto MULTI-seq (top) and SCMK (bottom) sample barcode space. n = 4032 cells from microfluidic lane #3. b Classification frequencies across all PBMC cell types following SCMK sample demultiplexing. c SCMK unclassified cells in CD4+ T cell gene expression space. n = 6879 CD4+ T cells

Article Snippet: PBMCs from 8 healthy HLA-mismatched donors (tubes on left) were barcoded with MULTI-seq LMOs (black double-helix hybridized to red DNA barcode) and BD single-cell multiplexing kit (SCMK) antibodies (black antibody conjugated to teal DNA barcode).

Techniques: In Silico, Expressing

Journal: Communications Biology

Article Title: Development of a sequencing system for spatial decoding of DNA barcode molecules at single-molecule resolution

doi: 10.1038/s42003-020-01499-8

Figure Lengend Snippet: a Schematic illustrations showing a procedure of sequencing of DNA barcoded antibody (Ab-Oligo) on our system. b A fluorescence image of individual CD55 Ab-Oligos visualized by the 1st virtual terminator incorporation. Scale bar represents 3 μm. Molecules identified as single fluorescence spots via image analysis are encircled with colors corresponding to those shown in d . c Extension traces of individual molecules; reads <5 nt were excluded. Each trace represents the extension of each molecule shown in b . d Average number of identified barcode molecules per FOV. Error bars represent SD ( N = 16). Note that for analyses shown in b – d , f reference to identify individual reads contained only CD55 barcode sequences. e Comparison of read lengths between different sequencing directions. f Comparison of identification efficiency between 6Q and 12Q. g Estimation of multiplexing ability. False positive identification (incorrect) was calculated by changing the number of barcode sequences in the reference (1–200 types of sequences). Up to 30 types in a reference, false positive identification was negligible. Colored ribbon plots represent SD.

Article Snippet: Fig. 4 Visualization and identification of DNA barcoded antibodies at single-molecule resolution. a Schematic illustrations showing a procedure of sequencing of DNA barcoded antibody (Ab-Oligo) on our system. b A fluorescence image of individual CD55 Ab-Oligos visualized by the 1st virtual terminator incorporation.

Techniques: Sequencing, Fluorescence, Comparison, Multiplexing