Fig. 2: single cell RNA seq of Index sorted HE and IAHC identify HSC-HE and T2-HSC.
From: Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate
A Scheme of the experimental set up. E10.5 and E11.5 AGM lysates were stained for the indicated cell surface markers and Index sorted for Smart seq 2 single cell RNA sequencing. The representative Flow cytometry plot highlights the sorted populations. CD31 + GFI1+cKIT- (GFI1 + HE), CD31 + GFI1+cKIT+ (GFI1 + IAHC) and CD31 + GFI1-cKIT+ (GFI1-IAHC). Cells were sorted in 2 independent experiments from a pool of 5 and 6 at E11.5 and 7 at E10.5 AGMs. B UMAP representation of all Index sorted and sequenced cells after quality control (n = 775 cells). The colors highlight the gate they were sorted with GFI1 + HE (green, n = 281 cells), GFI1 + IAHC (magenta, n = 356 cells) and GFI1-IAHC (blue, n = 138 cells). Green circle highlights the cluster of cells that consists only of cells sorted as GFI1-HE. C UMAP showing the 11 identified clusters. HSC-HE (n = 76 cells) and HSC (n = 109 cells) are designated. D Heatmap of selected HE and HSC specific genes across all clusters. E Forced directed graph layouts of all sequenced cells with colors highlighting individual clusters (left) and Jag1 normalized gene expression levels (right). F UMAPs showing the normalized expression levels of selected genes with annotation of HSC-HE and HSC (G) Dot plots showing the Fluorescence Activated Cell Sorting (FACS) Index levels (Mean Fluorescence Level, MFI) for NOTCH1, DLL4 and JAG1 considering all HSC-HE cells or HSC CD45+ or CD45- cells and distinguishing by developmental stage E10.5 or E11.5 (HSC-HE E10.5 n = 29; HSC-HE E11.5 n = 47; HSC E10.5 CD45- n = 43; HSC E11.5 CD45- n = 14; HSC E11.5 CD45+ n = 40). Group HSC E11.5 CD45+ was dropped since it only had three cells. Cells without CD45 MFI information were discarded (n = 9). Cells were labelled as CD45+ for those MFI values greater than Q1 in HSC E11.5 cells. Black dots indicate mean values and error bars refer to +/- standard deviation. (UMAP: Uniform Manifold Approximation and Projection; FA: Force Atlas).
