title: GSM6634638: VMO, scRNAseq; Homo sapiens; RNA-Seq
library_name: GSM6634638
library_construction_protocol: The thin transparent membrane was carefully removed from the bottom of the PDMS layer of the vascularized micro-organ to expose the tissue chambers. The chambers were washed with HBSS prior to adding TrypLE express enzyme in a drop-wise manner. After incubation at 37°C for 5 minutes, the chambers were flushed with a micropipette to collect ~10,000 cells into a centrifuge tube. Cells were then quenched with EGM-2. Next, cells were centrifuged (340xg for 3 min) and resuspended in an HBSS/collagenase type III solution (1 mg mL-1) to digest the matrix fully. The cells were then washed with EGM-2 and pushed through a 70 μm filter to create a single-cell suspension. Finally, a propidium iodide stain was used to remove dead and dying cells by FACS on a BD Flow assisted cell sorting AriaII The cellular suspensions were loaded onto a Chromium Single Cell Instrument (10X genomics) and processed to generate cDNA using 10X Genomics v2 chemistry according to the Chromium Single Cell 3' Reagents kits v2 user guide. After undergoing quantification and quality control of cDNA libraries using the Qubit dsDNA HS Assay kit (Life Technologies Q32851), high-sensitivity DNA chips (Agilent 5067-4626), and KAPA qPCR (Kapa Biosystems KK4824), cDNA were sequenced on an Illumina HiSeq4000 at the University of California Irvine's Genomics High Throughput Facility to achieve an average of 50,000 reads per cell.