GSM5067113: 10x70; Mus musculus; RNA-Seq
At embryonic day (E)13.5, mouse embryos were eviscerated, and the trunk region of the embryo was dissected from the skin, liver and intestine. The dissociation of cells was the same as for human fetal cells. Single EGFP+ cells were sorted on a BD Influx FACS equipped with a 140 µm nozzle and a cooling unit with a sample temperature of 4°C and collected into cold HBSS buffer. Collected cells were pelleted and subjected to RNA sequencing library preparation. On the day of 10x Chromium library preparation the cells were brought to +4°C and centrifuged at 1000 rcf for 10 min at 4°C. Supernatant was carefully removed and 500 uL of rehydration buffer (1X DPBS (Gibco 14190144) containing 1.0% BSA (Sigma, B4287) and 0.5U/?l RNAse Out (ThermoFisher Scientific, 10777019) was added to cell pellet, re-suspended and centrifuged at 1000 rcf for 10 min at 4°C. The wash with rehydration buffer was repeated one more time, after which cells were counted. If a lot of debris was observed during cell count the cell suspension was subjected for FACS to remove debris. After cell count or FACS cells were pelleted again and re-suspended in rehydration buffer to get 700-1200 cells/ul but no less than 50 uL. Library preparation was done in accordance with 10x Genomics Chromium Single Cell 3' (v3) aiming at recovering 5000-8000 cells at Illumina Novaseq with the help of Eukaryotic Single Cell Genomic Facility, SciLifeLab, Stockholm, Sweden.