10x-genomics/Chromium scRNA-seq
DemoSingle-cell gene expression with Chromium Next GEM — full workflow from cell suspension to sequencing-ready libraries.
This protocol covers the full single-cell RNA-seq workflow using the 10x Genomics Chromium platform. Starting from a single-cell suspension, it walks through GEM generation, barcoded cDNA synthesis, library construction, and QC — resulting in sequencing-ready libraries compatible with Illumina platforms.
Overview
The Chromium system partitions thousands of individual cells into nanoliter-scale Gel Bead-in-Emulsions (GEMs), where each cell’s mRNA is uniquely barcoded. After reverse transcription, the barcoded cDNA is pooled for amplification and library construction. A single library captures the transcriptomes of 500–10,000 cells.
Key considerations
- Cell viability: Input suspension should have >90% viability; dead cells inflate ambient RNA background
- Target cell recovery: Loading 10,000 cells typically yields ~6,000 recovered cells after filtering
- Sequencing depth: Recommend 20,000–50,000 reads per cell for standard gene expression
- Timing: Day 1 covers GEM generation through cDNA amplification; Day 2 covers library construction and QC
Materials
Chromium Next GEM Single Cell 3' Kit v4 (10x Genomics)
Chromium Controller or Chromium X
Next GEM Chip K (10x Genomics)
SPRIselect Reagent (Beckman Coulter)
Low TE Buffer
Bioanalyzer High Sensitivity DNA Kit (Agilent)
Qubit dsDNA HS Assay Kit (Invitrogen)