Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: Cells are loaded, adhered, and incubated in gelatin-coated nanovials functionalized with secretion capture antibodies. Secreted and captured proteins are labeled with oligonucleotide-barcoded detection antibodies. Nanovials loaded with single cells are then introduced into the 10X Chromium workflow for library preparation. Sequencing of resulting libraries results in matched secretion and transcriptomics data for downstream analyses.

Article Snippet: To detect secreted VEGF-A through a sequencing readout, we designed a custom VEGF-A detection antibody conjugated to a 10X Genomics compatible oligo-barcode ( ).

Techniques: Incubation, Labeling, Sequencing

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: a, Standard scRNA-seq equipment including Sony cell sorter, 10X Chromium scRNA-seq device, and Illumina sequencers are used in the different steps of the SEC-seq workflow (top to bottom). Checkmarks indicate the steps that were validated with corresponding equipment. b, Standard curve of VEGF-A on nanovials using recombinant VEGF-A, immobilized via the VEGF-A capture antibody, and detected with AF647-tagged anti-VEGF-A detection antibody shows a dynamic range across 3 orders of magnitude. Horizontal line represents the detection threshold (see ). c, (left) Schematic showing the steps of the VEGF-A secretion assay in single MSC-loaded nanovials. (middle) Flow cytometry histograms of VEGF-A secretion from single MSCs on nanovials after 0, 6, 12 hours of incubation. (right bottom) VEGF-A secretion assay on single MSC-loaded nanovials with and without VEGF-A capture antibody (Ab). More than 90% of cells in nanovials with capture antibody had fluorescence signal above the threshold (dotted line). (right top) The fluorescence microscopy image shows single MSCs on nanovials with secreted VEGF-A detected with a fluorescently (AF647)-tagged VEGF-A detection antibody (magenta) and cells stained with calcein AM (green). Scale bar is 50 µm. d, Bar plot shows cell viability measured by image analysis of live/dead stain (see ) following flow sorting of cells in suspension or loaded in nanovials. e, (top) Schematic of nanovial loading into droplets with 10X training gel beads in the absence of detergent (to prevent cell lysis). (bottom) Brightfield and fluorescence images of nanovials (red) with single MSCs (green) together with a gel bead in a droplet following emulsification. Scale bar is 50 µm. f , Graph showing the proportion of all nanovial-containing droplets with the indicated number of nanovials. g, After droplet formation and in the presence of lysis buffer, lysis of calcein (green)-labeled cells on nanovials was observed by diffusion of the green fluorescent signal throughout the droplets containing single-MSC loaded nanovials. Overlaid fluorescence and brightfield images of droplets generated with a 10X Genomics NextGEM kit. Scale bar is 50 µm. h, Distribution of species-specific reads from a scRNA-seq experiment with nanovials containing human MSCs or mouse fibroblasts pooled in a 1:1 ratio. Species identity was called by mapping to a joined genome contig and determining the ratio of reads from each species’ genome. i, Comparison of transcripts per cell for either suspended (unsorted) MSCs, suspended and sorted (sorted) MSCs, or MSCs loaded on nanovials and sorted (nanovial). j, Scheme explaining the experiment where MSCs cultured under normoxic and hypoxic conditions, respectively, were loaded on nanovials labeled with different oligo-barcoded streptavidin molecules (‘normoxic’ and ‘hypoxic’ barcodes) and analyzed in a 1:1 ratio in a single 10X channel. The scatter plot below depicts the assignment of cells based on the normoxic or hypoxic oligo-barcode attached to nanovials via streptavidin. Mixed cells have a signal for both barcodes. k , UMAP plots of the scRNA-seq data derived from the experiments in (j), where each cell is labeled by their oligo-barcode assignment. Mixed cells are excluded in UMAPs. l, The UMAP from (k) labeled by the hypoxic gene expression signature to identify MSCs cultured in hypoxic conditions.

Article Snippet: To detect secreted VEGF-A through a sequencing readout, we designed a custom VEGF-A detection antibody conjugated to a 10X Genomics compatible oligo-barcode ( ).

