GSM8170147: Meis2 cKO E13.5, scRNA-seq; Mus musculus; RNA-Seq
Embryos were harvested at stages E12.5 and E13.5 and genotyped. Brains were separated from dissected heads and craniofacial tissues were homogenized to single-cell suspension using TrypLE Express (Gibco). The highest efficiency was achieved by 3 rounds of incubation at 37 0C for 3 minutes, which was stopped in D-MEM with 10% FCS, followed by centrifugation at 1000 g for three minutes. Cells were resuspended in HBSS with 3% FCS before FACS. A total of 61% of cells were GFP-positive during FACS sorting. After cell sorting, 98.3% of cells were GFP-positive with the cell viability 99% .  For each condition, samples from 3 control embryonic heads (Wnt1-Cre2; Meis2 +/+; R26R-EYFPfl/+) and three conditional knockouts (Wnt1-Cre2; Meis2 fl/fl; R26R-EYFPfl/+) were pooled for scRNA-seq. Single-cell libraries were prepared using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 according to the manufacturer's protocol targeting 10,000 cells per sample. Briefly, GEM droplets with oligo-dT primers containing cell-barcode and UMI sequence were encapsulated together with cells. Cells were lysed inside droplets and RNA were transcribed into cDNA. The droplets were broken, and cDNA was amplified and fragmented. Adaptors were ligated to fragmented cDNA and amplified using Illumina specific adaptors with unique dual indices. Libraries were equimolarly pooled and sequenced using NovaSeq 6000 instrument with SP flow-cell in mode: read1 – 28 cycles, index1 – 10 cycles, index2 – 10 cycles, read2 – 90 cycles. About 250M reads per sample were obtained.