Illumina HiSeq 4000 sequencing; Single-cell RNA-seq of mouse endothelial cells from three distinct embryonic locations
Single-cell RNA-seq of mouse endothelial cells from three distinct embryonic locations
Timed-matings were set up between C57BL/6 mice, purchased from Charles River. Upon dissection, only embryos staged as Theiler Stage 12 according to Downs and Davies morphological criteria were further processed. Allantois, yolk sac and embryo proper were dissected and placed into separate tubes. Single-cell suspensions were prepared by incubating the embryos with TrypLE Express dissociation reagent (Life Technologies) at 37 °C for 7 min and quenching with heat inactivated serum. Single cells were subsequently stained with Flk1-PE antibody (1:100; Biolegend) and DAPI as viability stain (1µg/ml; Sigma). Live FLK1+ cells were isolated by fluorescence-activated cell sorting (FACS) using a BD Influx sorter into individual wells of a 96-well plate Live FLK1+ cells were isolated by fluorescence-activated cell sorting (FACS) using a BD Influx sorter into individual wells of a 96-well plate containing lysis buffer (0.2% (v/v) Triton X-100 and 2 U/μl RNase inhibitor (Clontech) and stored at −80 °C (1 plate per tissue was prepared). Plates were processed following the Smart-seq2 protocol. Libraries were generated using the Illumina Nextera XT DNA preparation kit.
Experimental Factor: organism part
embryo
Experimental Factor: single cell identifier
cell 119