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Updated: Jul 4, 2026

AQRNA-seq for Quantifying Small RNAs
Published on: February 2, 2024
Single-bacterium RNA-seq protocol to uncover heterogeneous expression of coding and noncoding genes in Bacteroides
Elise Bornet1, Alexander J Westermann2
1Department of Microbiology, Biocenter, University of Würzburg, Würzburg, Germany; Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research, Würzburg, Germany.
None:
Single-cell RNA sequencing is increasingly applied to bacterial model species, but a dedicated technique for anaerobic microbiota members of the Bacteroidota phylum has been lacking. Here, working with Bacteroides thetaiotaomicron, we describe experimental steps for transcriptome stabilization, fluorescence-activated cell sorting (FACS) collection, and optimized lysis of single cells from this group of organisms. We detail procedures for reverse transcription of RNAs via the multiple annealing and dC-tailing-based quantitative single-cell RNA sequencing (MATQ-seq) protocol, sensitive Cas9-based depletion of ribosomal sequences, and cDNA library generation. For complete details on the use and execution of this protocol, please refer to Bornet et al.1.
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