title: GSM7864436: HUVECs_WTinCT_scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Hydrogels were then collected using a spatula into a falcon tube and dissociated into single cells using an incubation period of 15 min in trypsin at 37°C to dissociate endothelial cells. Dissociated cells were then filtered in EGM2 growth medium and then centrifuged (1200 rpm for 5 min). The solutions were neutralized with a 1:10 volume of EGM-2 without antibiotics  and centrifuged for 5 min at 300 RCF, followed by 10 min at 500 RCF. After decanting the supernatant, the cell pellets were resuspended in 200 μL of EGM-2 and put on ice. Cells were isolated by fluorescence activated cell sorting (FACS) by flow cytometry on a Aria IIu (BD Biosciences). Discrimination of cell populations was based on fluorescence at 530/30 nm from 488 nm excitation and 610/20 nm from 561 nm excitation to identify respectively cells expressing Lifeact-GFP or Lifeact-RFP. Cells were run through a 100 um nozzle under 20 psi at a flow rate of 1 and recovered simultaneously in tubes in a 4-way purity mode. The sorted cells were labeled as WT, WT-CCM2, siCCM2 and frozen in EGM-2 + 40% FCS + 10% DMSO. After thawing the cells, their viability (>80%) and aggregation were tested before starting the single-cell library preparation. The concentration of freshly dissociated cells was adjusted to 1000 cells/μL in HBSS/0.05% BSA, and then the 10x Chromium Controller and the Chromium Single Cell 3′ Reagent kit V3.1 were used to perform the scRNAseq experiments. Libraries were constructed according to the manufacturer's instructions using Chromium™ Single Cell 3' v3.1 Reagent Kits and a Chromium instrument.