title: GSM8094009: scRNAseq_FL_E125_dCas9P300TSS; Mus musculus; RNA-Seq
library_name: GSM8094009
library_construction_protocol: Two replicates of forelimb buds of E12.5 of Pitx1-Inv1+/- embryos and one replicate of E12.5 Shox2-dCas9P300-ColA1-sgRNAs forelimb bud were micro-dissected and incubated for 12 minutes in 400l trypsin-EDTA 0.25% (Thermo Fischer Scientific, 25300062), supplemented with 40l 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 40m 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 1400cells/ul suspension was prepared. Single-cell libraries were prepared using the Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 following the manufacture's protocol (10X Genomics, PN-1000075). Briefly, Gel beads in EMulsion (GEMs) are generated by combining Single Cell 3ʹ v3.1 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.