Sometimes, doublets and multiplets in single-cell datasets can skew the protein visualization in FeaturePlot. Using a maximum cutoff (max.cutoff) bypasses the observed high level of protein expression from outliers and allows clear visualization. Use a large value for this parameter, such as "q90" or higher ("q95", "q99", etc.), as appropriate for your experiment. See the example in bold below:
FeaturePlot(object, features = c("protein_Phospho-S6(Ser235-236)-Ab"), max.cutoff = “q90”, min.cutoff = "q5")
It is also important to read the published literature and mine public databases to ensure that your protein is expressed in your sample, and learn what conditions may change that expression.
If a non-InTraSeq-validated antibody is used, insufficient antibody binding can skew visualization. Use CST® InTraSeq-validated antibodies in your single-cell experiments and follow the InTraSeq protocol precisely to ensure reliable and reproducible results.
Many signaling pathway targets are ubiquitously expressed and can be detected in multiple cell types. When analyzing and interpreting signaling pathway data, make sure to design your experiments with the appropriate controls (knockdowns, knockouts, stimulation, inhibition, etc.).
Using a color gradient in FeaturePlot helps facilitate the analysis and interpretation of single-cell signaling pathway data. See the example in bold below using blue (low expression), grey (mid expression), and red (high expression):
FeaturePlot(your_object, features = c("protein_Phospho-CREB(Ser133)-Ab"), max.cutoff = "q95", min.cutoff = "q5") + scale_color_gradientn(colours = c("#003C7B", "#73A9D2", "grey90", "#FDB393", "#b22222"))
CST scientists rigorously validate all InTraSeq reagents and protocols to ensure consistent and robust RNA preservation. Use InTraSeq-validated reagents to obtain high-quality RNA sequencing results and preserve cellular heterogeneity. InTraSeq antibody conjugates and protocols were developed and validated for use with methanol fixation and work best when following the InTraSeq protocol.
We designed and validated the InTraSeq reagents and protocol to be used with the 10x Genomics Chromium platform. After completing the InTraSeq protocol, scientists should use the 10x Genomics Chromium Single Cell 3' Kits with Feature Barcoding technology. For more information, see the appropriate user guides: CG000317, CG000206, or CG000732.
We offer a wide range of custom conjugation and labeling services for CST primary antibodies, including those for use in single-cell analysis.
It is important to note that custom-labeled antibodies with oligonucleotides are not validated for use with InTraSeq reagents. It is up to the customer to properly titrate and validate any custom conjugated antibody for use with InTraSeq technology.
For more details, please visit the Custom Conjugation and Labeling Service webpage, or contact your local sales department.
The rate at which the methanol fixation buffer is added can impact cell clumping. In Cell Fixation (Day 1) - step 7 of the InTraSeq protocol, it is very important to dispense the methanol over a period of at least 60 seconds. Failure to do so can result in doublets/multiplets.
If cell clumps are still observed after Washes and Count (Day 3) - step 8 of the InTraSeq protocol, additional pipette mixing is recommended before proceeding to the single-cell experiment.
When starting Step B. Blocking (Day 2) and continuing until the end of the protocol, it is crucial to centrifuge at 850 x g (not RPM). Centrifuging at lower speeds will result in cell loss.
Additionally, starting with Step C: Immunostaining (Day 2), it is essential to ensure the cell pellet is submerged in ~40 µL of fluid in every step that requires removing and discarding the supernatant. Attempting to discard all of the supernatant will result in cell loss.
While it is possible to stain live cells with conjugated antibodies or hashing reagents before an InTraSeq experiment, it is not recommended. Since the binding properties of antibodies may perform differently when using InTraSeq reagents, we highly discourage using non-InTraSeq validated 3’ conjugates in Step C. Immunostaining (Day 2).
If performing an immunostaining step before the InTraSeq 3’ protocol, we highly recommend beginning the immunostaining with at least 2 million cells to guarantee an adequate number of cells in Step 1.
The number of reads for the InTraSeq protein library should be at least 5,000 reads per cells.
Each antibody in the cocktail is titrated to guarantee a robust signal in the InTraSeq assay with minimal background.
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