GSM9135779: X1 695MB-PL4 Perilesion; Homo sapiens; RNA-Seq
One pair of stable nevus and perilesional melanocyte cultures were grown in MCBD 153 media supplemented as described above. Cells were grown to 75% confluency to ensure they were in the growth phase before being scraped, pelleted, and resuspended in media containing 12% FCS + 2mM EDTA. Sufficient cells were sorted with a BD FACSAria™ Fusion Flow Cytometer (BD Biosciences, New Jersey, U.S.) to achieve 200,000-300,000 viable cells. No cell surface markers were used since all cells were considered to be melanocytic in origin based upon cell morphology and growth characteristics. Only a cell viability marker was used to ensure a high proportion of live cells required for single cell Gel Bead-In-Emulsion (GEM) preparation. The single cell suspensions were loaded onto a 10X Genomics Single Cell A Chip and partitioned along with oligo-containing Gel Beads into nanolitre-scale droplets using the 10X Chromium Controller. Next, a reverse transcription reaction was performed using oligos contained within the Gel Bead. This process tagged each cDNA molecule with an identical 10X barcode and a random Unique Molecular Identifier (UMI). After the generation of full-length barcoded cDNA molecules inside the GEMs, the emulsion was broken and cDNA from single cells was pooled for subsequent sequencing library preparation. Single cell sequencing libraries, containing standard Illumina P5 and P7 sequencing adapters, were generated with the 10X Chromium 3' Single Cell Library Kit.  Each sample was given a unique sample index using the 10X Chromium i7 multiplex kit, allowing for multiplexing of different samples on a single sequencing run. Resulting sequencing libraries were assessed with an Agilent BioAnalyzer DNA High Sensitivity chip, pooled as desired, and quantified with a qPCR KAPA Library Quantification Kit. Denatured libraries were loaded onto an Illumina NextSeq-500 and sequenced using a 150-cycle High-Output Kit with the following protocol steps: 26bp (Read1), 8bp (i7 index), 98bp (Read2).  Read1 supplies the cell barcode and UMI, i7 the sample index, and Read2 the 3' sequence of the transcript.