title: GSM8951175: Day 17 vasc. HOs-on-chip HF; Homo sapiens; RNA-Seq
library_construction_protocol: For VNO single cell profiling, n = 3 independent microchannels were pooled together for each condition on-chip (low flow and high flow), and n = 3 wells of a 12-well plate, each containing about 50 VNOs, were used for the condition in well. For vasc. HOs-on-chip, n = 3 independent microchannels were pooled together. Organoids were dissociated enzymatically into single cells with 3 U/ml Dispase II (Gibco) and 2 U/ml Liberase (Roche) in PBS for 45 min at 37 °C. During incubation, organoids were mechanically disrupted every 10 min by vigorous pipetting with a P1000 tip. The dissociation reaction was stopped by adding 10 ml of inhibition medium containing DMEM/F12 with 10% FBS per ml of dissociation mixture. Next, the cell suspensions were centrifuged at 300 g for 8 min at 4 °C, resuspended in PBS containing 0.4% BSA, and filtered through a 70 µm cell strainer. Cell viability > 95% was assessed using a CellDrop FL cell counter (DeNovix), and final cell concentration was adjusted to 1,000 cells/µl. Single Cell Suspensions were loaded onto the 10x Genomics Chromium X for capture in droplet emulsion. Libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1, or the Chromium GEM-X Single Cell 3' Kit v4, and the standard protocol was followed for all steps. Libraries were then sequenced on an Illumina NextSeq 2000.