An efficient single-cell transcriptomics workflow for microbial eukaryotes benchmarked on Giardia intestinalis cells

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.
• 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.