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A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations
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Profiling low-mRNA content cells in complex human tissues using BD Rhapsody single-cell analysis.

Alexandra Scheiber1, Manuel Trebo2, Annabella Pittl1

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This study presents a protocol for efficiently recovering immune cells with low mRNA content using single-cell RNA sequencing (scRNA-seq). The method optimizes tissue dissociation and utilizes the BD Rhapsody platform for improved cell capture and analysis.

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Area of Science:

  • Immunology
  • Genomics
  • Biotechnology

Background:

  • Single-cell RNA sequencing (scRNA-seq) is vital for immune cell analysis.
  • Efficient recovery of low mRNA content immune cells is a major technical challenge.
  • Optimizing tissue dissociation and scRNA-seq technology is critical for success.

Purpose of the Study:

  • To develop and present a robust protocol for enhanced recovery of low mRNA content immune cells.
  • To validate the protocol using various tissue types including prostate, lung, and liver.
  • To enable deeper insights into immune cell populations through improved scRNA-seq.

Main Methods:

  • Optimized tissue dissociation techniques for prostate, lung, and liver tissues.
  • Utilized the BD Rhapsody single-cell RNA sequencing platform.
  • Incorporated Sample Tag antibody labeling, microwell-based cell capture, cDNA synthesis, library preparation, and data pre-processing.

Main Results:

  • Demonstrated efficient recovery of immune cells, including those with low mRNA content.
  • The protocol is applicable across multiple challenging tissue types.
  • Provided a comprehensive workflow from tissue dissociation to data analysis.

Conclusions:

  • The developed protocol significantly improves the recovery of low mRNA content immune cells for scRNA-seq.
  • This method enhances the capability to study diverse immune cell populations.
  • The protocol serves as a valuable resource for researchers in immunology and related fields.