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. 2021 Dec 21;1:82. doi: 10.1038/s43705-021-00078-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — A Flowchart of the experimental and bioinformatic analyses. Flash-frozen stool samples are divided into aliquots for subsequent biomolecular extractions. Int-DNA are obtained after elution of the lysate bound onto an AllPrep DNA spin column, the flow-through is loaded onto a RNeasy spin column for int-RNA isolation. To obtain the extracellular fractions, the supernatant is first filtered through a polyethersulfone (PES) membrane. Nucleic acid fractions are isolated using specific columns (NucleoSpin miRNA Plasma kit for ex-DNA and ex-sRNA, NucleoSpin RNA Blood kit for ex-lRNA). Ex-DNAs are subjected to an additional concentration step. All nucleic acid fractions are subjected to high-throughput sequencing. Ex-Prot are obtained from the resulting pellet after protein precipitation and analyzed by SDS-PAGE followed by LC-MS/MS. The sequencing information from the intracellular fractions allows for genome reconstruction by a DNA-RNA co-assembly using IMP [10]. This MG-MT reference allows further mapping and annotation of the extracellular fractions. Polar metabolites, SCFAs, and BAs are extracted from their respective aliquots by addition of specific internal standards (IS) and further processing of the supernatant (Supplementary Materials and Methods). The extracts are then analyzed by GC-MS, GC-MS, and LC-HRMS, respectively. B Masses of biomolecules extracted per mg of original stool sample (logarithmic scale). Error bars represent standard deviation on four independent samples. ex-DNA extracellular DNA, ex-sRNA extracellular small RNA, ex-lRNA extracellular large RNA, ex-Prot extracellular proteins, SCFAs short-chain fatty acids, BAs bile acids.