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InTraSeq™ Single Cell Analysis Overview

CD4+ and C8+ cluster visualization identifying cellular subpopulations in RNA and protein. CD4+ and C8+ cluster visualization identifying cellular subpopulations in RNA and protein.
Analysis of isolated CD4+ and CD8+ cells using InTraSeq intracellular and signaling pathway antibodies to identify cellular states that would be difficult to study using RNA alone. The InTraSeq assay offers additional insights into cellular subpopulations, by measuring PTMs and intracellular protein levels in single cells which can enable a deeper understanding of disease development mechanisms.

Seq What You’ve Been Missing

Intracellular protein and Transcriptomic Sequencing (InTraSeq) is a novel technology developed by CST that identifies signaling pathways and reveals molecular mechanisms in disease development in a single experiment. It enables simultaneous detection of RNA as well as both intracellular and surface proteins in thousands of cells, allowing researchers to investigate signaling pathways with the transcriptome—all at a single-cell resolution.

InTraSeq facilitates deeper understanding of cell biology by:

  • Uncovering biological insights not revealed with traditional single-cell RNA sequencing (scRNA-seq) methods
  • Highlighting the dynamics between transcription and translation in single-cell datasets
  • Dissecting cell signaling mechanisms by quantifying post-translational modifications at the single-cell level

InTraSeq 3’ technology is developed and validated by CST, using the 10x Genomics Chromium Single Cell 3' Reagent Kits with Feature Barcoding technology.

What makes InTraSeq unique?

Graphic showing that InTraSeq™ Single Cell Analysis detects surface and intracellular proteins while guaranteeing a robust RNA signal, as opposed to other technologies attempting to capture intracellular protein in single-cell assays at the cost of RNA integrity.
A)Most single-cell analysis techniques currently available detect RNA and use barcoded antibodies to measure surface proteins for phenotyping purposes. B) Many have tried developing methods to detect intracellular proteins, but these methods typically suffer from significant RNA loss and degradation. C) InTraSeq™ Single Cell Analysis detects RNA alongside intracellular and surface proteins while preserving RNA.

Most currently available single-cell analysis technologies are limited to phenotyping a sample because they only identify RNA and, occasionally, surface marker expression levels. Additionally, other technologies are not able to determine and measure important signaling cascades activated during a disease state or by pharmacologic perturbation. By combining transcriptomic, protein, and post-translational modifications quantification into a single assay, InTraSeq technology goes beyond phenotyping and dissects the cellular mechanisms that distinguish cellular differences—all in one experiment. With InTraSeq technology, scientists can readily explore multiple signaling pathways along with genetic expression changes to efficiently identify key molecular mechanisms underlying physiologic responses and discern downstream protein modifications.

Single-Cell Analysis (SCA) for Intracellular Proteins and Post-Translational Modifications

Single-cell RNA sequencing (scRNA-seq) is commonly utilized to determine cellular responses to pharmacologic and environmental perturbations. However, protein expression levels often do not directly correlate with RNA expression, leaving a gap in the understanding of complex physiologic systems. Determining how single-cell protein expression and post-translational modifications differ from single-cell gene expression can offer functional insight into pathway activation that cannot be obtained from scRNA-seq alone.

Post-translational modifications (PTMs) can alter protein activity, longevity, and expression levels. Understanding the interplay between genetic expression and protein activity is crucial to determine the underlying cellular heterogeneity and functional state of complex biological systems. The InTraSeq 3' Conjugate Antibody Cocktail is formulated with 31 of our most popular antibodies that bind to both human and mouse targets, including PTMs. Scientists can quantify dozens of critical signaling proteins in a single assay with ease and without bias.

Co-Quantification of Single-Cell PTMs and mRNA

Many cells can exhibit similar levels of RNA expression, but some may differ in which proteins are ultimately expressed and subsequently modified to regulate activity after pharmacologic or environmental perturbations. Co-quantification of RNA, protein, and post-translational modifications facilitates a more thorough understanding of complex physiologic interactions that RNA expression alone cannot elucidate.

RNA Does Not Necessarily Correlate with Protein Levels
A scatterplot graph showing expression levels of mRNA and proteins. A scatterplot graph showing expression levels of mRNA and proteins.
Co-quantitation of mRNA and cellular proteins with InTraSeq reveals differential expression levels of mRNA and protein where the two values often do not correlate.

InTraSeq Single Cell Analysis Advantages

Reduced Hands-On Time with a Streamlined Workflow

A Straightforward, Four-Step Protocol
Graphic showing the four steps in the InTraSeq protocol, as described elsewhere in the text.
The InTraSeq™ 3’ Assay Kit is optimized to provide a robust mRNA and protein signal in a straightforward protocol that requires minimal bench time. The two convenient stopping points allow for flexibility and sample storage for up to seven days, providing maximum workflow flexibility.

