GSM5129495: NS14Unt2; Homo sapiens; RNA-Seq
To generate single cell suspensions, the cells were trypsinized for 3 mins and following neutralization passed through a FlowmiTM tip strainer (40 µm porosity, catalog number H13680-0040, SP Bel-Art, Wayne, NJ, USA). Cells were counted on a hemocytometer prior to diluting cells to 800 cells/µL in 0.1% BSA/PBS buffer for scRNA-seq library preparation with Chromium Single Cell 3ʹ Reagent Kits v3 (catalog number PN-1000075, 10X Genomics, Pleasanton, CA, USA). scRNA-seq libraries were generated according to 10X Genomics User Guide CG000183 Rev C. Briefly, a reaction mix containing 12,800 cells was loaded into a Chromium Chip B, targeting cell recovery of 8000 cells. After generating Gel Beads-in-emulsion (GEMS) on the Chromium controller, GEMS were transferred to PCR tubes (catalog number 951010022, Eppendorf, Hamburg, Germany) for GEM-Reverse Transcription (GEM-RT). After cleanup with Dynabeads MyOne Silane (catalog number PN-2000048, 10X Genomics), cDNA was amplified with 11 cycles. Amplified cDNA was cleaned with 0.6X SPRISelect reagent (catalog number B23318, Beckman Coulter, Pasadena, CA, USA). Cleaned cDNA was then quality checked on an Agilent Tapestation 4150 (catalog number G2992AA, Agilent, Santa Clara, CA, USA) using a High Sensitivity D5000 ScreenTape (catalog number 5067-5592, Agilent) and quantified using Qubit dsDNA High Sensitivity Assay Kit (catalog number Q32851, Thermo Fisher Scientific) read on a Qubit 4 Fluorometer (catalog number Q33238, Thermo Fisher Scientific). An aliquot of 25% of the cleaned cDNA was used for library construction (fragmentation, end repair, A-tailing, adaptor ligation), according to the manufacturer's instructions. Libraries were indexed (catalog number PN-220103, 10X Genomics) using 10 cycles of PCR. Amplified libraries were cleaned with SPRISelect reagent using a double-sided size selection protocol. Cleaned libraries were quality checked on an Agilent Tapestation 4150 using a High Sensitivity D1000 ScreenTape (catalog number 5067-5584, Agilent) and quantified using Qubit dsDNA High Sensitivity Assay Kit. Libraries were diluted to 4nM prior to sequencing on a NovaSeq 6000 S1 PE50 flow cell.