Single-cell RNA sequencing analysis of left atrial appendage tissue following magnetogel-based occlusion in a porcine model
Single-cell RNA sequencing analysis of left atrial appendage tissue following magnetogel-based occlusion in a porcine model
Left atrial appendage (LAA) tissues were collected from Bama miniature pigs (Sus scrofa) following a survival period of over ten months post-magnetofluid occlusion. Tissues were dissected into two regions: the contact zone (tissue in direct contact with the implanted magnetogel) and the non-contact zone (adjacent tissue not in direct contact). All procedures were approved by the Institutional Animal Care and Use Committee. Single-cell suspensions were prepared from harvested LAA tissues through meticulous mincing and enzymatic dissociation using a defined cocktail of collagenase and DNase. The cell suspension was filtered through a cell strainer to remove debris. Cell viability and concentration were assessed using standard Trypan Blue staining and counting methods. The resulting single-cell suspension was used directly for single-cell library construction without intermediate RNA extraction. Single-cell RNA sequencing libraries were constructed using the 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kit according to the manufacturer's instructions. Single-cell suspensions were combined with Master Mix and loaded onto a Chromium Chip to generate gel beads-in-emulsion (GEMs). Following GEM cleanup and cDNA amplification, the libraries were constructed through enzymatic fragmentation, end-repair, A-tailing, adapter ligation, and sample index PCR. Final libraries were quantified and quality-controlled to confirm the expected library size distribution.