Illumina HiSeq 4000 sequencing; Single-cell RNAseq of additional control chimeric mouse embryos at embryonic day 8.5 of mouse development
Single-cell RNAseq of additional control chimeric mouse embryos at embryonic day 8.5 of mouse development
tdTomato-expressing mouse embryonic stem cells (ESC) were derived as previously described(http://dx.doi.org/10.1101/pdb.prot094086) from E3.5 blastocysts obtained by crossing a male ROSA26tdTomato (Jax Labs – 007905) with a wildtype C57BL/6 female. The cells were expanded under the 2i+LIF conditions(https://dx.doi.org/10.1038%2Fnature06968) and transiently transfected with a plasmid expressing Cre using Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific # L3000008) according to manufacturer's instructions. Single GFP+ cells were sorted 48h post-transfection into 96-well plates. Individual clones were allowed to grow and were manually picked for expansion. A tdTomato+, male, karyotypically normal line, competent for chimera generation as assessed using morula aggregation assay, was  injected into C57BL/6 E3.5 blastocysts. Chimeric embryos were subsequently transferred into recipient females at 0.5 days of pseudopregnancy following mating with vasectomised males, as described previously(https://doi.org/10.1242/dev.096875). Chimeric embryos were harvested at E7.5 and E8.5, dissected, pooled, and 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. Cells were resuspended in in PBS with 0.4% BSA and cell counts were assessed with a haemocytometer for subsequent 10X scRNA-seq library preparation (version 2 chemistry) 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: genotype
ROSA26tdTomato