Single-cell RNA sequencing analysis of human aortic smooth muscle cells under mechanical stretch and inflammatory stimulus
Single-cell RNA sequencing analysis of human aortic smooth muscle cells under mechanical stretch and inflammatory stimulus
Human aortic SMCs (ATCC: PCS-100-012) cultured in smooth muscle cell growth medium (SMC-GM) containing 5% fetal bovine serum (FBS), bFGF [2 ng/mL], EGF [0.5 ng/mL], insulin [5 µg/mL], gentamicin [50 µg/mL], and amphothericin B [50 ng/mL] (Provitro, 211-0601) at 37 ºC and 5% CO2. Only cells in passages 4-6 were used for the experiments.  Cells were seeded onto BioFlex 6-well plates precoated with COL1 (Flexcell, BF-3001C) and treated following one of the growth protocols. SMCs from each condition were harvested and resuspended as single-cell suspensions in PBS.  Protocol \"Static\". At ~80% confluency, cells were cultured for 18+6 hours (24 h in total) in a static condition. Protocol \"TNF stimulation\". Recombinant human tumor necrosis factor-alpha (TNF) protein (10 ng/mL, R&D systems, 210-TA-005) diluted in PBS containing 0.1 % BSA was added to the SMC-GM. SMCs were treated at a confluency of ~80% for 24 h.  Protocol \"Stretch 10%\". At ~80% confluency, cells were cultured under static conditions for 18 h and subjected to 10% cyclic equibiaxial stretch for 6 h. The Flexcell tension system (FX-5000 T) applied a sinusoidal waveform stretch at a frequency of 1Hz. We followed the BD Rhapsody™ HT Single-Cell Analysis System (23-24257(01)) and Single-Cell Capture and cDNA Synthesis (23-24252(01)) protocols. For each condition, 40,000 cells were loaded per lane, capturing ~28,000 cells with ~7–8% multiplet rate. Beads containing cell barcodes, UMIs, and oligo-dT sequences were loaded into each lane and used to capture polyadenylated mRNA after cell lysis directly in the microwells, followed by cDNA synthesis. Library preparation of ~14,000 cells (half of each condition) was performed according to the BD Rhapsody™ Whole Transcriptome Analysis (WTA) protocol (23-24117(02)), and quality was confirmed using an Agilent Bioanalyzer. As forward primer for the index PCR, we used the TruSeq D501 primer, while the Nextera N709, N710, and N707 were used as reverse primers for the static, TNF, and 10% stretch libraries, respectively.