smartseq2 single cell rna-seq  (fluidigm)

Journal: Cell systems

Article Title: Single-cell analysis reveals regulatory gene expression dynamics leading to lineage commitment in early T cell development

doi: 10.1016/j.cels.2019.09.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: C1TM-Fluidigm Smartseq2 Single Cell RNA-seq ETP-DN2a cells were purified as a continuum as described above ( Fig. S1c ), except that no DN3 cells were pooled in for C1 analysis.

Techniques: Virus, Recombinant, Amplification, Multiplex Assay, Gene Expression, Software, Flow Cytometry, Microscopy, Fluorescence, Inverted Microscopy

Journal: Cell systems

Article Title: Single-cell analysis reveals regulatory gene expression dynamics leading to lineage commitment in early T cell development

doi: 10.1016/j.cels.2019.09.008

Figure Lengend Snippet: High T-cell precursor frequency in ETP cells and bulk population gene expression comparison with DN2a cells. a) Schematics of early T-cell developmental stages, checkpoints, associated key developmental markers, and previously unresolved questions addressed in this study. b) Diagram of clonal culture and imaging methods for following the development of individual sorted ETP cells and a representative false color image of the progeny of an ETP clone (top). Histogram plots showing the numbers of ETP clones with different percentages of CD25+ (magenta) or Bcl11b+ (cyan) cells on day 6 of culture (n = 66 viable clones) (bottom). c-d) Heatmaps of bulk RNAseq measurements on Flt3+ and Flt3− ETP and Bcl11b− (uncommitted) and Bcl11b+ (committed) DN2a sorted populations. Color scales indicate raw expression levels as log(FPKM+0.1), without row normalization. Some samples were sequenced with pre-amplification, indicated (o) (see Methods). c) Clustered expression heatmap of bulk RNAseq measurements for genes differentially expressed between all ETP and committed Bcl11b+ DN2a cells (n≥3, adj. pval<0.05, fold change ≥ 2 either way, also see Table S1). Representative non-T or stem/progenitor genes are labeled. d) Selected key genes involved in T development, on the same populations as in (c).

Article Snippet: C1™-Fluidigm Smartseq2 Single Cell RNA-seq ETP-DN2a cells were purified as a continuum as described above ( Fig. S1c ), except that no DN3 cells were pooled in for C1 analysis.

Techniques: Expressing, Imaging, Clone Assay, Amplification, Labeling

Journal: Cell systems

Article Title: Single-cell analysis reveals regulatory gene expression dynamics leading to lineage commitment in early T cell development

doi: 10.1016/j.cels.2019.09.008

Figure Lengend Snippet: Semi-supervised C1 Fluidigm (C1) analysis of single cells in the ETP-DN2a developmental continuum supports co-expression hierarchy of T-lineage and progenitor-associated genes. a) Principal component (PC) loading of first 2 PCs of the analysis based on genes that are differentially expressed in bulk RNAseq shown in Table S1. b) PC1–2 display of 193 cells measured by C1, colored by stage categorization of Flt3, Il2ra (ETP vs. DN2a), and Bcl11b positivity. c) tSNE display of C1 data with SLM clusters color projected. Both tSNE and clustering with SLM were performed with PC 1–10. d) tSNE display with expression patterns of specific genes as indicated overlaid in red. e) Heatmap of expression patterns of selected genes (‘non-T’ genes and ‘T-associated’ genes). The clusters are ordered by approximate T developmental order, according to c) and d). Also see Table S4 for the list of feature genes that are enriched in individual clusters. f) Bi-plots of expression patterns of two non-T lineage markers Irf8 and Mpo, against T specification genes Tcf7 and Bcl11b, showing the pattern of overlap of Mpo and both T specification genes. Irf8, on the other hand, overlaps with early T specification gene, Tcf7, but minimally with Bcl11b, which is expressed at a later stage. The dots are colored by expression of Il2ra (CD25) on a log transformed color scale. g) Co-expression patterns of stem and progenitor genes and T specification genes Tcf7, Gata3 and Bcl11b. n= 228 total cells measured, n= 193 cells were shown in this figure after filtering for single cells with a minimum of 3600 genes and a mitochondrial gene fraction under 0.11.

Article Snippet: C1™-Fluidigm Smartseq2 Single Cell RNA-seq ETP-DN2a cells were purified as a continuum as described above ( Fig. S1c ), except that no DN3 cells were pooled in for C1 analysis.

Techniques: Expressing, Transformation Assay

Journal: Cell systems

Article Title: Single-cell analysis reveals regulatory gene expression dynamics leading to lineage commitment in early T cell development

doi: 10.1016/j.cels.2019.09.008

Figure Lengend Snippet: Summary of key findings in this study Data imply sequential sub-stages within the ETP compartment before transition to DN2a, not only marked by asynchronous downregulation of progenitor genes but also by transient activation of gene waves as the cells progress toward commitment. The frequency of T lineage potential is very high in ETPs overall, and although some transiently activated genes are otherwise associated with non-T fates (multilineage priming), alternative lineage potential in pro-T cells decreases monotonically as the cells progress from Flt3+ ETP to Flt3− ETP to DN2a to commitment.

Article Snippet: C1™-Fluidigm Smartseq2 Single Cell RNA-seq ETP-DN2a cells were purified as a continuum as described above ( Fig. S1c ), except that no DN3 cells were pooled in for C1 analysis.

Techniques: Activation Assay

Journal: Cell systems

Article Title: Single-cell analysis reveals regulatory gene expression dynamics leading to lineage commitment in early T cell development

doi: 10.1016/j.cels.2019.09.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: C1™-Fluidigm Smartseq2 Single Cell RNA-seq ETP-DN2a cells were purified as a continuum as described above ( Fig. S1c ), except that no DN3 cells were pooled in for C1 analysis.

Techniques: Recombinant, Amplification, Multiplex Assay, Expressing, Software, Flow Cytometry, Microscopy, Fluorescence, Inverted Microscopy