Getting Started with Single-Cell RNA-Seq

Why Single-Cell?

Bulk RNA-seq averages expression across thousands of cells, hiding rare populations. Single-cell RNA-seq (scRNA-seq) measures expression in each cell independently, allowing us to discover new cell types and track developmental trajectories.

The 10x Genomics Workflow

The most common platform, 10x Genomics, uses microfluidics to encapsulate cells in oil droplets with unique barcodes. This allows we to trace every read back to its cell of origin.

Analysis Steps with Seurat

• QC: Filter distinct cells (doublets) and dead cells (high mitochondrial content).
• Normalization: Adjust for sequencing depth differences.
• HVG Selection: Focus on Highly Variable Genes that drive biological difference.
• Clustering: Group cells by spectral similarity.
• Annotation: Use known markers to label clusters (e.g., CD3+ = T-Cells).