TotalSeq™-B or -C Cell Hashtag Staining with 10x Chromium Single Cell Flex Gene Expression

 

The following protocol describes TotalSeq Hashtag staining for use with the 10x Genomics Single Cell Flex Gene Expression assay. This protocol is specifically for users wishing to stain with hashtags only, prior to starting the Flex Gene Expression assay. DO NOT combine TotalSeq-B and TotalSeq-C hashtags in a single experiment.

 

After completing cell hashtag staining and sample pooling, users should proceed directly to the 10x Genomics Fixation of Cells and Nuclei for Chromium Fixed RNA Profiling user guide to fix cells before advancing to the appropriate Chromium Fixed RNA Profiling Reagent Kit. Cells must be stained with hashtags prior to fixation.

 

It is essential to review the Fixation of Cells and Nuclei for Chromium Fixed RNA Profiling user guide before staining to ensure that necessary buffers are prepared for fixation following hashtag staining.

 

Read through this protocol in its entirety and the associated 10x Genomics user guides for your 10x Genomics kit. The 10x Genomics Fixed RNA Profiling Reagent Kit user guide required is dependent on the TotalSeq format you will be using.

 

10x Genomics Fixation user guide required for any TotalSeq format:

• Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling

 

10x Genomics kit user guide compatible with TotalSeq-B:

• Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples with Feature Barcode technology for Protein

 

10x Genomics kit user guides compatible with TotalSeq-C:

• Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples with Feature Barcode technology for Protein using Barcode Oligo Capture
• Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples with Feature Barcode technology for Protein using Barcode Oligo Capture
• GEM-X Flex Gene Expression Reagent Kit for Singleplex samples with Feature Barcode technology for Protein Expression
• GEM-X Flex Gene Expression Reagent Kit for Multiplex samples with Feature Barcode technology for Protein Expression
• GEM-X Flex v2 For Singleplexed Samples with optional Feature Barcode technology for Protein
• GEM-X Flex v2 For Multiplexed Samples with Feature Barcode technology for Cell Surface Protein

 

 

Disclaimers

• 10x Genomics has not validated the use of TotalSeq hashtags with Gene Expression Flex and will provide limited support to customers on use of them.
• If you intend to use TotalSeq-C hashtags in combination with 10x Antibody Multiplexing barcodes to multiplex samples, BioLegend has not validated this multiplexing method and will only be able to provide limited guidance for demultiplexing the data.

 

Protocol Overview

 

 

Reagent and Consumable List

• Cell Staining Buffer (BioLegend, Cat. No. 420201)
• Human TruStain FcX™ (Fc Receptor Blocking Solution) (BioLegend, Cat. No. 422301)
• TruStain FcX™ PLUS (anti-mouse CD16/32) (BioLegend, Cat. No. 156603)
• Low Protein Binding Microcentrifuge Tubes (ThermoFisher Scientific, Cat. No. 90410 or equivalent)
• 12 x 75 mm Falcon™ Round-Bottom Polystyrene Tubes (Fisher Scientific, Cat. No. 14-959-1A or equivalent)
• TotalSeq-B Hashtags OR TotalSeq-C Hashtags

 

Best Practices and Important Considerations for Best Results

Surface Staining

 

Cell Viability

• High cell viability prior to staining is essential for optimal performance. Ideal cell viability is >95%. Contact BioLegend Technical Services with any questions regarding cell viability. Low cell viability is associated with poor single-cell sequencing data. If low cell viability is observed, users may need to enrich live cells or repeat cell suspension preparation. For samples with low viability, we recommend using BioLegend’s MojoSort™ Human or Mouse Dead Cell Removal Kits to enrich for viable cells prior to staining.

 

Cell washing

• When washing cells, it is extremely important to thoroughly decant the wash buffer, and, upon the addition of new wash buffer, that the cell pellet is resuspended either with pipette mixing or gentle vortexing. When decanting, pour off the wash buffer in a single firm (but not forceful) motion. Following decanting, continue to hold the tube inverted and remove remaining droplets on the lip of the tube by gently dabbing with a clean paper towel before returning the tubes to an upright position. This technique should be used during all cell washes.

 

Optimal cell staining with TotalSeq antibodies

• Our liquid, single-antibody TotalSeq conjugates require optimization of staining concentration (titration) to obtain best results, as performed by the end user of the antibodies. Optimization of antibody staining concentration is essential to obtain good quality data in antibody-based applications such as surface staining using TotalSeq antibodies.
• Antibody titration for sequencing-based applications using TotalSeq antibodies is best performed using matching procedures as much as possible. This means titration-by-sequencing if possible. In the absence of titration-by-sequencing, it may be possible to replicate this protocol using fluorescent antibodies of the same clone and titrate via flow cytometry. BioLegend can provide recommended concentration ranges for most TotalSeq antibodies, and these can be obtained by contacting BioLegend Technical Services.
• This protocol has been optimized using fresh human PBMCs isolated using density gradient centrifugation. Whole blood or lysed whole blood is not recommended. If using cells isolated with a different procedure, users may need to verify the antibody staining pattern using alternative methods.
• BioLegend has not tested this protocol using single-cell suspensions derived from enzymatically digested tissue. Enzymatic digestion may result in alterations of surface protein epitopes and impact staining with TotalSeq antibodies. Optimization of staining conditions and concentrations may be required.

