Optimizing single-cell RNA sequencing methods for human colon biopsies: droplet-based vs. picowell-based platforms.

Area of Science:

• Gastroenterology
• Molecular Biology
• Bioinformatics

Background:

• Single-cell RNA sequencing (scRNA) provides insights into gastrointestinal microenvironments.
• Droplet-based scRNA (D-scRNA) requires immediate processing of human biopsies, risking epithelial cell damage.
• Picowell-based scRNA (P-scRNA) allows short-term frozen storage, offering a potential advantage for biopsy processing.

Purpose of the Study:

• To compare the performance of P-scRNA and D-scRNA platforms using human colon biopsies.
• To evaluate the impact of frozen storage on P-scRNA data quality.
• To assess cell recovery, cell typeability, gene coverage, and expression consistency between platforms.

Main Methods:

• Human rectosigmoid mucosal biopsies were used to generate single-cell suspensions.
• Parallel experiments compared fresh P-scRNA, frozen P-scRNA (-20°C and -80°C), and fresh D-scRNA.
• Over 10,000 cells per participant were processed for each platform.

Main Results:

• P-scRNA showed significant cell recovery for fresh (80.9%) and -80°C frozen (48.5%) samples, while D-scRNA achieved 76.6% recovery.
• D-scRNA captured a higher percentage of typeable cells (71.5%) compared to fresh (15.8%) and -80°C frozen (18.4%) P-scRNA.
• D-scRNA yielded higher gene coverage but also higher mitochondrial read fractions; D-scRNA cells showed more consistent gene expression profiles within cell types.

Conclusions:

• P-scRNA and D-scRNA platforms possess non-overlapping strengths for analyzing colon biopsies.
• Innovation is needed to achieve high-fidelity capture of colonic epithelial cells, especially with P-scRNA.
• Platform-specific variations present challenges for rigor and reproducibility in multi-study analyses.