GSM9759907: patient 23 human aortic valve leaflet cells, female, diseased; Homo sapiens; RNA-Seq
For single cell RNA sequencing: Aortic valve leaflets were excised from the aortic valve by cutting along the valve base and rinsed with Earle's Balanced Salt Solution (EBSS) (Gibco, Cat. No. 24010043). All 3 leaflets per specimen were cut in half with 3 halves placed on ice for spatial transcriptomics protocols and three halves digested for single cell isolation and sequencing. Tissues were minced in Liberase TM (Sigma-Aldrich, Cat. No. 5401119001) in EBSS at 0.25 WU/mL and digested on an orbital shaker at 175 RPM for 30 mins at 37°C. DNAse I (Sigma Aldrich, Cat. No. DN25) was added to digestion medium at 100 µg/µL to prevent cell aggregation from genomic DNA. After digestion cells were immediately quenched with 10% Fetal Bovine Serum (FBS, Thermo Scientific, Cat. No. 16000069) in Medium 199 (Thermo Fisher Scientific, Cat. No. 11043023). Cells were strained in 3 successive straining steps with 100 µm, 70 µm, and 30 µm strainers and centrifuged at 400 rcf for 10 minutes. The cell pellet was resuspended in red blood cell lysis buffer (Thermo Scientific, Cat. No. 00433357) and rinsed using 10% FBS in media. After a final centrifugation step, cells were resuspended in 200 µL of 10% FBS media and counted using a cell hemacytometer prior to cell encapsulation in gel-beads for single cell RNA sequencing. For spatial transcriptomics: Aortic valve leaflets embedded in OCT medium were cryosectioned at 10 μm in thickness at -20°C. Spatial transcriptomics protocols (10X Genomics) were performed on tissue sections with an RNA Integrity Number (RIN) > 7 measured on the Agilent TapeStation for all samples. For single cell RNA sequencing: Following the manufacturer's protocol (Single Cell 3′ v2, 10x Genomics), human aortic valve cells isolated from valve leaflets were suspended in master mix and inserted into a microfluidic chip together with partitioning oil and gel beads to form gel bead-in-emulsions (GEMs). Within each GEM, poly-A RNA from the cell lysate was reverse transcribed into cDNA incorporating an Illumina R1 primer sequence, a Unique Molecular Identifier (UMI), and the 10x barcode. The barcoded cDNA was then purified with Silane DynaBeads, PCR-amplified, and size-selected using SPRIselect reagent. During library assembly, the Illumina R2 primer sequence was added, along with paired-end constructs containing P5 and P7 sequences and a sample index.  For spatial transcriptomics: Spatial gene expression libraries were prepared using the Visium Spatial Gene Expression Kit (10x Genomics) following the manufacturer's instructions for fresh frozen tissue. Briefly, cryosectioned tissue was placed on Visium Gene Expression Slides, fixed, and stained with hematoxylin and eosin (H&E) for imaging. Permeabilization released mRNA, which hybridized to spatially barcoded capture oligonucleotides on the slide. Reverse transcription generated spatially barcoded cDNA, which was then collected, purified, and amplified by PCR. The amplified cDNA was enzymatically fragmented, end-repaired, A-tailed, and ligated to sequencing adapters containing sample indices. Final libraries were amplified, quantified, and assessed for quality prior to sequencing.