Microbial composition of the rectal fecal samples collected from rats 10 days after MCAO/R treated with or without FMT using 16S rRNA gene sequencing analyses.

<strong>1. Method of profiling of the gut microbiota</strong> Using a previously described protocol, rectal feces were processed for the total DNA extraction using the cetyltrimethylammonium bromide/sodium dodecyl sulphonate method. DNA concentration and purity were monitored on 1% agarose gel, before being diluted to 1 ng/μl using sterile water. Subsequently, 16S ribosomal (r)RNA genes of distinct regions (16S V3-V4) were amplified using a specific primer pair (forward: 5’-CCTAYGGGRBGCASCAG-3’; reverse: 5’-GGACTACNNGGGTATCTAAT -3’). All PCR reactions were performed using Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Inc.). Equal volumes of 1X loading buffer (SYB green) were then mixed with the PCR products and subjected to electrophoresis on 2% agarose gel for detection. Samples with bright main strips that are 400 to 450-bp long (16S) and 100 to 400-bp long (ITS) were chosen for further experiments. PCR products were first mixed in equidensity ratios. The mixture of PCR products was then purified using the Qiagen Gel Extraction Kit (Qiagen GmbH). Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, Inc.) according to the manufacturer’s protocols, before the index codes were added. Library quality was assessed using the Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) and the Agilent Bioanalyzer 2100 system (Agilent Technologies, Inc.). The library was then sequenced on an Illumina NovaSeq 6000 platform (Illumina, Inc.), where 250 bp paired-end reads were generated. The raw tags were double-ended reads, which could be accessed through fastq join (v1.3.1; https://code.google.com/p/ea-utils/) and pear (v0.9.11). Since the original sequence contained two primer sequences, Cutadapt (v1.18) was then used to isolate the sequences without primers and cut out the fully sequenced primers from the reads. Q30. Usearch (version 11.0.667) with no ambiguous bases was used to cluster according to 97% similarity. