High‐throughput Profiling of Pseudouridines in Microbiome‐derived Bacterial RNA

Pseudouridine (Ψ) is a widespread RNA modification that influences RNA stability, structure, and translation. However, its role in bacterial mRNA, particularly within complex microbiomes, remains poorly defined. Here, we describe a bisulfite-based sequencing workflow coupled with a scalable computational pipeline for base-resolution, quantitative mapping of pseudouridine in microbiome transcriptomes. The protocol is optimized for low-input, high-complexity samples and includes strategies for efficient RNA extraction, ribosomal RNA depletion, bisulfite conversion, library preparation, and sequencing. The accompanying analysis pipeline enables detection and quantification of Ψ sites from chemically induced signatures, with modules for read alignment, site calling, and filtering in mixed-bacterial datasets. This approach addresses key challenges in microbiome transcriptomics, including limited biomass, high rRNA content, and community heterogeneity. The protocol can be applied to samples from diverse microbial ecosystems to generate pseudouridylation profiles, enabling investigation of pseudouridine's role in post-transcriptional regulation across microbial communities. Published 2026. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microbiome sample collection and processing Basic Protocol 2: mRNA enrichment and bisulfite treatment Basic Protocol 3: cDNA synthesis and multiplexing of samples for sequencing Basic Protocol 4: Computational pipeline for pseudouridine analysis.