Dec 19, 2022
UPitt TriState SenNet TMC Cell Hashing of single cell suspension for scRNAseq in 5' workflow (10x Genomics)
- Tracy Tabib1,
- Oliver Eickelberg1,
- Melanie Königshoff1,
- lafyatis Lafyatis1
- 1Division of Pulmonary and Critical Care Medicine. Department of Medicine. School of Medicine University of Pittsburgh
Protocol Citation: Tracy Tabib, Oliver Eickelberg, Melanie Königshoff, lafyatis Lafyatis 2022. UPitt TriState SenNet TMC Cell Hashing of single cell suspension for scRNAseq in 5' workflow (10x Genomics). protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjdy5vk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 15, 2022
Last Modified: December 19, 2022
Protocol Integer ID: 74066
Funders Acknowledgement:
TriState SenNET (Lung and Heart) Tissue Map and Atlas consortium – NIA
Grant ID: U54AG075931
Abstract
TotalSeq™-C antibodies can be used for Cell Hashing in the 5' workflow, and can be purchased from BioLegend directly. To enable cell hashing, the Demonstrated Protocol for Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols (10x Genomics) must be modified. The required changes are described below. The recommended number of cells for this modified protocol is 0.2-2 x 10^6 cells per sample.
Antibody Mix Supernatant Preparation
Antibody Mix Supernatant Preparation
Buffers – Preparation
For samples containing >70% viable cells
• Chilled (4°C): PBS + 1% BSA
• Chilled (4°C): PBS + 0.04% BSA
For samples containing <70% viable cells
• Chilled (4°C): PBS + 10% FBS
Prepare Antibody Mix Supernatant
• Add appropriate/manufacturer's recommended amount of antibody-oligonucleotide conjugates to a 1.5-ml microcentrifuge tube.
• If using a custom lyophilized antibody: Resuspend the antibody-oligonucleotide conjugates in an appropriate volume of labeling buffer.
• Centrifuge the mix at 14,000 rcf for 10 min at 4°C.
• Transfer the supernatant (containing Antibody Mix) to a new tube and maintain at 4°C
Use TotalSeq-C (Biolegend) for Single Cell 5' v1, v1.1 & v2 protocol with Feature Barcode technology for Cell Surface Protein.
- TotalSeq™pre-pooled antibody panels, such as the TotalSeq™ Universal cocktails are lyophilized and need reconstitution before proceeding with staining cells.
- Equilibrate the lyophilized panel vial to room temperature for 5 minutes.
- Rehydrate lyophilized panel in PBS + 10% FBS using appropriate reconstitution volume needed for the number cells
- For 0.2-2 x 10^6cells per sample**, add 25-50ul of PBS + 10% FBS to the lyophilized panel. Vortex and incubate for 10min at room temperature. Then, centrifuge the vial at 14000 rcf for 10min at 4°C.
- Transfer the supernatant (containing Antibody Mix) to a low-bind tube and maintain at 4°C.
**Please note: Staining with >500k cells is not covered by BioLegend's product performance guarantee.
Prepare FACS Antibody Pool
• Add appropriate/manufacturer's recommended amount of fluorophore antibodies to a 1.5-ml microcentrifuge tube on ice.
• Gently pipette mix and maintain at 4°C. Avoid light exposure.
Cell Surface Protein Labeling Protocol
Cell Surface Protein Labeling Protocol
Transfer cells to a new 5-ml tube and add chilled PBS + 0.04 % BSA for a total 1 ml volume.
For samples containing <70% viable cells, use chilled PBS + 10% FBS.
Centrifuge cells at 4°C. Use of swinging-bucket rotor is recommended for higher cell recovery. Centrifugation speed and time depends upon the sample type.
| Sample Type | Centrifugation Conditions | |
| Samples containing >85% viable cells, e.g., PBMCs | 300-400 rcf for 5 min | |
| Samples containing <85% viable cells, e.g., tumor cells | 150-300 rcf for 5-10 min |
Table 2. Sample Type Specific Centrifugation Conditions
Remove the supernatant without disturbing the pellet.
Resuspend cell pellet in 50 μl chilled PBS + 10% BSA.
For samples containing <70% viable cells, resuspend in chilled PBS + 10% FBS.
Add 5 μl Human TruStain FcX. Gently pipette mix.
Incubate for 10 min at 4°C.
Add the prepared Antibody Mix supernatant (TotalSeq™ cell surface antibodies + hashing antibody). Each sample should be stained with a different hashing antibody before pooling the samples.
Add chilled PBS + 1% BSA to the cells to bring the total volume to 100 μl. Gently pipette mix 10x (pipette set to 90 μl).
For samples containing <70% viable cells, add chilled PBS + 10% FBS.
Incubate for 30 min at 4°C. If using FACS antibodies, incubate without light exposure.
Recommended incubation temperature for most sample types is 4°C. However, incubation temperature is sample type dependent and should be chosen accordingly
Recommended Cell Wash Protocol
Recommended Cell Wash Protocol
Buffers – Preparation
• Chilled (4°C): PBS + 1% BSA
Wash 1
Wash by adding 3.5 ml chilled PBS + 1% BSA to the labeled cells. Gently pipette mix.
Centrifuge at 4°C. Centrifugation speed and time depends upon the sample type. Use Table 2 for guidance.
Remove the supernatant without touching the bottom of the tube to avoid dislodging the pellet. Leaving behind excess supernatant may cause non-specific binding, which may result in increased background reads during sequencing.
Wash 2
Using a pipette tip, resuspend the pellet or cells in 3.5 ml chilled PBS + 1% BSA and transfer to a new 5-ml tube.
Centrifuge at 4°C. Centrifugation speed and time depends upon the sample type. Use Table 2 for guidance.
Remove the supernatant without touching the bottom of the tube to avoid dislodging the pellet.
Wash 3
Using a pipette tip, resuspend the pellet or cells in 3.5 ml chilled PBS + 1% BSA.
Centrifuge at 4°C. Centrifugation speed and time depends upon the sample type. Use Table 2 for guidance.
Remove the supernatant without touching the bottom of the tube to avoid dislodging the pellet
(OPTIONAL)
For enrichment of labeled and viable cells by FACS:
• Based on starting concentration and assuming ~50% cell loss, add an appropriate volume chilled PBS + 2% FBS (including a dead cell marker) to obtain a final cell concentration of 5-10 x 106 cells/ ml and proceed to FACS.
• After FACS, determine cell concentration and viability using an Automated Cell Counter or a hemocytometer.
• Proceed immediately to relevant Chromium Single Cell RNA Sequencing protocols with Feature Barcode technology
If not performing FACS:
• Based on starting concentration and assuming ~50% cell loss, add an appropriate volume chilled PBS + 1% BSA to obtain a concentration of 700- 1,200 cells/μl.
• Determine cell concentration and viability using an Automated Cell Counter or a hemocytometer.
• Proceed immediately to relevant Chromium Single Cell RNA Sequencing protocols with Feature Barcode technology.