Expanding your range of sample access

Fresh samples are the most commonly used sample types in research due to their capacity to provide a wide array of information for various research questions. However, their vulnerable and easily degradable nature necessitates timely processing. This issue places heavy burdens on sample processing infrastructure and requires effective coordination between relevant parties. Besides, unpredictable transport time prevents researchers from using the fresh specimens in their own space or adjusting their experimental design to align with their research interests.

In response to retrospective studies corresponding to a cohort of patients, FFPE samples, which are preserved in hospital bio-banks with patients’ medical histories around the world, are extracted. Despite their tremendous potential, these samples were traditionally incompatible with 10x Genomics assays like Chromium 3’ and 5’ Gene Expression.

This is where Single cell fixed RNA profiling steps in to make what previously impossible possible for single cell studies, allowing you to uncover critical cellular insights from fresh, frozen, and fixed sampleseven FFPE.

Unprecedented throughput

What sets our Chromium Fixed RNA Profiling apart is its outstanding throughput and versatility. For singleplex samples, you can efficiently partition 500–10,000 cells per channel, running up to 8 samples and 80,000 cells per chip. For multiplex samples, you can multiplex up to 16 samples and 128,000 cells per channel, running 1–128 samples or up to 1 million cells each run. Additionally, this assay offers cell size flexibility with no lower limits and is compatible with fresh or PFA-fixed tissues, cells, and nuclei as well as FFPE samples.

Furthermore, our technology supports cell surface protein expression compatible with whole cells, using both singleplex and multiplex workflows. With high cell capture rates of approximately 65% and low rates of undetected multiplets (0.8% per 1,000 cells, up to 8% at max load per sample), our Fixed RNA Profiling assay should be fully entrusted with delivering reliable and comprehensive results.

Figure 1. Workflow. Cells are fixed using 10x Genomics’ Fixation Buffer containing 4% formaldehyde followed by hybridization with probe sets, each of which goes with a unique Probe Barcode (depicted in different colors) to enable sample multiplexing. After overnight hybridization, samples are pooled, washed, and partitioned in the Chromium iX/X instrument, where the probes are ligated along with addition of a 10x GEM Barcode, followed by library construction, sequencing, and data analysis.

Probe-based technology with exceptional sensitivity and robustness

Chromium Fixed RNA profiling assay has brought the probe-based technology to the next level with the design of whole transcriptome probe sets covering over 18,000 gene transcript. This unmatched quality is achieved by employing up to three probe pairs to tile more than 90% of genes. Besides, our probe set enhances accuracy and reliability by managing noise signals, excluding certain gene families, common variants and minimizing off-target ligations.

The superior features of our probe set are validated through a comparative analysis of gene expression profiles generated using both the Chromium Single Cell 3’ v3.1 Gene Expression assay and the Fixed RNA Profiling assay. Our probes demonstrate high specificity and sensitivity, as evidenced by the differential expression correlation between the three tiled probe pairs and across the two aforementioned assays (see Figure 2).

Figure 2. Probe specificity and sensitivity is demonstrated for 8 genes by comparing their expression patterns in dissociated tumor cells (DTCs). Breast cancer DTCs were analyzed using the Chromium Single Cell 3’ v3.1 assay (fresh), and were concurrently fixed and assessed using the Chromium Fixed RNA Profiling assay. The two datasets, integrated by the Harmony package, had overlapping UMAPs indicating comparable sensitivity and specificity.

Sparkling new possibilities in research with integration of spatial technologies

Single cell and spatial technologies have independently emerged as key methods, allowing researchers to delve deeper into the complex biological systems, yet there’s still more horizon to be explored. With single cell analysis, researchers can characterize specific cells present in a sample and, with spatial information, identify where and how those cells are organized and interact with one another. Together, the synergy between these two complementary technologies empowers scientists to identify and localize novel cell types and states, unraveling the underlying mechanisms of development and disease pathology.

To exemplify, ductal carcinoma in situ (DCIS) has long been recognized as an earliest stage of breast cancer, so unraveling its tumor microenvironment is a cornerstone to refine our understanding of its pathogenesis in women. This is where the power of all three technology platforms —Chromium Gene Expression Flex, Visium CytAssist, and Xenium In Situ—is harnessed to perform the deep characterization of the tumor microenvironment of a human FFPE DCIS tissue block (7). In this publication, by leveraging single cell (scFFPE-seq) and Visium spatial analysis on adjacent tissue sections, they created a comprehensive map of the cellular composition and spatial organization of the tumor sample. This integrated approach localized cell types and disease states across the tumor section, including the spatial location of three tumor domains—two molecularly distinct types of DCIS (DCIS #1 and #2) and an invasive tumor region—and other immune, stromal, and adipocyte cells (see Figure 3).

Figure 3. Characterization of an FFPE-preserved breast cancer sample using whole transcriptome single cell and spatial technologies. (a) Dimension reduction of the scFFPE-seq data yielded a t-SNE projection with 17 unsupervised clusters. (b) t-SNE projection of Visium spots also identifies 17 clusters. Based on differential gene expression analysis, ten clusters could be unequivocally assigned to cell types, while the others were mixtures of cell types. (c) H&E staining conducted pre-CytAssist is shown for reference alongside the spatial distribution of clusters in (b). Cell-type-specific marker genes are expressed as log2(normalized UMI counts). The Visium data elucidated the spatial location of two molecularly distinct DCIS and invasive subtypes and the general locations of immune, myoepithelial, adipocytes, and stromal cells. Additionally, Visium features mitochondrial probes (e.g., MT-ND1), and their spatial distribution correlates with the invasive region of the tissue section. This experiment was performed on two serial sections, with one representative section shown here. Image adapted from Figure 2 from Janesick et al. 2023. (CC BY 4.0). Find more information about this article here

Dive deeper into Chromium Fixed RNA profiling, Visium Spatial Transcriptomics, Xenium Spatial Transcriptomics with GeneSmart- an official distributor of 10x Genomics in Vietnam.

References

1. Kohlway, A., Hill, A., Abousoud, J., Cui, F., Lund, P., Sponer, N., Arthur, J., Alexis, M., Jackman, S., Maheshwari, S., Chatterjee Bhattacharjee, S., Chen, K., Hui, W., Tran, K., Sauzade, M., Stott, R., Giangarra, V., & Pfeiffer, K. (2022).

2. High sensitivity single cell RNA profiling of fixed cells via in situ RNA-templated ligation [Poster presentation]. Single Cell and Spatial Technologies at 2022 AGBT General Meeting, California, June 7, 2022.Janesick A, et al.

3. High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis. Nat Commun (2023). 14: 8353. doi: 10.1038/s41467-023-43458-x

4. Olivia Habern. Two platforms, one powerful spatial biology toolkit: When and how researchers are using Visium and Xenium. 10x Genomics Blog. (2023) https://www.10xgenomics.com/blog/two-platforms-one-powerful-spatial-biology-toolkit-when-and-how-researchers-are-using-visium-and-xenium