GSM5151766: scRNAseq-HL-E125-delPitx1; Mus musculus; RNA-Seq
Two replicates of fore and hindlimb buds of E12.5 wildtype embryos and hindlimb buds of mutant embryos (Pitx1Pen-/Pen-, Pitx1-/-) were micro-dissected and incubated for 12 minutes in 400ul trypsin-EDTA 0.25% (Thermo Fischer Scientific, 25300062), supplemented with 40ul of 5% BSA. During incubation tissues were disrupted by pipetting after 6 minutes of incubation and at the end of the 12 minutes. Trypsin was then inactivated by adding 2x volume of 5% BSA and single cell suspension was obtained by passing cells in a 40um cell strainer. Cells were then spun at 250g for 5 minutes at 4° and resuspended in 1%BSA in PBS. Cells were then counted using an automatized cell counter and a 1% BSA 700cells/ul suspension was prepared. 10ul of this solution was used as input for the 10X Genomics library preparation. Single-cell libraries were prepared using the Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 following the manufacture's protocol (10X Genomics, PN-1000075). Briefly, Gel beads in EMulsion (GEMs) are generated by combining Single Cell 3ʹ v3 Gel Beads, a Master Mix containing cells, and Partitioning Oil onto Chromium Chip B. Incubation of the GEMs produced from the poly-adenylated mRNA barcoded, full-length cDNA. Immediately, gel beads are dissolved and cDNA is amplified via PCR followed by library construction and sequencing. Libraries were paired-end sequenced on a HiSeq 4000. On average, 7000 cells were loaded on the Chromium Chip and between 25000-35000 mean reads were obtained.