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. 2020 Mar 3;10:3950. doi: 10.1038/s41598-020-60862-1

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© The Author(s) 2020

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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The workflow used in this study. (A) chronic restraint-induced stress was applied to Sprague Dawley rats during the critical adolescent period, while a control group remained in their home cages. Rats were sacrificed immediately after stress (PD 44) or 3 weeks after stress (PD 64) as a model of adolescent and young adulthood. (B) DNA isolation. (C) Gut microbial metabolites extraction. (D) PCR amplification of the 16S rDNA region (E) Apply targeted metabolic profiling for cecum samples. (F) The next generation sequencing library was quantitated by KAPA qPCR reaction and Agarose Gel Analysis were applied as quality control for size selection of genomic DNA. (G) Apply statistical analysis using MetaboAnalyst 4.0. (H) Next generation sequencing using the MiSeq platform. (I) Post-processing sequencing data were analyzed using QIIME. (J) Statistical analysis of metabolite profiles. (K) Principal co-ordinate plot represented the beta diversity of four groups. (L) Significant metabolite-microbial associations generated by Spearman correlation analysis.