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Single Cell Biology | EK26


Advances in single cell whole transcriptome analysis: single nucleus RNAseq and simultaneous protein and mRNA profiling using the BD RhapsodyTM Single-Cell Analysis system and BD® AbSeq


Mar 17, 2021 12:00am ‐ Mar 17, 2021 12:00am

Description

Advances in single cell whole transcriptome analysis: single nucleus RNAseq and simultaneous protein and mRNA profiling using the BD RhapsodyTM Single-Cell Analysis system and BD® AbSeq Hye-Won Song1, Gretchen Y. Lam2, Margaret Nakamoto2, Punya Narayan2, Ian Taylor3 and Stefanie Mortimer2 1. BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 2. BD Biosciences, 2350 Qume Drive, San Jose, CA 95131 3. FlowJo LLC, 385 Williamson Way, Ashland, OR 97520 Advances in high-throughput single cell whole transcriptome analysis (WTA) have enabled the discovery of biomarkers and cellular pathways critical to resolving diversity in diseases. Despite these advances, challenges still remain in comprehensively profiling single cells. Here we address two challenges, limited use of frozen tissues, and inability to resolve post-transcriptional differences from cell to cell. Single-cell RNAseq often requires intact single cells to get good resolution of signal, preventing the use of archival frozen samples. To overcome this limitation, we have tested the possibility to use dissociated nuclei that can be isolated from frozen samples in the BD RhapsodyTM Single-Cell Analysis system. As a proof of concept, we compared high throughput single-nucleus WTA data to that produced from single-cell RNAseq from the same cell type. Our data showed that mRNAs from single nuclei can be captured, and amplified using the BD RhapsodyTM system. Furthermore although the number of molecules detected is lower in nucleus, probably due to lower mRNA content, their expression profiles are well correlated with Pearson’s correlation coefficient over 0.9. This opens up the possibility to conduct single cell transcriptomic analysis on the large pool of archived frozen specimens, expanding the variety and number of disease samples that can be examined at the single cell level in the BD RhapsodyTM system. A second limitation of WTA analysis is that it only provides information about gene regulation at the transcript level. Given that regulation critical for cellular pathways is often found not only at the transcriptional level but also at the post-transcriptional level, quantitative analysis of proteins and mRNAs at the single cell level can provide deeper understanding of the disease cells. Here, we used DNA-barcoded antibodies BDT® AbSeq to enable multiomic analysis, examining protein alongside mRNA expression and enabling simultaneous transcriptional and post-transcriptional gene profiling in single cells. Our data show that upon activation human peripheral blood mononuclear cells and isolated T cells undergo regulation of markers at the protein level that cannot be resolved at the mRNA level. Several key markers used to define activation states including CD69 and L-Selectin were found to be regulated at the protein level confirming that addition of protein analysis to WTA can generate more comprehensive cell profiling. These approaches offer flexibility and choice in experimental design and allow users to utilize archived frozen samples or to obtain mRNA and protein profiles from single cells. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

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