TotalSeq™-D with Mission Bio Tapestri® Single-Cell DNA + Protein Protocol

 

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™–D antibodies, to enable protein detection with the Mission Bio Tapestri Single Cell DNA + Protein Kit V3.

 

Please read the entire protocol below and the Mission Bio Tapestri Single Cell DNA + Protein Sequencing V3 User Guide before starting the experiments.

 

It is strongly recommended that you titrate TotalSeq™-D antibodies for each application before use.

 

If you are planning to spike-in antibodies to the TotalSeq™–D Human Heme Oncology Cocktail please reach out to BioLegend Tech Services for guidance.

 

Required Consumable Reagent List

• Human TruStain FcX (Fc Receptor Blocking Solution, Cat. No. 422301)
   
• Cell Staining Buffer (BioLegend Cat. No. 420201)
   
• Tapestri Single-Cell DNA + Protein Core Kit (Mission Bio)
   
• Flowmi™ Cell Strainer (Bel-Art, H-B Instrument, Cat# H13680-0040)
   
• 1.5 mL DNA low-bind Microcentrifuge Tubes (Eppendorf Cat # 0030108035)
   
• 15 mL DNA low-bind conical (Eppendorf Cat# 30122208)
   
• 200 μL Wide bore tip, rack, sterile (USA Scientific Cat# 1011-8410)
   
• 1000 μL Wide bore tip, rack, sterile (USA Scientific Cat# 1011-9410)

 

Antibodies
TotalSeq™-D Antibodies
Note: Only TotalSeq™ D antibodies are compatible with the Mission Bio Tapestri platform.

 

Protocol
I) Sample and Solutions Preparation

• Prepare single cell suspension by following the Cell Handling Guidelines and the Prepare Cell Suspension sections of the Mission Bio Tapestri Single Cell DNA + Protein Sequencing V3 user guide.
   

II) Cell labeling for Mission Bio Tapestri Platform

1. In a 15 mL low-bind conical Eppendorf tube gently resuspend 1 million cells in 40 µL Cell Staining Buffer with a 200 µL wide bore tip.
    
2. Combine the following reagents with the cell suspension:

Reagent	Volume (µL)
Human TruStain FcX	5.0
Blocking Buffer (orange cap, Mission Bio Tapestri Protein Staining Kit)	5.0

3. Gently mix with a 200 µL wide bore tip and incubate for 15 minutes on ice.
    
4. While cells are incubating with blocking reagents, prepare antibody pool using titrated amounts (up to 1 µg) of each TotalSeq™ antibody.
   Note: If antibody pool volume is less than 50 µL, add Cell Staining Buffer up to 50 µL.
    
5. To maximize performance, centrifuge the antibody pool at 14,000 x g at 2 – 8°C for 10 minutes before adding to the cells.
    
6. Carefully aspirate 50 µL of the TotalSeq™ antibody pool, avoiding the bottom of the tube, and add to the blocked cell suspension for a total volume of 100 µL. Gently mix with a 200 µL wide bore tip. 
    
7. Incubate for 30 minutes on ice.
    
8. Add 14 mL of pre-chilled cell staining buffer and centrifuge at 400 x g for 10 minutes at 4°C. Carefully aspirate 13.5 mL of supernatant, leaving approximately 0.5 mL of supernatant, and discard. Avoid touching the bottom and sides of the tube. Do not disturb or resuspend the cell pellet.
    
9. Repeat wash 2 more times for a total of 3 washes, centrifuging at 400 x g for 5 minutes each at 4°C.
    
10. After final wash, remove and discard supernatant, leaving approximately 100 µL. Add 900 µL of Cell Staining Buffer to the cell pellet bringing the total volume to 1 mL. Gently resuspend the cell pellet using a 1 mL wide bore tip.
     
11. Filter cells through 40 µm Flowmi™ Cell Strainer into a 1.5 mL DNA low-bind Eppendorf tube.
     
12. Centrifuge at 400 x g for 5 minutes at 4°C.
     
13. Inspect the cell pellet and carefully remove all supernatant with a P-200. Do not disturb the cell pellet.
    Important: Failure to remove all Cell Staining Buffer from the cell pellet may reduce the stability of emulsions during the cell encapsulation step of the Mission Bio DNA + Protein protocol.
     
14. Resuspend the pellet in 60 µL of Cell Buffer (Mission Bio) by pipetting up and down several times.
     
15. Count cells using an automated cell counter and dead-cell exclusion dye (e.g., Trypan Blue or Propidium iodide) according to the manufacturer’s instructions. Assess both single-cell suspension and cell viability.
     
16. Dilute cell suspension to 3,000 to 4,000 cells/µL using Cell Buffer.
    Important: Mission Bio’s Cell Buffer contains density gradient medium. Cells that are resuspended in Cell Buffer are difficult to pellet via centrifugation.
    Important: Use of cell concentrations outside the range of 3,000 to 4,000 cells/µL or viability < 90% may adversely affect results. If the minimum concentration of 3,000 cells/µL cannot be met in a total volume of 50 µL, the total volume of Cell Suspension may be reduced to as low as 35µL.
     
17. Place cell suspension on ice. Do not keep cell suspension on ice longer than 30 minutes.
     

 

Proceed to:
Section 2 – Encapsulate Cells, of the Mission Bio Tapestri Single Cell DNA + Protein Sequencing V3 User Guide.