Fast Encapsulation of Microbes into Dissolvable Hydrogel Beads Enables High‐Throughput Microbial Single‐Cell RNA Sequencing of Clinical Microbiome Samples

Abstract Microbial single‐cell RNA‐seq (mscRNA‐seq) can achieve resolution at the cellular level, enhancing the understanding of microbial communities. However, current high‐throughput mscRNA‐seq methods are limited by multiple centrifugation steps, which can lead to microbial loss and bias. smGel‐seq is reported, a high‐throughput single‐microbe RNA sequencing method for clinical microbiome samples that employs hydrogel beads to encapsulate individual microbes to reduce microbial loss and input requirements. In this method, a novel microchannel array device is implemented for encapsulating single microbe in dissolvable hydrogel beads (smDHBs), along with an optimized automated microfluidic platform to co‐encapsulate barcoded beads and smDHBs, enabling high‐throughput barcoding of individual microbes. smGel‐seq significantly increases the microbial recovery rate in a gut microbiome sample from 8.8% to 91.8%. Furthermore, this method successfully processes clinical microbiome samples with microbial inputs 20 times lower than those required by previous methods. Notably, smGel‐seq enables the first mscRNA‐seq in a clinical sputum microbiome sample, revealing a specific microbial subpopulation that may play a key role in environmental adaptability, antibiotic resistance, and pathogenicity. These results highlight the compatibility of smGel‐seq with clinical microbiome samples and demonstrate its potential for widespread application in diverse clinical and research settings.