The InTraSeq protocol requires approximately one hour of hands-on bench time and includes multiple stopping points, providing maximum time flexibility when performing single-cell experiments. Samples can be stored for up to seven days after the fixation step without compromising RNA integrity. Unlike currently available single-cell analysis protocols, the convenience offered by InTraSeq eliminates the need to coordinate staff and equipment with long single-day protocols. The ability to store samples for longer periods of time can also facilitate high-throughput applications by allowing bulk sample and single-cell processing protocols to be performed on sequential days without compromising quality.

The straightforward immunostaining protocol includes the following steps:

Step 1: Fix the cells overnight.

  • ~5 min hands-on benchwork
  • Cells can be stored in the freezer for up to seven days.

Step 2: Incubate with scBlock.

  • ~10 min hands-on benchwork
  • This step is optimized to obtain high-quality single-cell readout of both RNA and proteins.

Step 3: Add CST® InTraSeq™ 3’ Conjugate Antibody Cocktail, and incubate overnight.

  • ~5 min hands-on benchwork

Step 4: Wash the cells.

  • ~20 min hands-on benchwork
  • At this point the cells are ready for a single-cell 10x Genomics 3’ experiment.

Investigate Intracellular Protein Signaling while Guaranteeing Robust RNA Signal

Preserving RNA integrity in single-cell multiomic experiments has been a persistent and significant challenge. Many single-cell assays utilize harsh chemicals to disrupt the membrane resulting in RNA degradation and loss. InTraSeq reagents have been rigorously optimized to gently permeabilize the cell and nuclear membranes. This allows the conjugated antibodies to enter the cell and bind to their targets while maintaining RNA levels throughout the assay. This enables accurate quantitation of both RNA and protein expression.

InTraSeq Preserves the Transcript Abundance in the Sample Compared to Live Cell Control
Two side-by-side scatterplot graphs comparing live PMBCs to InTraSeq PMBCs expression levels. Two side-by-side scatterplot graphs comparing live PMBCs to InTraSeq PMBCs expression levels.
  Live PBMCs InTraSeq™ PBMCs
Relative Median Genes/Cell (matched reads/cell) 100% 86.2%
Fraction Reads in Cells 94.9% 85%
Antibody Reads in Cells N/A 60%
Reads Mapped Confidently to Genome 89.9% 85.5%
Average gene expression was quantified in live and InTraSeq prepared samples of PMBCs. InTraSeq reagents preserve the genetic profile of live cells in difficult samples such as PBMCs, with minimal loss of mRNA.

Identify Hard-To-Detect Cells with Unbiased Depth-of-Coverage

The power of InTraSeq single-cell analysis technology lies in the ability to detect and quantify both intracellular and surface proteins in conjunction with single-cell gene expression to differentiate heterogeneous cell populations. In contrast to CITE-Seq, which only detects cell surface proteins, InTraSeq provides scientists with tools to study complex intracellular signaling pathways without bias.

InTraSeq Method Preserves Cellular Heterogeneity
A colorful UMAP visualization that compares live PBMCs and PBMCs after the inTraSeq protocol, showing the proportions of each cell type were consistent between samples, demonstrating mRNA preservation.
UMAP displaying cell types in PBMCs from the same donor before and after the InTraSeq protocol. The split UMAP shows that the proportion of each cell type was similar and not impacted by the InTraSeq protocol.

Explore Multiple Molecular Mechanisms In One Experiment

Combined with CST expertise in intracellular signaling and post-translational modification antibodies, the InTraSeq technology enables researchers to study and quantify intricate intracellular, nuclear, and surface protein interactions together with gene expression. Simultaneously measuring transcriptomics changes and protein expression, including post-translational modifications, generates more comprehensive data sets, enriching the knowledge of complex signaling systems in a way not possible with other assays and deepens our understanding of disease mechanisms.

Identifying Cell States within CD4+ and CD8+ Cells Using Single-Cell Intracellular Protein Readout
CD4+ and C8+ cluster visualization identifying cellular subpopulations in RNA and protein. CD4+ and C8+ cluster visualization identifying cellular subpopulations in RNA and protein.
Analysis of isolated CD4+ and CD8+ cells using InTraSeq intracellular and signaling pathway antibodies to identify cellular states that would be difficult to study using RNA alone. The InTraSeq assay offers additional insights into cellular subpopulations, by measuring PTMs and intracellular protein levels in single cells which can enable a deeper understanding of disease development mechanisms.

Of the 31 validated antibodies in InTraSeq 3' Conjugate Antibody Cocktail, 2 target surface proteins, and the other 29 target intracellular proteins, including PTMs. The antibody cocktail is designed for unbiased quantitation of proteins along signaling pathways commonly studied in immunology and cancer research providing accurate and reliable data sets.

