GSE139052
Single-cell RNA-seq of sorted E11.5 AGM HSCs
The aim of this study was to analyse the heterogeneity within the first haematopoietic stem cells generated during mouse development using single-cell RNA-Seq. Freshly dissected aorta-gonads-mesohenphros (AGM) regions from E11.5 mouse embryos were dissociated by collagenase and the cells stained for the cell surface markers EPCR and CD45. EPCR+CD45+ cells were index-sorted as single cells into 96-well plates and processed based on the SmartSeq2 protocol. PCA and tSNE analyses identified 2 main subpopulations which are segregated based on differences in cell cycle. Known HSC markers such as Gata2 and Runx1 show little correlation. Overall design: EPCR+CD45+ E11.5 AGM cells, which contain all HSCs, were analysed by single-cell RNA-Seq to generate HSC2 cells. Freshly dissected aorta-gonads-mesohenphros (AGM) regions from E11.5 mouse embryos were dissociated by collagenase and the cells stained for the cell surface marker Ngfr. Ngfr+Pdgfrb- cells were index-sorted as single cells into 96-well plates and processed based on the SmartSeq2 protocol. This was done separately for Ngfr+Pdgfrb- cells isolated from the ventral side of the dorsal aorta and from the dorsal side of the aorta, but the two data sets were analysed together. This generated Dorsal and Ventral cell populations. tSNE analysis identified 6 subpopulations which differentially express markers that define specific maturation steps, thus allowing a reconstruction of the differentiation pathway. Pdgfrb+Ngfr- E11.5 AGM cells, which were isolated from the ventral side of the aorta and which contain subaortic mesenchymal cells, were analysed by single-cell RNA-Seq. This population are the Mesenchymal cell population.