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PCR was carried out in a final volume of 50 l with 40 ng of DNA, 600 M dNTP and 2.5 U Amplitaq gold polymerase

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Hierarchical shifts in gut microbiota composition Family-level analysis (Figure S1A) confirmed pronounced dysbiosis in the VPA-ASD group, which was enriched with pathogenic families such as Clostridiaceae and Erysipelotrichaceae and exhibited severe depletion of beneficial families including Muribaculaceae

The media were replaced with fresh media every 2 or 3 d

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