GSM7434423: FM08 Split A, Primary melanocytes, no treatment; Homo sapiens; RNA-Seq
RNA was extracted as per the manufacter's protocol using a 10x Genomics v3 kit. We generated gel beads-in-emulsion (GEMs) using the 10X Chromium system and a Chromium Next GEM Single Cell 3' Reagent Kit v3.1 as per the manufecturer's protocol, aiming for 10,000 cells for recovery. We used the 10X Genomics single-cell RNA-seq kit v3 to sequence cells. We resuspended the cells (targeting ~10,000 cells for recovery/ sample) in PBS and followed the protocol for the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 as per manufacturer directions (10X Genomics, Pleasanton, CA). Briefly, we generated gel beads-in-emulsion (GEMs) using the 10X Chromium system, and subsequently extracted and amplified (11 cycles) barcoded cDNA as per post-GEM RT-cleanup instructions. We then used a fraction of this amplified cDNA (25%) and proceeded with fragmentation, end-repair, poly A-tailing, adapter ligation, and 10X sample indexing per the manufacturer's protocol. We quantified libraries using the High Sensitivity dsDNA kit (Thermo Fisher Q32854) on Qubit 2.0 Fluorometer (Thermo Fisher Q32866) and Bioanalyzer 2100 (Agilent G2939BA) analysis prior to sequencing on a NextSeq 500 machine (Illumina) using 28 cycles for read 1, 55 cycles for read 2, and 8 cycles for i7 index. A subset of FateMap sequencing runs (NRAS melanoma samples and Metronomic therapy experiments), we used NextSeq 2000 (Illumina) using 26 cycles for read 1, 124 cycles for read 2, and 8 cycles for i7 index.