GSM6202171: E8.5_WT_tdTomato-_1; Mus musculus; RNA-Seq
GSM6202171
10x Genomics Single Cell Gene Expression Single embryos were dissociated into single-cells using 200μl of TriplE Express for 10 minutes at 37°C on a shaking incubator then quenched with 1ml of ice-cold 10% FCS in PBS. Cells were filtered using a 40μM Flowmi cell strainer, span down at 300g for 5 minutes then resuspended in 50μl of PBS containing 0.04% BSA. Cells were counted and viability assessed using trypan blue staining on a Countess II instrument (Invitrogen). >95% of cells were negative for trypan blue indicating high sample quality. embryos were pooled and dissociated as above then flow-sorted using the BD Influx High-Speed Cell Sorter (BD Biosciences) or a BD FACSAriaTM system (BD Biosciences) in a biosafety cabinet, collecting DAPI negative singlets into two 1.5ml tubes, one for tomato positive (knockout cells) and one for tomato negative (host cells). Cells were spun down at 300g for 5 minutes and resuspended in 50ul of PBS containing 0.04% BSA then counted as above. embryos were pooled and dissociated as above then flow-sorted using the BD Influx High-Speed Cell Sorter (BD Biosciences) or a BD FACSAriaTM system (BD Biosciences) in a biosafety cabinet, collecting DAPI negative singlets into two 1.5ml tubes, one for tomato positive (knockout cells) and one for tomato negative (host cells). Cells were spun down at 300g for 5 minutes and resuspended in 50ul of PBS containing 0.04% BSA then counted as above. scRNA-seq was performed using 10x Genomics 3' v3 following manufacturers instructions and loading 16,000 cells. Sequencing was performed using an Illumina Novaseq using the recommended read lengths.