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Updated: Dec 17, 2025

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights
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An efficient single-cell transcriptomics workflow for microbial eukaryotes benchmarked on Giardia intestinalis cells.

Henning Onsbring1, Alexander K Tice2, Brandon T Barton2

  • 1Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, 75123, Uppsala, Sweden.

BMC Genomics
|July 1, 2020
PubMed
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This summary is machine-generated.

This study optimized the Smart-seq2 protocol for single-cell RNA sequencing in protists. Adding freeze-thaw cycles improved transcript recovery, making protist cell biology exploration more efficient.

Area of Science:

  • Microbiology
  • Genomics
  • Eukaryotic diversity

Background:

  • Microbial eukaryotes (protists) represent vast eukaryotic diversity but are underrepresented in genomic studies due to cultivation challenges and biased sequencing.
  • Single-cell genomics offers a cultivation-independent approach but faces limitations like low genome recovery and complex assembly.
  • Single-cell transcriptomics bypasses genome assembly issues by focusing on transcribed regions.

Purpose of the Study:

  • To optimize the Smart-seq2 protocol for robust and cost-efficient single-cell RNA sequencing in protists.
  • To identify modifications that improve transcript recovery and reduce costs for protist single-cell analysis.

Main Methods:

  • Adaptation and testing of the Smart-seq2 protocol for single-cell RNA sequencing.
Keywords:
Giardia intestinalisMicrobial diversityMicrobial eukaryotesProtistsRNAseqSingle cell genomicsSingle-cell RNA sequencingSmart-seq2TranscriptomeTranscriptomics

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  • Inclusion of freeze-thaw cycles, reduction of reaction volumes, and alternative cDNA purification methods.
  • Benchmarking modifications using the sequenced genome of Giardia intestinalis.

    • Main Results:

    • Addition of freeze-thaw cycles significantly increased transcript recovery in protists.
    • Reducing reaction volumes or using alternative purification methods did not improve results and sometimes decreased gene detection.
    • Other tested modifications, including 5' biotinylation and altered oligo-dT concentration, showed no significant impact on gene detection.

    Conclusions:

    • The modified Smart-seq2 protocol, incorporating freeze-thaw cycles, provides an efficient method for single-cell RNA sequencing in protists.
    • This workflow enhances the exploration of protist diversity and cell biology at the single-cell level.