Cell Surface Labeling With TotalSeq™-B or TotalSeq™-C reagents for Single Cell RNA Sequencing Applications

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The following protocol describes surface protein staining with TotalSeq™–B and TotalSeq™–C reagents, to enable protein detection in addition to Single Cell 3’ v3 and Single Cell V(D)J Feature Barcoding technology from 10x Genomics.

Please read the entire protocol before starting the experiments.

Reagent and Instrument List
  • Human TruStain FcX (Fc Receptor Blocking Solution, Cat. No. 422301)
  • Cell Staining Buffer (BioLegend Cat. No. 420201)
  • Dextran Sulfate Sodium Salt (MP Biomedicals, Cat. No. 101516 or equivalent)
  • DNA LoBind Tubes (Eppendorf, Cat# 022431021)
  • TotalSeq™–B antibodies for Single Cell 3' v3 protocol with Feature Barcoding technology for Cell Surface Protein
  • TotalSeq™–C antibodies for Single Cell V(D)J protocol with Feature Barcoding technology for Cell Surface Protein
I) Sample and Solutions Preparation
  • Prepare single cell suspension following a suitable protocol
  • Prepare Dextran Sulfate solution: 1% w/v (10 mg/ml) Dextran Sulfate Sodium Salt in Nuclease-free Water
II) Cell labeling for 10x Genomics platforms
1. Carefully count all cells to ensure accurate quantitation.
  • Make note of cell viability (>95%) and also include dead cells in the total cell count.
  • If high cell death is observed, live cell enrichment (e.g. by Flow Cytometry) is recommended.
2. Resuspend 1–2 million cells in 50 µl Cell Staining Buffer.  
3. Add 5 µl of Human TruStain FcX™ Fc Blocking reagent and 2 µl of Dextran Sulfate solution  
4. Incubate for 10 minutes at 4°C.
5. While cells are incubating in Fc Block, prepare antibody pool using 1 µg (or titrated amounts) of each TotalSeq™ antibody.  
6. To maximize performance, centrifuge the antibody pool at 14,000xg at 2 – 8°C for 10 minutes before adding to the cells.

Note: If antibody cocktail volume is less than 50 µl, add Cell Staining Buffer up to 50 µl, then centrifuge
7. Carefully pipette out the liquid, avoiding the bottom of the tube, and add the TotalSeq™ antibody cocktail to the cell suspension.  
8. Incubate for 30 minutes at 4°C.
9. Wash cells 3 times with 1 mL of Cell Staining Buffer, spin 5 minutes 350g at 4°C.

Note: It has been observed in some cases that various factors, including cell/sample type, tube manufacturer, rotor type, wash buffer, etc., may result in an excessive number of cells coating the side of the tube. Please ensure that staining and washing conditions are appropriate for your sample type.
10. Resuspend cells in PBS supplemented with 0.04% BSA.

Note: Based on starting cell concentration and assuming approximately 50% cell loss, calculate volume to achieve a final cell concentration of 700 – 1,200 cells/µl.
11. Filter cells through 40 µm strainers.  
12. Verify cell concentration and viability by counting on hemocytometer after filtration.  
Proceed to:
  • Chromium Single Cell 3' Reagent Kits v3 User Guide with Feature Barcoding technology for Cell Surface Protein (CG000185 Rev B) for TotalSeq–B reagents    OR
  • Chromium Single Cell V(D)J Reagent Kits User Guide with Feature Barcoding technology for Cell Surface Protein (CG000186 Rev A) for TotalSeq–C reagents
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