Buyer is solely responsible for determining whether Buyer has all intellectual property rights that are necessary for Buyer's intended uses of the BioLegend TotalSeq™ products. For example, for any technology platform Buyer uses with TotalSeq™, it is Buyer's sole responsibility to determine whether it has all necessary third party intellectual property rights to use that platform and TotalSeq™ with that platform.

The following protocol describes surface protein staining with TotalSeq™–B and TotalSeq™–C antibodies and/or hashtag antibodies, 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

Protocol

I) Sample and Solutions Preparation

• Prepare single cell suspension following a suitable protocol
• Note: We no longer recommend the use of dextran during blocking/staining. If you have any questions please contact BioLegend Technical Support. 

II) Cell labeling for 10x Genomics platforms

1. 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. 2. Resuspend 1–2 million cells in 50 µl Cell Staining Buffer.  


3. 3. Add 5 µl of Human TruStain FcX™ Fc Blocking reagent or 0.5 μl of TruStain FcX™ PLUS (anti-mouse CD16/32) Blocking reagent.


4. 4. Incubate for 10 minutes at 4°C.


5. 5. While cells are incubating in Fc Block, prepare antibody pool using 1 µg (or titrated amounts) of each TotalSeq™ and/or hashtag or biotinylated antibody.  


6. 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. 7. Carefully pipette out the liquid, avoiding the bottom of the tube, and add the TotalSeq™ antibody cocktail to the cell suspension.  


8. 8. Incubate for 30 minutes at 4°C.


9. 9. Add 3.5 mLs of cell staining buffer and spin 5 minutes 400g at 4°C. Repeat wash 2 more times for a total of 3 washes.


10. 10. If using biotinylated antibodies, incubate with the appropriate oligo barcoded streptavidin at the recommended amount specified in the product technical datasheet for 20 minutes. Then add 3.5 mLs of cell staining buffer and spin 5 minutes 400g at 4°C. Repeat wash 2 more times for a total of 3 washes.


11. 11. Resuspend cells in 500 µL of PBS to get a concentration of 1x10⁶ cells/mL.


12. 12. Filter cells through 40 µm Flowmi™ Cell Strainer.


13. 13. Verify cell concentration by counting on hemocytometer after filtration.
    Note: We highly recommend determining cell viability. Ideally the viability should be >90% for optimal capture rate. The presence of a large number of non-viable cells can decrease the efficiency of the cell partitioning and recovery within the 10x Chromium chip.


14. 14. Dilute cells as necessary for appropriate input into the 10X Chromium chip.

Proceed to:

• Chromium Single Cell 3' Reagent Kits v3 User Guide with Feature Barcoding technology for Cell Surface Protein (CG000206 Rev D) for TotalSeq–B reagents    OR
• Chromium Single Cell V(D)J Reagent Kits User Guide with Feature Barcoding technology for Cell Surface Protein (CG000208 Rev D) for TotalSeq–C reagents