GSM8423234: CD45+ cells from resting neonate and adult skin HTO library; Mus musculus; RNA-Seq
Skins from neonate mice or female adult mice were cut and weighted, around 1.5X2cm sized skin were minced in 2ml PBS with 55um b-mercaptoethanol, 10mM HEPES, 250ug/ml liberase TL and incubated with shaking for 2.5 hours in 37C. Digestions were stopped with adding EDTA to 12.5mM, and rinsed with PBS containing 4% FBS and 10mM HEPES (FACS buffer) through 70um strainers. After washing, single-cell suspensions were preincubated with anti-CD16/32 prior staining with surface fluorescent conjugated antibodies and/or oligo-tagged antibodies at predetermined concentrations. Stained cells were resuspended in 4′,6-diamidino-2-phenylindole (DAPI) in FACS buffer prior to FACS sorting. For resting condition hashtag labeled CD45+ cells were sorted from neonate and adult mice fo library preparation. For PBS or HDM inoculated neonate mice, CD301b+cDC2 cells were enriched from each group to mix with other cell types to get enough representations. Final ratio of different cell types is CD301b+cDC2: Other CD45+ cells: Epidermal cells: Lineage negative cells=1:2:2:2. Cells from each treatment were sequenced independently. Library construction were performed according to manufacter's  instruction  for single cell 3' 10x genomic