GSM9476545: pt4_CoronaryDiseased; Homo sapiens; RNA-Seq
Artery digestion protocol: Immediately after collection, fresh coronary artery tissues were separated into equal 50mg pieces. These samples were then placed in a digestion solution comprising Liberase TM (10.4U/mL working concentration in HBSS, Sigma-Aldrich 05401127001) and elastase (8U/mL, Sigma E7885 in HBSS) followed by finely minced on ice using iris scissors. The resulting digested product was transferred to a 2.0ml Eppendorf tube and subjected to constant agitation at 650RPM in a heated Eppendorf Thermomixer (Model 5382). To enhance heat exchange, distilled water was added to the heating block. The digestion mixture was monitored at 30 and 45 minutes under the microscope to assess for single cell suspension, with vigorous pipetting (3 times) performed if undigested cell clumps were observed. After 60 minutes of digestion at 37°C, the digested products were vigorously pipetted 10 times and then filtered through a 70μm strainer. The filtered solution was centrifuged at 500g for 10 minutes in V-bottom plates to optimize cell collection. The resulting cell pellet was resuspended in fresh, cold HBSS. Finally, the samples underwent FACS (Fluorescence-Activated Cell Sorting) using Calcein green (ThermoFisher) for live/dead cell discrimination. The sorted cells were used immediately for single-cell capture and subsequent library preparation. Single cell captures and library preparation were performed using chemistry version 3 (V3) according to 10x Genomics instructions. Cells were loaded into 10x Genomics microfluidics chip G for encapsulation with their proprietary barcoded gel beads in the 10x Genomics Chromium X controller. Single-cell libraries were contruscted according to manufacturer's instructions with index labeling using the Dual Index Kit TT set A. This allows for library pooling into one lane for sequencing on the Illumina NovaSeq6000 platform with a targeted read depth of 75,000 reads per cell.