GSM6588547: CI0000035270, cell line; Homo sapiens; RNA-Seq
Cellular suspensions were loaded on a GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs. GEM-RT was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad; P/N 1851197): 55 °C for 2 h, 85 °C for 5 min; held at 4 °C. After RT, GEMs were broken and the single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; P/N 37002D) and SPRIselect Reagent Kit (0.6 × SPRI; Beckman Coulter; P/N B23318). cDNA was amplified using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98 °C for 3 min; cycled 14 × : 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C. Amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6 × SPRI). The cDNA was subsequently sheared to ∼200 bp using a Covaris M220 system (Covaris; P/N 500295). A single-cell RNA library was generated using the GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) and Single Cell 3' Library & Gel Bead Kit v2 and Chip Kit (P/N 120236 P/N 120237 10x Genomics). The sequencing ready library was purified with SPRIselect, quality controlled for sized distribution and yield (LabChip GX Perkin Elmer), and quantified using qPCR (KAPA Biosystems Library Quantification Kit for Illumina platforms P/N KK4824).