GSM6215855: SH_S1_02132019; Homo sapiens; RNA-Seq
The PDMS membrane was carefully removed from the bottom of the platform to expose the VMT tissue chambers. Chambers were washed with HBSS before adding TrypLE express enzyme drop wise to each VMT unit and incubated at 37 °C for 5 minutes to loosen the extra-cellular matrix from the device. Chambers were flushed with a pipette to collect cells into a conical tube and washed once with EGM2 medium to stop the digestion reaction. Next, cells were subjected to centrifugation (340×g for 3 min) and briefly resuspended in an HBSS/collagenase type III solution (1 mg mL−1) to fully digest the matrix. Finally, the single cell suspension was washed with EGM2 and put through a 70 μm filter. Cells were processed for sorting within an hour and kept on ice. Sorting was performed on the BD flow assisted cell sorting (FACS) AriaII with BD FACS Diva software version 8.0.1. Samples were sorted using a 100 μm nozzle at 20 psi with a gating strategy to select for live cells. Flow cytometry sorted cells were washed in PBS with 0.04% BSA and re-suspended at a concentration of ∼1000 cell per μL. Cellular suspensions were loaded onto a Chromium Single Cell Instrument (10X Genomics) to generate single-cell gel beads in emulsion (GEMs). Library construction was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). GEMs were processed to generate cDNA libraries by using 10X Genomics v2 chemistry according to the Chromium Single Cell 3' Reagents kits v2 user guide: CG00052 Rev B. Quantification of cDNA libraries was performed using Qubit dsDNA HS Assay kit (Life Technologies Q32851), high-sensitivity DNA chips (Agilent 5067-4626) and KAPA qPCR (Kapa Biosystems KK4824). Libraries were sequenced on an Illumina HiSeq4000 to achieve an average of 50 000 reads per cell.