GSM8462811: Arm, Young, Patient 1; Homo sapiens; RNA-Seq
To generate a single cell suspension from adult skin, the process involved cutting healthy skin into thin strips in PBS. The top 200 μm layer was isolated using a dermatome with a Pilling Wecprep blade and a .008 gauge Goulian guard. Grid slits were introduced into the skin sheets for enzymatic access, followed by a 1-hour treatment with 2U/ml dispase II in RPMI at 37°C. Subsequently, the epidermis was peeled from the dermis, and both fragments were separately digested in a petri dish at 37°C 5% CO2 in RF-10 media with 1.6 mg/ml type IV collagenase (Worthington, CLS-4) overnight (12 hours). The work was conducted within class II biological safety cabinets using autoclave-sterilized equipment. The media was collected using a serological pipette and filtered through a sterile 100 μm cell strainer (BD Falcon, 352360). After washing the petri dish and strainer with RF-10 to gather any remaining cells, centrifugation was employed to pellet the cells at 500 x g for 5 minutes. The supernatant was discarded, and the pellet was gently resuspended in 1 ml RF-10 through pipetting up and down. Cell counting was performed using a hemocytometer after staining 10 μl of the sample with a 1:1 mixture of 0.4% trypan blue (Sigma, T8154) to identify dead cells. scRNA-seq libraries were generated following the manufacturer's instructions using the Chromium 10x Single-Cell Instrument (10x Genomics) and 10x Genomics Chromium Single Cell 3' GEM Library and Gel Bead Kit v2. Each channel was loaded with cells, aiming for 5,000 cells per sample, with cell concentration measured by Moxi GO II (Orflo Technologies). The cDNA amplification and sample identification were performed in the Bio-Rad C1000 Touch Thermal cycler with 96 Deep-Well Reaction Module, utilizing 12 cycles. Amplified cDNAs and final libraries were assessed using a Fragment Analyzer (AATI) with a High Sensitivity NGS Analysis Kit (Advanced Analytical) to determine the average fragment length of the 10x cDNA libraries. Quantification of the libraries was done via qPCR using the Kapa Library Quantification kit. Subsequently, the libraries were diluted to a final concentration of 2 nM and pooled together for each run of NovaSeq sequencing. The NovaSeq 6000 Sequencing System (Illumina) was used for sequencing all the libraries.