NextSeq 500 sequencing; Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing
Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing
iPSC: iPSC - Cells were harvested using ReleSRTM (Stem Cell Tech) and dissociated into a single cell suspension. Cells were counted and assessed for viability with Trypan Blue using a Countess II automated counter (Thermo Fisher Scientific), then pooled at a concentration of 391-663 cells/µL (3.91x105 - 6.63x105 cells/mL).   TMWC: TMWCs were plated in T75 flasks and transfected with a pooled single guide RNA (sgRNA) library lentivirus containing sgRNA for 128 targets, 10 of which were control genes. Cells were harvested 7 days after virus transduction and were FACS sorted for EGFP-positive and viable cells (propidium iodide-negative cells) before applying to the Chromium System (10x Genomics) single cell RNA-sequencing workflow.   PBMC1 and PBMC2: Peripheral blood samples were collected in Vacutainer Cell Preparation Tubes containing sodium heparin and ficoll (BD Biosciences: 362753), and were processed according to the manufacturer's recommendations. Following separation, PBMCs were cryopreserved and stored. Samples were subsequently thawed, and each library contained a pool of PBMCs from 14 donors, with 40,000 cells loaded to achieve a targeted 20,000 cells per library.  iPSCs were maintained with StemFlex (ThermoFisher Scientific), while TMWCs were plated in T75 flasks. Samples were loaded on to the 10x Genomics Chromium Controller (10x Genomics, USA) for partitioning and barcoding. cDNA were extracted from all samples using the Single Cell 3' Library and Gel Bead Kit (V2; 10x Genomics; PN-120237).
Experimental Factor: single cell identifier
SI-GA-D4
Experimental Factor: cell type
trabecular meshwork cell
Experimental Factor: sequencing platform
NextSeq 500