GSM8898827: Skin CD45+ cells-1; Homo sapiens; RNA-Seq
Samples were processed using a dermatome to isolate the top layer of the epidermis at a thickness of 0.6 µM and subsequently cut into pieces no larger than 4mm by 4mm. Skin pieces were cryopreserved with FCS with 10% DMSO at -80°C until thawing and processing. Tissue pieces were thawed with complete media (RPMI 1640, 10% FCS, 1% PSG, 1% HEPES) and rinsed with fresh complete media to thoroughly remove residual DMSO from the freezing media. Skin tissue was digested in resting media (89% RPMI 1640, 10% FCS and 1% Pen-Strep-Glut) containing 750 ug/ml Collagenase IV and 20 ug/ml DNase I for 2 hours at 37°C. Cells were filtered through a 100 µm strainer, centrifuged at 500g for 5 minutes at 4°C, counted, and resuspended for flow cytometry staining. Cells were transferred to a 96-well v-bottom plate and stained with LIVE/DEAD Fixable Aqua (1:500 in 1X PBS, 20 million cells per 1mL). Cells were washed with 150uL of sort buffer (2mM EDTA, 2% FBS in 1X PBS), centrifuged at 500g for 5 minutes at 4°C, and incubated with the antibody surface cocktail for 30 minutes at 4°C. Cells were washed with 150uL of sort buffer and centrifuged at 500g for 5 minutes at 4°C. Live CD45+ cells were sorted with FACSAria Fusion Flow Cytometer. Cells from four donors were evenly pooled (approximately 14K cells per donor) and encapsulated using the Chromium Next GEM Single Cell 5' Reagent Kits V2 (Dual Index) according to the manufacturer's instructions. Libraries were amplified with 13 cycles of cDNA amplification. Sequencing was performed on the NovaSeq X Plus instrument at the UCSF Center for Advanced Technology.
GEO
GSM8898827_r1