Single Cell Transcriptomics

Single Cell 3' and 5' Workflow

10x single-cell 3' and 5' assays partition individual cells into GEMs that uniquely barcode hundreds to thousands of cells with a capture efficiency of 65%. 3’ or 5' end counting determines gene expression and characterizes cells of a heterogeneous population. In addition to expression profiling, the 5' assay enables immune profiling by enriching barcoded cDNA for V(D)J sequences of T or B cells. Both the 3' and 5' assays can be combined with "Feature Barcoding" technology which determines expression of cell-surface proteins through oligo-labelled antibodies. For more information about these assays, please visit 10xGenomics.Single-cell v2 protocol

Cell Sample Prep

10x’s cell prep guide covers the main steps used to wash and re-suspend cells for use on the Chromium System. Please follow these guidelines carefully as the assay is sensitive to large amount of protein contamination from buffers. A low percentage of BSA (.04%) should be used. The Bauer Core Cell Prep Worksheet includes washing instructions and can be used to record counts. Our flow cytometry core can assist with increasing viability or obtaining accurate cell counts.   

Reagent Kits

For staff-run experiments, the core will provide all required reagents. For self-run experiments, use this guide to determine what to purchase and what the core provides.

Sequencing for Single-cell Assays

Libraries generated with 10x kits are compatible with any of the core's Illumina Sequencers. Sequencing depth is dependent the particular kit used, number of cells captured & experimental design. Here are our standard recommendations for read lengths and # of reads/cell:

1. Single cell 3' v3 expression sample: 20-50K reads/cell; Read 1: 28bp, Index: 8bp, Read 2: 91bp (may truncate Read 2 to 58bp)

2. Single cell 3' v3 Feature Barcoding sample: 5K reads/cell; Read 1: 28bp, Index: 8bp, Read 2: 91bp (may truncate Read 2 to 25bp)

3. Single cell 5' v1 expression sample: 20-50K reads/cell; Read 1: 26bp, Index: 8bp, Read 2: 91bp (may truncate Read 2 to 58bp)

4. Single cell 5' v1 VDJ enrichment sample: 5K reads/cell; Read 1: 150bp, Index: 8bp, Read 2: 150bp

5. Single cell 5' v1 Feature Barcoding sample: 5K reads/cell; Read 1: 26bp, Index: 8bp, Read 2: 25bp 

6. Single Cell ATAC v1: 25K reads/nucleus; Read 1: 50bp, Index 1: 8bp, index 2: 16 bp, Read 2: 50bp (may truncate Read 2 to 49bp)