GSM4848109: Unwounded Dorsal Skin scRNA-seq; Mus musculus; RNA-Seq
Single cells were isolated from excised tissue from naïve animals (pooled from 3 wounds) using a collagenase/dispase digestion. Cells were stained with antibodies against CD45 (hematopoietic), CD31 (endothelial) and Ter119 (red blood cells); live cells were discriminated with DAPI. At least 100,000 live, CD45-CD31-Ter119- cells were subjected to single cell capture and droplet formation following instructions in the online Drop-seq protocol v.3.1 (http://mccarrolllab.org/download/905/) and those published in the original Drop-seq paper. In brief, cells at 150,000 cells/ml, barcoded beads at 175,000 beads/ml and droplet generation oil were co-flowed at a rate of 4, 4, and 15 ml/hour, respectively, in a PDMS microfluidics chip to generate oil droplets containing beads and lysed cells. Post flow, droplets were broken and reverse transcription performed. Complementary DNA was PCR amplified, magnetically cleaned up and subjected to tagmentation and sequencing library construction. Prepared libraries were cleaned up and sequenced via Illumina HiSeq4000 or NovaSeq. Drop-seq protocol v.3.1; uses Illumina reagents