Microscopic-scale gut microbiota dataset of Kunming mice revealed by 16S rRNA gene high-throughput sequencing

Gut microbiota profoundly influence host physiology, yet most studies rely on bulk-scale intestinal samples, overlooking microbial heterogeneity at the micro-scale. Here, we present a dataset generated from 378 colonic micro-scale grains (20-40 µm) and 20 bulk samples collected from four Kunming mice. Using Illumina NovaSeq 6000 sequencing of the 16S rRNA V4 region, we obtained over 8.0 million raw reads, processed into 1506 amplicon sequence variants (ASVs) spanning 19 phyla and 203 genera. <i>Firmicutes</i> and <i>Bacteroidetes</i> were the dominant phyla across scales, while marked differences were observed in ASV richness (<i>p</i> < 0.001, Wilcoxon test) and network complexity (number of edges, <i>p</i> = 0.03, Wilcoxon test) between bulk samples and micro-scale grains. The dataset, deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1249752, provides a high-resolution view of gut microbial spatial organization at the sub-100 µm scale. These data advance current understanding of microbial assembly processes, interspecies interactions, and scale-dependent community structure, thereby serving as a valuable resource for microbiome ecology and future mechanistic studies.