GSM8827481: Patient 1 unaffected skin_scRNA-seq; Homo sapiens; RNA-Seq
The skin punch biopsies were incubated in 200 μl of Dispase solution (2mg/mL) for 30 minutes at  37 C. Following the incubation, the epidermis was peeled from dermis with curved forceps, washed in PBS and successively minced into < 1 mm3 pieces, using scalpel in a serum free RPMI 1640 media with DNase I (0.2 mg/mL), 20 mM HEPES and 0.25 mg/ml Liberase TM, Roche. The suspension was incubated for 90 minutes at 37 C. The digestion was stopped by adding 100 μl FBS and 3 μl of 0.5 M EDTA and filtered through a 70-mm cell strainer. The cells were pelleted and washed twice with PBS containing 1% BSA. Finally the cells were resuspended in PBS containing 0.04% BSA and an aliquot was taken for counting. Library was performed according to the manufacturer's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion. The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.