GSM8108430: iFlpMosaic-TomatoN123_KO; Mus musculus; RNA-Seq
For RNA-seq analysis, embryos were dissociated using Miltenyi Biotec Tissue Dissociation Kit 2 following the instructions. A volume of 1,2ml of dissociation solution was used per embryo inside a 2ml round bottom tube. Following a 5 minutes incubation in a 37C water bath, tubes were put inside a pre-warmed 50ml falcon and incubated for 25 minutes in the MACSmix Tube Rotor. After cells were resuspended with a Gilson P1000 pipet for 30 times. Starting slow and increasing speed gradually. To the 1.2ml of cells, 3ml of sorting buffer (10% FBS in DPBS -Ca2-Mg2-) was added and this solution transferred to a 5ml syringe. This volume was pressed slowly through a small 70 μm strainer to remove clumps. Each embryo gave around 30 million cells at this stage. Cells suspensions were spinned at 400g for 6 minutes. Pellet was detached and ressuspended in 300μl sorting buffer containing DAPI. DAPI negative, single, and live Tomato+ and YFP+ cells were sorted to Eppendorf tubes containing 300ul of Sorting buffer. 66k Tomato+ and 30k YFP+ cells were sorted using a 100 μm Nozzle, 20 PSI and high purity scale and low flow rate (1) to preserve cell viability and decrease contamination. At the end sorted cells were spinned at 500g for 5min, and resuspended in 30-40μl Cell Capture Buffer (DPBS (-)Ca2+ (-)Mg2+ with 0.04%BSA). After counting cells in Countess III, two independent 10x genomics ports were loaded with either 16k Tomato+ cells (90% vaibility) or 16k YFP+ cells (88% viability). Single cell suspensions of freshly isolated ECs were resuspended in Cell Capture buffer to a final concentration of 800 to 1600 cells/μl. scRNA-seq libraries were prepared using the Chromium Single Cell 3´ Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer's instructions.