GSM8004988: HHT VMO; Homo sapiens; RNA-Seq
The thin transparent membrane was carefully removed from the bottom of the PDMS layer to expose the tissue chambers for two microfluidic devices containing 24 device units. 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 then 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