GSM7109021: F376 E3, YAP/TAZ Osx-cKO, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Embryonic forelimbs were isolated on ice and trimmed to removed excess and loose skin/soft tissue and the handpaw. Individual embryonic tails or yokesacs were placed aside for genotyping. A single cell susupension was generated in the presence of the transcriptional inhibitor Actinomyosin Actinomycin D (MP Biomedicals). Briefly, cells from individual samples were disassociated from the tissue with a digestion media including DMEM, fetal bovine serum (vendorSigma), Collagenase II (Gibco Thermo Fisher Scientific), Pronase (Sigma Aldrich), and Actinomyosin Actinomycin D (2 mg/ml) for 75 minutes at 37oC. Following, cells were washed with PBS/0.01% BSA and passed through 70 mm cell strainers. Then, red blood cells were lysed with a Red Blood Cell Lysis kit (Miltenyi Biotec, 130-094-183), per manufacturer instructions. Dead cells were then removed with a Dead Cell Removal kit (Miltenyi Biotec 130-090-101) and MACS MS columns (Miltenyi Biotec), and a MiniMACS Magnetic Cell Separator (Miltenyi Biotec). Following dead cell removal, each sample was confirmed to have >95% viability. To allow time for genotyping, cells were then processed according to 10x Genomics 'Methanol Fixation of Cells for Single Cellingle-cell RNA Sequencing protocol, Rev D'. Cell suspensions were resuspended per 10x Genomics 'Methanol Fixation of Cells for Single Cellingle-cell RNA Sequencing protocol, Rev D' and processed for sequencing. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Samples OsxF289_E4, OsxF289_E5, OsxF289_E6, OsxF289_E8, and F350 E1 were processed by Chromium Next GEM Single Cell 3' Reagent Kits v3.1 with Single Index Kit T Set A. All others were processed with Chromium Next GEM Single Cell 3' Reagent Kits v3.1 with Dual Index Kit TT Set A