Extended Data Fig. 4: Microbial small RNA on lipoproteins is not depleted in germ-free mice.

a) Plasma from two cohorts of adult mice - specific pathogen free (SPF; n = 6 mice total) and facility-matched germ-free (GF; n = 17 mice total) fed a chow diet were harvested at the National Gnotobiotic Rodent Resource Center (NGRRC; North Carolina, USA) for lipoprotein sRNA-seq b) Plasma was fractionated by size-exclusion chromatography (SEC) and cholesterol-rich fractions corresponding with HDL were selected for sRNA-seq. c) Relative percentage of reads aligned to host and non-host databases, as well as reads too short for analysis or reads that failed to align to either database (unmapped). d) Percentage of sRNA reads aligned to host miRNA, host tRNA and host rRNA transcripts. e) Percentage of reads aligned to the non-host rRNA database and tRNA database. f) Percentage of reads aligned to genomes of fungi and algae. g) Percentage of reads aligned to bacterial genomes associated with a human microbiome (HMB) database. h) Reads per million total reads (RPM) mapped to indicated bacterial phyla within the HMB database. i) Percentage of reads aligned to bacterial genomes within an environmental bacteria (ENV) database. j) Differential abundance (log₂) of bacterial sRNA (dots represent individual genomes of the HMB and ENV databases) between GF and SPF mice categorized by phyla. Gray bar represents a 1.5 fold change. Data are mean ± SEM. (d-g, i) Statistical differences between GF and SPF were assessed by Mann-Whitney U-test, but no evaluations were statistically significant. j) Differences in abundance of individual genomes within each database were assessed between groups by the Wald Test, but applying False Discovery Rate correction (α = 0.05) resulted in no differentially abundant genomes. Numerical source data, statistics, exact p values and q values are provided.

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