BGISEQ-500 paired end 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.  cDNA libraries were created in using the 10x Genomics Single Cell 3' Library and Gel Bead Kit (V2; 10x Genomics; PN-120237). For compatibility with BGI sequencers, the libraries underwent a conversion step via the MGIEasy Universal Library Conversion kit (App-A) (Part Number: 1000004155).
Experimental Factor: single cell identifier
SI-GA-D4
Experimental Factor: cell type
trabecular meshwork cell
Experimental Factor: sequencing platform
MGISEQ-2000