GSM4186888: validation - 5w - replicate 1; Mus musculus; RNA-Seq
After sacrifice, the back of the mice was shaved and the dorsal skin was isolated and floated on HBSS + 0.04% BSA. The skin was then cut into smaller pieces (around 1 mm in width) and incubated in 0.2% collagenase at 37°C for one hour. The partly digested skin was placed in a 70 µm cell strainer, smashed with a piston and rinsed three times with HBSS + 0.04% BSA. The flow-through was saved on ice, while the undigested tissue remaining in the cell strainer was incubated in 0.05% trypsin-EDTA for 15 minutes at 37°C. The trypsin-digested tissue was smashed once more and then passed through a 70 µm cell strainer. The flow-through of the collagenase digestion (containing mainly stromal cells) and the flow-through of the trypsin digestion (containing mainly epidermal cells) were pooled, spun down and subsequently filtered through a 40 µm cell strainer. The viability of the cell suspension was determined using trypan blue. Single-cell cDNA libraries were prepared using the 10X Genomics Chromium Single Cell 3' kit (main dataset: version1; validation dataset: version 3) according to the manufacturer's instruction. The prepared libraries were sequenced on an Illumina HiSeq2500 (main dataset) or Illumina NovaSeq 6000 (validation dataset) respectively.