Question: How can I optimize my TotalSeq™ antibody labeling protocol?
Answer: There are a few critical workflow steps for staining with Cell Surface Proteins that should be highlighted for optimal assay performance.
(Note: These guidelines apply to both the TotalSeq panel and user-conjugated antibodies)
- Co-staining with antibodies for Feature Barcoding and FACS. Cells can be stained with antibodies for use with both flow sorting and Feature Barcoding simultaneously. To do this, we recommend preparing the pool separately, then adding them to the cell suspension at the same time. Please note that distinct antibody clones need to be selected for FACS and cell surface protein labeling if staining for the same cell surface protein for both workflows.
- Removal of antibody aggregates. Over time, antibodies tend to clump together and form aggregates. These aggregates will result in very high antibody UMI counts for the cells that they bind to, and they will appear in the downstream data as 'Antibodies in Barcodes with High UMI counts'. While these aggregates are removed from downstream analysis and therefore should not impact the rest of the data quality, they will make up a significant portion of your final library, resulting in wasted sequencing. In order to prevent the presence of aggregates, after making your antibody mix, centrifuge it at 14,000 rcf for 10 min at room temperature. Transfer the supernatant (containing Antibody Mix) to a new tube and maintain at 4C. This step will leave the antibody aggregates at the bottom of the original tube.
- Thorough washing of your sample post-antibody staining. Thorough washing of your sample post-stain is critical to obtaining good data. Failure to fully remove the unbound antibody will result in high background, making it impossible to look for differential expression of the antibodies. We recommend a minimum of 3 washes, performed in a volume that is sufficient to thoroughly dilute out the antibody after each wash, in order to fully remove all the antibody that did not bind to cells.
The following plot shows that a minimum of 3 washes is required in order to obtain a high percentage of antibody reads in cells.
Please see the infographics below for guidelines on proper washing of cell suspensions stained with antibodies:
If 1.5 ml of the buffer is used for the wash solution, then almost all of the supernatant must be removed during the washes (leaving only ~10 ul of wash buffer on the cell pellet). This may require multiple pipette draws from the tube, first with a P1000, then subsequent draws with a P200 or P20 to ensure complete removal of wash buffer. If this is not feasible, then a larger volume of wash buffer can be used, which allows for leaving ~100 ul of media on the pellet:
For more guidance on cell surface protein staining, please see our Demonstrated Protocol: Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols.
Products: Single Cell Gene Expression, Single Cell Immune Profiling