GSM8155798: scMm_Shox2trac_HL_E125_rep1; Mus musculus; RNA-Seq
Two replicates of hindlimb buds of E10.5, E11.5, and E12.5 and one replicate of E13.5 Shox2trac embryos were micro-dissected and incubated for 8, 9, 12 or 15 min respectively in 400ul of trypsin-EDTA 0.25% (Thermo Fischer Scientific, 25300062),supplemented with 40ul of 5% BSA. During incubation tissues were disrupted by pipetting after the first 6 minutes of incubation and at the end of the rest of the incubation time. 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. Single-cell libraries were prepared using the Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.0 or v3.1 following the manufacture's protocol (10X Genomics, PN-1000075 or PN-1000121). In brief, Single-Cell 3' Gel Beads were combined with the Master Mix containing single cells, and Partitioning Oil onto Chromium Chip B, to generate Gel Beads in Emulsion (GEMs). Full-length cDNA was produced, during beads incubation, from the poly-adenylated mRNA barcoded. Right after, gel beads were dissolved, cDNA was amplified via PCR. This cDNA was used to construct the library and then sequenced on an Illumina HiSeq 4000 or an Illumina NovaSeq 6000. On average, 7000 cells were loaded on the Chromium Chip and between 25000-35000 mean reads were obtained.