 

Protocol

Cell Hashtag Staining

1. Prepare cell suspension with preferred or recommended method

   Notes:

   • This protocol is optimized for fresh human PBMCs isolated by density gradient centrifugation. Whole blood or lysed whole blood is not recommended. Single cell suspensions from enzymatically digested tissue have not been tested and may alter epitope integrity; staining conditions may require optimization. If using other isolation methods, verify antibody staining independently.
2. Count and assess cell viability
   1. Using your preferred method, carefully count all cells and assess cell viability. It is recommended to stain at least 1 x 10⁶ cells.

      Note:

      Contact BioLegend Technical Services with any questions regarding cell viability. Ideal cell viability is >95%. Low cell viability is associated with poor single-cell sequencing data. If low cell viability is observed, users may need to enrich live cells or repeat cell suspension preparation.

3. Dilute cells and Fc receptor blocking

    

   Cell Type	Cell #	Cell Staining Buffer	Human TruStain FcX	Total Staining Volume
   Human Cells	1 - 2 x 106	45 μL	5 μL	50 μL

    

   Cell Type	Cell #	Cell Staining Buffer	TruStain FcX Plus	Total Staining Volume
   Mouse Cells	1 - 2 x 106	49.5 μL	0.5 μL	50 μL

    

   1. For human cells, dilute 1 - 2 x 10⁶ cells in 45 μL of Cell Staining Buffer in a 12 x 75 mm flow cytometry tube, then add 5 μL of Human TruStain FcX™ to the 1 - 2 x 10⁶ cells in 45 µL of Cell Staining Buffer (total volume = 50 μL).
   2. For mouse cells, dilute 1 - 2 x 10⁶ cells in 49.5 μL of Cell Staining Buffer in a 12 x 75 mm flow cytometry tube, then add 0.5 µL of TruStain FcX™ PLUS (anti-mouse CD16/32) to the 1 - 2 x 10⁶ cells in 49.5 µL of Cell Staining Buffer (total volume = 50 μL).
   3. Incubate for 10 minutes at 4°C.
   4. While cells are incubating in Fc Block, proceed to step 4 – Stain cells with hashtag antibodies.
4. Staining individual samples with TotalSeq hashtag antibodies

    

   
   Figure 1: Staining individual samples with hashtag antibodies followed by pooling.

    

   1. Make a unique hashtag staining solution for each sample using a titrated amount of each hashtag in Cell Staining Buffer. Add the calculated amount of each hashtag antibody to a low protein-binding microcentrifuge tube and bring up the total volume to 50 µL with Cell Staining Buffer.

      Note:

      The recommended starting amount for titration is ≤ 1.0 µg per million cells in 100 µL volume. It is strongly advised to titrate this reagent to your specific sample type to ensure optimal staining performance.

   2. Centrifuge the hashtag solution at 14,000 x g at 2 - 8°C for 10 minutes before adding to the cells to ensure removal of protein aggregates.
   3. Carefully pipette out the prepared hashtag solution, avoiding the bottom of the tube, and add it to the 50 µL blocked cell suspension. The total staining volume will be 100 µL.
   4. Incubate for 30 minutes at 4°C.
   5. Wash cells, add 3 mL of Cell Staining Buffer, mix by gently pipetting 5 times. Centrifuge at 4°C for 5 minutes at 400 - 600 x g depending on your sample type.
   6. Repeat wash twice for a total of 3 washes.

      Note:

      It is extremely important to thoroughly decant the wash buffer and resuspend the cell pellet either with pipetting or gentle vortexing. Discard supernatant with a single firm (but not forceful) motion. Proceed to absorb any remaining liquid on the lip of the tube with a clean paper towel.

   7. After the final wash, resuspend each sample in 100 µL of Cell Staining Buffer, and verify cell concentration and assess cell viability for each sample.
   8. Based on each sample’s cell concentration, combine an equal number of cells from each sample to achieve 1 - 2 x 10⁶ cells total in a low protein binding microcentrifuge tube.
   9. Bring the total volume of the combined samples up to 500 µL with Cell Staining Buffer, and gently resuspend cells by pipetting. Then centrifuge at 4°C for 5 minutes at 400 - 600 x g.
   10. After the final wash, using a pipette, carefully remove 400 µL of Cell Staining buffer taking care to not disturb cell pellet. Gently mix the cells by pulse vortexing the cell pellet at medium intensity in the remaining volume.
   11. Proceed immediately to Sample Fixation, Step c of the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol.
       • Step c: Add 1 mL Fixation Buffer to the labeled cells and pipette mix five times. Fixation Buffer preparation and fixation protocol are listed in Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol.
       • To store fixed samples, after fixation follow the guidance provided in the Appendix, Fixed Sample Storage Guidance, of the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol.

Recommended sequencing depth for hashtag library

 

To obtain sufficient read coverage for hashtag libraries, follow recommended library loading and pooling specifications provided in 10x Genomics user guides. See table below for BioLegend sequencing depth recommendations for Hashtag libraries.

 

Library Type	Minimum Sequencing Depth (reads/cell)
Hashtag Library (HTO)	500