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. 2024 Dec 20;5(4):103475.
doi: 10.1016/j.xpro.2024.103475. Epub 2024 Dec 9.

Profiling low-mRNA content cells in complex human tissues using BD Rhapsody single-cell analysis

Alexandra Scheiber  1 , Manuel Trebo  2 , Annabella Pittl  1 , Isabel Heidegger  3 , Theresa Hautz  4 , Rupert Oberhuber  4 , Zlatko Trajanoski  5 , Florian Augustin  6 , Sieghart Sopper  2 , Dominik Wolf  1 , Andreas Pircher  7 , Stefan Salcher  8
Affiliations

Profiling low-mRNA content cells in complex human tissues using BD Rhapsody single-cell analysis

Alexandra Scheiber et al. STAR Protoc. .

Abstract

The successful recovery of immune cells, particularly those with low mRNA content, by single-cell RNA sequencing (scRNA-seq) remains a significant challenge. Tissue dissociation and selection of the appropriate scRNA-seq technology are crucial. Our protocol efficiently recovers low-mRNA content immune cells using the BD Rhapsody scRNA-seq platform. It includes optimized tissue dissociation for prostate, lung, and liver tissues, cell labeling with Sample Tag antibodies, microwell-based single-cell capture, cDNA synthesis, library preparation, and data pre-processing with basic quality control analysis. For complete details on the use and execution of this protocol, please refer to Salcher et al.1.

Keywords: Bioinformatics; Cancer; RNAseq; Sequencing.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Schematic overview of wet lab workflow
Figure 2
Figure 2
GentleMACS tube with dissociated tissue
Figure 3
Figure 3
Cell straining with plunger
Figure 4
Figure 4
Sample calculator of BD Rhapsody Scanner for cartridge loading Ratio of samples adjusted to viability.
Figure 5
Figure 5
BD Cartridge image metrics
Figure 6
Figure 6
TapeStation High Sensitivity D1000 sample traces of purified RPE PCR product
Figure 7
Figure 7
Qualitative analysis of WTA and Sample Tag index PCR products TapeStation High Sensitivity D1000 sample traces of WTA (A) and (optional) Sample Tag (B) library preparation.
Figure 8
Figure 8
QC metrics of prostate (benign/tumor), lung (normal/tumor) and liver (normal) datasets generated with BD Rhapsody (A) Uniform manifold approximation and projection (UMAP) color-coded by cell type. (B) UMAP color-coded by total transcript counts. (C) nCounts quality metrics of filtered prostate, lung and liver cells.
Figure 9
Figure 9
Image of the microwells in the BD Rhapsody Cartridge after cell load Multiplet indicated with an arrow.
Figure 10
Figure 10
Results of erythrocyte lysis Cell count image before (A) and after (B) additional erythrocyte lysis.
Figure 11
Figure 11
Qualitative analysis of PCR product purification TapeStation profiles of purified WTA index PCR product before (A) and after (B) additional purification.
See this image and copyright information in PMC

References

    1. Salcher S., Heidegger I., Untergasser G., Fotakis G., Scheiber A., Martowicz A., Noureen A., Krogsdam A., Schatz C., Schäfer G., et al. Comparative analysis of 10X Chromium vs. BD Rhapsody whole transcriptome single-cell sequencing technologies in complex human tissues. Heliyon. 2024;10 doi: 10.1016/j.heliyon.2024.e28358. - DOI - PMC - PubMed
    1. Salcher S., Sturm G., Horvath L., Untergasser G., Kuempers C., Fotakis G., Panizzolo E., Martowicz A., Trebo M., Pall G., et al. High-resolution single-cell atlas reveals diversity and plasticity of tissue-resident neutrophils in non-small cell lung cancer. Cancer Cell. 2022;40:1503–1520.e8. doi: 10.1016/j.ccell.2022.10.008. - DOI - PMC - PubMed
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    1. Hautz T., Salcher S., Fodor M., Sturm G., Ebner S., Mair A., Trebo M., Untergasser G., Sopper S., Cardini B., et al. Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver. Nat. Commun. 2023;14:2285. doi: 10.1038/s41467-023-37674-8. - DOI - PMC - PubMed
    1. Heidegger I., Fotakis G., Offermann A., Goveia J., Daum S., Salcher S., Noureen A., Timmer-Bosscha H., Schäfer G., Walenkamp A., et al. Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer. Mol. Cancer. 2022;21:132. doi: 10.1186/s12943-022-01597-7. - DOI - PMC - PubMed

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