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.