GSM9351973: HUVEC with uninfected PMA-differentiated U937 cells, 8 h, rep1; Homo sapiens; RNA-Seq
Cell samples were then gently washed 1x with PBS, followed by the addition of 200 µL per well of 0.05% Trypsin-EDTA for 5 min to allow cell detachment. Next, 800 µL of cell culture medium containing 5% DMSO was added. Cells were frozen overnight at –80°C and subsequently transferred to –120 °C until shipment on dry ice to the company carying out the scRNA-seq. GEM generation, library preparation and sequencing were performed by Lexogen NGS Services, Lexogen GmBH, Austria. In brief, cell viability and counting were performed with AO/PI staining and LUNA FX7 cell counter (Logos Biosystems). Approximately 8000 cells per sample were subjected to single-cell RNA sequencing (scRNA-seq) on the 10X Chromium platform. Single nuclei were loaded onto a Chromium Next GEM Chip G Single Cell Kit (10 × Genomics) and processed through a Chromium Controller to generate GEMs (Gel Beads in Emulsion). The sequencing libraries were prepared with the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10 × Genomics) following the manufacturer's protocol. Libraries were sequenced on an Illumina NovaSeq X platform, at 20000 reads per cell per sample. Sequencing read mode applied was: Read 1 28bp; Read 2 90bp; Index Reads 1 and 2 10 bp each.