title: RNA-Seq of human retinal endothelial cells
design: Human retinal endothelial cells (Cell Systems, Kirkland, WA) were maintained on gelatin-coated culture dishes in complete human endothelial cell media with supplement kit containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution (Cell Biologics, Chicago, USA). Confluent cells were serum starved for 4 hours prior to treatment and IL-6 trans-signaling was activated with IL-6 (50ng/mL) and sIL-6R (150ng/mL; PeproTech, Rocky Hill, NJ, USA) overnight. To inhibit the IL-6 trans-signaling, cells were pre-treated for 1 hour with sgp130Fc (10ug/mL; R&D Systems, Minneapolis, MN, USA) before IL-6 trans-signaling activation. After treatment, cells were harvested and pelleted at 300 x g for 5 minutes. All RNA samples had an RNA integrity number > 8.0. Samples were prepared for sequencing using the NEBNext Ultra RNA Library Prep Kit (Illumina; San Diego, CA) to enrich for mRNA and prepare cDNA libraries. Library quality was assessed using the 2100 Bioanalyzer. Sequencing was performed using a 150 bp paired reads protocol on the NovaSeq 6000 (Illumina).