GSM8447652: HUVECs, exposed to healthy RBC (hRBC) flow; Homo sapiens; RNA-Seq
Endothelial cells were removed from the microfluidic devices and resuspended in media. The suspension was passed through 70 and 40 μm cell strainers and centrifuged at 500xg at 4°C for 10 minutes. For RBC lysis, ACK buffer was added. For immediate single-cell capture, the cells were pelleted at 400xg at 4°C for 5 minutes and resuspended in PBS containing 1.0% BSA to get a concentration of 500 cells/μl. For single-cell RNA sequencing (scRNA-seq), barcoded gel bead-in-emulsions (GEMs) were generated from single-cell suspensions with gel beads and reverse transcription (RT) mix on the Chromium Controller, following the 10x Genomics workflow. Following RT and cDNA amplification, the Chromium Next GEM 5'reagent kit v2 (10x Genomics) was used to generate 5' gene expression libraries. Gene expression libraries were prepared using the Chromium Next GEM 5'reagent kits v2 (10x Genomics). GEMs were generated and barcoded by loading single-cell suspensions along with gel beads and reverse transcription master mix onto Chromium Next GEM Chip K and running on the 10x Genomics chromium controller. Following reverse transcription, the cDNA was amplified and used to generate 5' gene expression libraries. The cDNA and gene expression libraries were quantified using a Qubit 3.0 fluorometer, and quality was assessed using HS DNA chips and 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed using massively parallel sequencing on the Illumina Novaseq S4 platform, targeting ~50,000 reads/cell to capture the expression of ~1000–2000 transcripts/cell.