Illumina HiSeq 2500 sequencing; Single-cell RNAseq of Tal1 knockout chimeric mouse embryos at embryonic days 7.5 and 8.5 of mouse development.
Single-cell RNAseq of Tal1 knockout chimeric mouse embryos at embryonic days 7.5 and 8.5 of mouse development.
Mouse embryonic stem cells with inactivated Tal1 were expanded under 2i+LIF conditions and stably transfected with a constitutively expressed H2B-tdTomato construct under the control of a CAG promoter. They were then injected into C57BL/6 E3.5 blastocysts, and the resulting chimeric embryos were subsequently transferred into recipient females at 2.5 days of pseudopregnancy following mating with vasectomised males. Chimeric embryos were harvested at E7.5 and E8.5 of development, dissected, and single-cell suspensions were generated from pools of embryos. 7 embryos were harvested at E7.5, and 11 embryos at E8.5. 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-cell suspensions were sorted into tdTomato+ and tdTomato- samples using a BD Influx sorter with DAPI at 1µg/ml (Sigma) as a viability stain for subsequent 10X scRNA-seq library preparation. Steps were performed according to manufacturers' instructions (10X Genomics, v2 chemistry) Steps were performed according to manufacturers' instructions (10X Genomics, v2 chemistry). Specific produce numbers were: Chromium Single Cell 3' Library kit v2 P/N-120234; Chromium Single Cell 3' Gel Bead kit v2 P/N-120235;  Chromium Single Cell 3' Chip kit v2 P/N-120236; Chromium i7 Multiplex kit v2 P/N-120262.
Experimental Factor: developmental stage
embryonic day 8.5
Experimental Factor: genotype
Tal1 -/-; CAG-H2B-tdTomato