Phospho-Stat3 (Ser727) heatmap showing PTM levels correlated with RNA and protein and Phospho-p44/42 MARK (Erk1/2) (Tyr202/Tyr204) heatmap showing PTM levels correlated with RNA and protein. Phospho-Stat3 (Ser727) heatmap showing PTM levels correlated with RNA and protein and Phospho-p44/42 MARK (Erk1/2) (Tyr202/Tyr204) heatmap showing PTM levels correlated with RNA and protein.
PBMCs were stimulated with LPS and processed using the InTraSeq™ Assay Kit, RNA, total protein, and PTMs were quantified with scRNA-seq and the InTraSeq™ 3' Conjugate Antibody Cocktail. The heatmaps display the PTM levels of Stat3 and MAPK before and after stimulation, as well as which RNA and proteins correlate with those shifts in phosphorylation levels.

How does InTraSeq support my research?

InTraSeq can be a unique and powerful screening tool when starting a project. It enables researchers to understand a specific genetic phenotype by offering an unbiased approach that determines which signaling pathway is perturbed and can help determine where to focus.

InTraSeq can also measure acute cellular perturbations that occur within short periods of time (< 20 minutes) since PTMs responses occur before mRNA. These acute responses would otherwise not be observed in RNA alone in single cells and outlines the importance and uniqueness of InTraSeq technology.

Trust Your Results with the CST-Validated InTraSeq™ 3' Conjugate Antibody Cocktail

CST scientists rigorously validate and optimize all InTraSeq antibodies and reagents to provide consistent, high-quality results. The InTraSeq™ 3' Conjugate Antibody Cocktail is formulated such that all the included antibodies and reagents are at optimal concentrations and ready to use in your experiments. InTraSeq removes the guesswork and enables scientists to collect trustworthy data without the need for time-consuming optimization experiments.

InTraSeq Single Cell Analysis Product Offerings

Find the right solution for your workflow below.

InTraSeq™ 3' Conjugate Antibody Cocktail

Contains 31 primary antibodies against popular signaling pathway proteins and reacts with both human and mouse samples.

InTraSeq™ 3' Assay Kit

A buffer kit specially formulated for single-cell analysis.

InTraSeq™ 3' Conjugates

Besides the thirty-one 3' conjugated antibodies included in the InTraSeq 3' Conjugate Antibody Cocktail 1 (human and mouse reactive), seven additional InTraSeq 3' conjugates (human reactive) are also available individually.

Surface Targets Species Reactivity Included in Cocktail?
CD3 (UCHT1) Mouse mAb (InTraSeq™ 3' Conjugate 3028) H No
CD4 (RPA-T4) Mouse mAb (InTraSeq™ 3' Conjugate 3029) H No
CD8α (SK1) Mouse mAb (InTraSeq™ 3' Conjugate 3030) H No
CD19 (Intracellular Domain) (D4V4B) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3010) H, M Yes
CD68 (D4B9C) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3012) H No
NCAM1 (CD56) (E7X9M) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3025) H, M Yes
T-bet/TBX21 (D6N8B) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3009) H No
Intracellular Targets (non-PTM) Species Reactivity Included in Cocktail?
Aiolos (D1C1E) Rabbit mAb #15103 (InTraSeq™ 3' Conjugate 3027) H, M Yes
FoxO1 (C29H4) Rabbit mAb (InTraSeq™ 3' Conjugate 3037) H, M Yes
GAPDH (14C10) Rabbit mAb (InTraSeq™ 3' Conjugate 3007) H, M Yes
Iba1/AIF-1 (E4O4W) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3003) H, M Yes
Ikaros (D6N9Y) Rabbit mAb #14859 (InTraSeq™ 3' Conjugate 3026) H, M Yes
NFAT1 (D43B1) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3032) H, M Yes
S100A9 (D5O6O) Rabbit mAb (InTraSeq™ 3' Conjugate 3004) H No
TCF1/TCF7 (C63D9) Rabbit mAb (InTraSeq™ 3' Conjugate 3013) H, M Yes
Vimentin (D21H3) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3011) H, M Yes
Isotype Control Species Reactivity Included in Cocktail?
Mouse (G3A1) mAb IgG1 Isotype Control (InTraSeq™ 3' Conjugate 3001) H No
Controls Included In Assay Kit (not for individual sale) Species Reactivity Included in Cocktail?
Histone H3 (D1H2) XP® Rabbit mAb (InTraSeq™ 3' Conjugate 3002) H, M No
Rabbit (DA1E) mAb IgG XP® Isotype Control (InTraSeq™ 3' Conjugate 3000) H, M No

Technical Support

Visit the Technical Support page to search InTraSeq Single Cell Analysis-related troubleshooting information, answers to technical questions, and how to contact us.

Cell Signaling Technology, CST, and InTraSeq are trademarks of Cell Signaling Technology, Inc. All rights reserved. 10x Genomics, 10x, Feature Barcode, and Chromium are the trademarks or registered trademarks of 10x Genomics, Inc. All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
Subject to patents licensed from 10x Genomics, Inc., for use with single-cell (i.e., Chromium) 10x products.
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