Abstract
ABSTRACT
Sediment microbiomes drive global biogeochemical cycling, but their functional heterogeneity and transcriptional activity are hardly resolved at single‐cell resolution. We optimized a scalable single‐microbe RNA sequencing workflow for environmental muddy sediment samples, achieving high‐throughput single cell sequencing across environmental gradients. The method integrates iohexol‐based microbial enrichment, optimized enzymatic digestion step, droplet‐based single‐cell barcoding, and RNA sequencing, then generating a transcriptomic atlas of 55 859 high‐quality microbial cells from 8 composite samples covering freshwater, estuarine and nearshore marine habitats. Comparison with metagenomic data showed a high consistency in species composition, supporting the reliability of single‐microbe RNA sequencing in community profiling. Taxonomic analysis revealed site‐specific microbial communities associated with methane oxidation, sulfur cycling and anaerobic nitrogen metabolism. 15 distinct functional clusters were identified transcriptionally, showing functional heterogeneity within sedimentary microbiome. Co‐expression network analysis further resolved coordinated gene modules, with module activities varying across samples. Overall, this work establishes a practical framework for single‐microbe transcriptomics in sediment systems and demonstrates the potential of single‐cell resolution to uncover functional heterogeneity within complex microbial communities.