design: scRNA-seq of VSMCs isolated from human carotid plaques
library_construction_protocol: Carotid atherosclerotic plaque specimens were collected from the operating room and immediately placed in ice cold sterile phosphate-buffered saline (PBS). All samples were processed within 30 minutes of collection. Each specimen was weighed, and a small portion from the area of maximal stenosis was kept for histological analysis to grade atherosclerotic plaque stability using Hematoxylin and Eosin, as previously described10-11. Plaque instability was determined based on two gold-standard classification methods: (i.) the American Heart Association (AHA) classification by Stary et al., and (ii) the semiquantitative scale by Lovett et al. Evaluating pathologists were blinded to the patients' cerebrovascular symptomatic status, demographic details, and all pertaining clinical information. Single-cell suspensions were prepared and loaded into the 10x Genomics Chromium Controller or Chromium X instrument using the Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1, following the manufacturer's protocol. Cells were encapsulated into Gel Beads-in-Emulsion (GEMs) containing barcoded oligonucleotides. Within each GEM, cells were lysed, and mRNA was reverse transcribed into barcoded cDNA. Post reverse transcription, GEMs were broken, and cDNA was purified using Dynabeads. The cDNA underwent amplification via PCR, followed by fragmentation, end-repair, A-tailing, and adaptor ligation. Indexed libraries were then amplified and purified using SPRIselect beads. Library quality and quantity were assessed using LabChip GX and qPCR, respectively.