A Microcapsule‐Integrated smRandom‐seq Platform Enables Fixation‐Free Single Microbe RNA Sequencing

Single‐microbe RNA sequencing (smRNA‐seq) emerges as a powerful approach for resolving microbial heterogeneity at single‐cell resolution, but current high‐throughput workflows still depend on chemical fixation to preserve cell‐of‐origin transcriptomic information during lysis, permeabilization, and downstream processing. Although fixation is integral to current smRNA‐seq workflows, its broader impact on overall performance remains unresolved because a practical alternative is lacking. Here, we present an smRNA‐seq strategy that integrates aqueous two‐phase system microcapsules with our previously developed smRandom‐seq method. Using polyethylene glycol diacrylate‐ and dextran‐based microcapsules, our method physically confines bacterial cells and released nucleic acids within individual microcompartments, thereby enabling single‐cell transcript capture in both fixation‐based and fixation‐free workflows. With two model species, Escherichia coli and Acinetobacter baumannii , we show that fixation‐free processing improves smRNA‐seq performance, increasing per‐cell gene detection sensitivity by approximately 50% relative to fixed controls while preserving similar overall transcriptional profiles. To our knowledge, this microcapsule‐integrated smRandom‐seq platform achieves the highest transcriptomic capture sensitivity among current high‐throughput smRNA‐seq platforms. This framework provides a practical alternative to fixation in high‐throughput smRNA‐seq and opens new prospects for the development of single‐microbe omics technologies.