TotalSeq™-B or -C with 10x Feature Barcoding Technology

 

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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’ v3.1 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) TruStain FcX™ PLUS (anti-mouse CD16/32) (BioLegend, Cat# 156603/156604) Cell Staining Buffer (BioLegend Cat. No. 420201) Flowmi™ Cell Strainer (Bel-Art, H-B Instrument, Cat# H13680-0040) 12 x 75mm Falcon™ Round-Bottom Polystyrene Tubes (Fisher Scientific, Cat# 14-959-1A or equivalent)

Format-Specific Reagents

	TotalSeq™-B	TotalSeq™-C
Antibodies	TotalSeq™-B antibodies and/or hashtag reagents	TotalSeq™-C antibodies and/or hashtag reagents
	(For use only with the Single Cell 3’ v3.1 Feature Barcoding kit)	(For use only with the Single Cell V(D)J Feature Barcoding kit)
Biotin (optional)	A biotinylated antibody and TotalSeq™-B barcoded streptavidin	A biotinylated antibody and TotalSeq™-C barcoded streptavidin
Single Index Kit	Chromium Single Cell 3′ Feature Barcode Library Kit 16 rxns 1000079	Chromium Single Cell 5’ Library Construction Kit 16 rxns 1000020
OR Dual Index Kit	Dual Index Kit NT Set A (for Feature Barcode Libraries) 96 rxns 1000242	Dual Index Kit TN Set A (for Feature Barcode Libraries) 96 rxns 1000250
NOTE: The TotalSeq™ antibodies used will vary based on the nature of the experiment. DO NOT combine TotalSeq™-B and TotalSeq™-C antibodies in a single experiment.

 

Protocol

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1. Prepare cell suspensions.

    

   • This protocol has been optimized using fresh human PBMCs isolated using Ficoll gradients and mouse splenocytes prepared using mechanical dissociation. If using cells isolated from whole lysed blood or other sample types, users may need to optimize staining concentrations.
   • BioLegend has not tested this protocol using single cell suspensions derived from enzymatically digested tissue. Enzymatic digestion may result in cleavage of epitopes and result in reduced staining with TotalSeq™ antibodies. Optimization of staining conditions and concentrations may be required.
2. Assess Cell Viability. Carefully count all cells to ensure accurate quantitation and assess cell viability.  Ideal cell viability is ≥ 95%.

    

   • Low cell viability is associated with generation of poor data and is not ideal for single cell experimentation. If low cell viability is observed, users may need to enrich live cells or repeat cell suspension preparation.
   • Contact Technical Services with any questions regarding cell viability. BioLegend uses a Countess II for counting and assessing cell viability, however other methods for assessing cell viability are suitable. For more information about the protocol used by BioLegend, see the following link, details can be found under “PBMC viability assessment—general methods”.
3. Dilute cells in an appropriate volume prior to staining.

    

   • If working with human cells, dilute 1 million cells in 45 μL of Cell Staining Buffer in 12 x 75mm flow cytometry tubes.
   • If working with mouse cells, dilute 1 million cells in 49.5 μL of Cell Staining Buffer in 12 x 75mm flow cytometry tubes.
4. Block cells.

    

   • Add 5 µL of Human TruStain FcX™ Fc Blocking reagent or 0.5 µL of TruStain FcX™ PLUS (anti-mouse CD16/32) antibody. The final blocking volume should be 50 µL.
   • Incubate for 10 min at 4°C.
   • While cells are incubating in Fc Block, proceed to step 5.
5. Prepare antibody pool using titrated amounts (up to 1 µg) of each TotalSeq™, Cell Hashing, and/or biotinylated antibody. For more information regarding TotalSeq™ antibody concentrations, please reach out to BioLegend Tech Services.

    

   • When performing dual staining with cell hashing antibodies and TotalSeq™ antibodies, we recommend adding cell hashing antibodies into each respective sample’s TotalSeq™ antibody pool. If sequentially staining samples, stain with cell hashing antibodies first and then pool your hashtag labeled samples for TotalSeq™ antibody staining. Please note that in some cases cell recovery may be diminished.   
   • If using biotinylated antibodies, we recommend staining with your primary antibody first followed by staining with streptavidin TotalSeq™ conjugates. Do not stain with more than 1 unique biotinylated antibody for detection.
6. If the antibody cocktail volume is less than 50 µL, add Cell Staining Buffer up to 50 µL, then centrifuge the antibody pool at 14,000 x g at 2 – 8°C for 10 minutes before adding to the cells. If the volume of the pool is above 50 µL, no volume adjustment is necessary.

    

7. Carefully pipette out the prepared antibody pool, avoiding the bottom of the tube, and add the TotalSeq™ antibody cocktail to the 50 µL blocked cell suspension.

    

8. Incubate for 30 minutes at 4°C.

    

9. Add 3 mL of Cell Staining Buffer and spin at 4°C for 5 minutes at 400 x g -600 x g depending on your sample type. Repeat wash 2 more times for a total of 3 washes.

    

10. If using a biotinylated primary antibody, incubate the stained cells with the appropriate oligo barcoded streptavidin at the recommended amount specified on the product technical datasheet for 20 minutes. Repeat step 9, then proceed to step 11.

     

11. Resuspend cells in 500 μL of Cell Staining Buffer.

     

12. Slowly filter cells through a 40 µm Flowmi™ Cell Strainer.

     

13. Verify cell concentration and viability 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.
    • Dilute cells as necessary for appropriate input into the 10X Chromium chip.

 

 

Proceed to:
 

For TotalSeq™-B reagents:

 

If using v3.1 (single index) of the Single Cell Gene Expression Solution, proceed to Chromium Next GEM Single Cell 3' Reagent Kits v3.1 with Feature Barcoding technology for Cell Surface Protein user guide (CG000206).

 

If using v3.1 (dual index) of the Single Cell Gene Expression Solution, proceed to Chromium Next GEM Single Cell 3' Reagent Kits v3.1 with Feature Barcoding technology for Cell Surface Protein user guide (CG000317).

 

 

For TotalSeq™-C reagents:

 

If using v1.1 of the Single Cell Immune Profiling Solution, proceed to the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 with Feature Barcoding technology for Cell Surface Protein user guide (CG00208).

 

If using v2 of the Single Cell Immune Profiling Solution, proceed to the Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) with Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping user guide (CG000330).

 

 

Recommended Sequencing Depth for Cell Surface Protein Library:

To obtain sufficient read coverage for Cell Surface Protein libraries follow recommended library loading and pooling specifications provided in 10x Genomics user guides. See table below for sequencing depth recommendations for Cell Surface Protein libraries which are applicable to both the Single Cell Gene Expression Solution and the Single Cell Immune Profiling Solution.

 

Library Type	Minimum Sequencing Depth (reads/cell)
Cell Surface Protein Library <100 ADT panel	5,000
Cell Surface Protein Library ≥100 ADT panel	10,000
Cell Hashing Libraries	500

 

 

Library Pooling:

 

Given that the recommended sequencing read configurations differ between single index and dual index Chromium Single Cell 3’ libraries, we recommend sequencing single index and dual index libraries separately. However, it may be possible to optimize pooling single and dual index libraries together for sequencing.