Techniques: Recombinant, Flow Cytometry, Incubation, Fluorescence, Microscopy, Staining, Suspension, Lysis, Emulsification, Labeling, Diffusion-based Assay, Generated, Comparison, Cell Culture, Derivative Assay, Gene Expression

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: a, Schematic of the detection of secreted VEGF-A protein and corresponding global gene expression for individual MSCs using the SEC-seq method. b, UMAP dimensionality reduction based on transcriptomes from SEC-seq experiments on normoxic and hypoxic MSCs in nanovials. Cells are labeled according to the culture condition. c, UMAP displaying VEGF-A secretion level, shown as log transformation of the UMI collapsed anti-VEGF-A oligo-barcode reads per cell. d, UMAP displaying VEGFA transcript levels, shown as normalized transcripts per cell. e, Distribution of VEGF-A secretion for cell-loaded nanovials in normoxic and hypoxic conditions, detected by FACS using a fluorescent anti-VEGF-A antibody (top) or by the SEC-seq experiment in (b) using the oligo-barcoded anti-VEGF-A antibody (middle). The last plot shows distribution of VEGFA transcript levels from the SEC-seq experiment cells in (b) (bottom). f, UMAP displaying cluster assignment. g, Violin plots by cluster showing VEGFA transcripts and VEGF-A secretion levels for all cells in the normoxic clusters (N1-N5, red shades), hypoxic clusters (H1–4, blue shades), and mixed clusters (M1–3, green shades) from (f). For mixed clusters, the levels are shown separately for normoxic and hypoxic cells. The dashed line represents the mean across all cells for each plot. Data below this threshold are lightened to highlight differences. h, Scatter plots showing the relationship between VEGFA transcript and VEGF-A secretion levels for individual normoxic (left) and hypoxic (right) cells from the experiment in (b). Best fit regression lines and Pearson correlation coefficients are shown. i, Plot showing the ranking of all detected genes based on the correlation of their transcript levels to the VEGF-A secretion level per cell for normoxic (top) and hypoxic (bottom) MSCs. The rank of the VEGFA gene is highlighted, and the top three genes per sample are also noted.

Article Snippet: To detect secreted VEGF-A through a sequencing readout, we designed a custom VEGF-A detection antibody conjugated to a 10X Genomics compatible oligo-barcode ( ).

Techniques: Gene Expression, Labeling, Transformation Assay

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: a, Scatter plot of the transcript to VEGF-A secretion correlation for all genes from SEC-seq experiments for normoxic and hypoxic MSCs from . The 10 most highly correlating genes based on both experiments are labeled. b, Table giving the ranking (based on average correlation), gene name, correlation to secretion in normoxic and hypoxic cells, and the average of those two values for the ten top genes from (a). c, UMAPs showing VEGF-A secretion levels and expression of 5 select correlated genes from (b) in normoxic and hypoxic MSCs from . The VEGF-A secretion UMAP is given from for comparison. d, As in (c), for a separate SEC-seq experiment performed on MSCs in the normoxic culture condition. e, Cell clusters projected onto the UMAP of the replicate SEC-seq experiment f, Heatmap of the top 10 differentially expressed genes from each cluster (indicated on top) of the SEC-seq experiment in (c,d) (rows=genes, columns=individual cells). Displayed at the top are the log transformed VEGF-A secretion and VEGFA transcript levels. Right: top 3 genes differentially expressed gene for each cluster. g, Heatmap of the top GO terms found for all of the differentially expressed genes from the clusters in (e). The (–logP) value indicates if the term was enriched for a given cluster. h , Venn diagram showing the overlap of differentially expressed genes from the highly secreting cluster in 3 SEC-seq experiments (top left: normoxic MSCs from (3b), top right: hypoxic MSCs from (3b), bottom: normoxic MSCs from (4e)). Overlapping genes form the Vascular Regenerative Signal (VRS). i, Gene ontology analysis for VRS genes from (h). Similar terms were collapsed. j , Average of the normalized transcripts level of VRS genes per cell, displayed for the SEC-seq experiment in (e) and (3b). k, As in (j), for MSCs loaded in oligo-barcoded nanovials (see - ). l, As in (j), for a standard scRNA-seq experiment on unsorted, suspended MSCs. m, Comparison of gene type classification for VRS genes and genes differentially expressed in all clusters in (e) except for those from cluster c5. n, Enrichment of possible TF regulators of the VRS genes based on the TRRUST database. o, Consensus rank of VEGF-A secretion to gene correlation based the SEC-seq experiments used in (h), with red dots displaying all VRS genes. p, Schematic depicting the heterogeneity of VEGF-A secretion in MSCs under normoxic and hypoxic conditions, highlighting the importance of the VRS genes for marking high VEGF-A secretion.

Article Snippet: To detect secreted VEGF-A through a sequencing readout, we designed a custom VEGF-A detection antibody conjugated to a 10X Genomics compatible oligo-barcode ( ).

Techniques: Labeling, Expressing, Comparison, Transformation Assay

Journal: bioRxiv

Article Title: Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

doi: 10.1101/2023.01.07.523110

Figure Lengend Snippet: List of reagents and resources

Article Snippet: To detect secreted VEGF-A through a sequencing readout, we designed a custom VEGF-A detection antibody conjugated to a 10X Genomics compatible oligo-barcode ( ).

Techniques: Conjugation Assay, Recombinant, Modification, Cell Culture, Enzyme-linked Immunosorbent Assay, Sequencing, Software