GSM4396314: AML1566_AraC_scRNAseq; Homo sapiens; RNA-Seq
Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 3' v3 Reagent Kit (10× Genomics) following the directions of the manufacturer. Briefly, the initial step consisted in performing an emulsion where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10× cell barcodes, unique molecular identifiers (UMI), and poly(dT) sequences. GEM-Reverse Transcription (53 °C for 45 min, 85 °C for 5 min; held at 4 °C) was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad, Hercules). After RT reaction, GEMs were broken and the single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, Waltham, MA). The cDNA was amplified for 12 cycles (98 °C for 3 min; 98 °C for 15 s, 63°C for 20 s, 72 °C for 1).  Quality of the cDNA was assessed using an Agilent Bioanalyzer 2100 (Santa Clara, CA), obtaining a product of about 1400bp. This cDNA was enzymatically fragmented, end repaired, A-tailed, subjected to a double-sided size selection with SPRIselect beads (Beckman Coulter, Indianapolis, IN) and ligated to adaptors provided in the kit. A unique sample index for each library was introduced through 12 cycles of PCR amplification using the indexes provided in the kit (98 °C for 45 s; 98 °C for 20 s, 54 °C for 30 s, and 72 °C for 20 s x 14 cycles; 72 °C for 1 min; held at 4 °C).