GSM6598855: Healthy control patient 3; Homo sapiens; RNA-Seq
6 mm punch biopsy specimen was first incubated in RPMI 1640 medium (Gibco) containing 5% fetal bovine serum (5% FBS/RPMI) and 10 mg/ml Dispase II (Sigma D4693-1G) for 45 minutes at 37° C shaking at 200-250 rpm. The sample was then removed from the media and minced with sterile iris scissors, followed by digestion with Liberase TM (Sigma) 0.5 mg/ml and DNase I 30 Units/ml in 5% FBS/RPMI for 45 minutes at 37° C shaking at 200-250 rpm. The resulting single cell suspension was then filtered through a 70 mm nylon membrane and washed. Live cells were sorted on a FACSAria and their final concentration and viability quantified with Trypan blue on a hemacytomer. The cells were pelleted and suspended in phosphate buffered saline containing 0.04% bovine serum albumin between 500-1000 cells/ml. 3000-6000 cells with greater than 80% viability were submitted to the Yale DNA Sequencing facility for generation of single-cell cDNA libraries using the Chromium Single Cell Controller (10x Genomics). per 10x Genomics protocol using Single Cell 3' v2. Each cDNA library was sequenced paired-end on 1 lane with 75 base-pair read length using the Illumina HiSeq 2500 System generating at least 75,000 reads per cell.