TotalSeq™-B or -C with 10x Feature Barcoding Technology
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
|Antibodies||TotalSeq™-B antibodies and/or hashtag reagents||TotalSeq™-C antibodies and/or hashtag reagents|
|(For use only with the Single Cell 3’ v3 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|
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.|
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. 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 or 0.5 μl of TruStain FcX™ PLUS (anti-mouse CD16/32) Blocking reagent.
- 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™ and/or hashtag or biotinylated 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. 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. 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. Resuspend cells in 500 µL of PBS to get a concentration of 1x106 cells/mL.
- 12. Filter cells through 40 µm Flowmi™ Cell Strainer.
- 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. Dilute cells as necessary for appropriate input into the 10X Chromium chip.
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 Rev D).
If using v3.1 (dual index) of the Single Cell Gene Expression Solution, proceed to Chromium Next GEMS Single Cell 3' Reagent Kits v3 with Feature Barcoding technology for Cell Surface Protein user guide (CG000317 Rev A).
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_Rev_D).
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_Rev_A).
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.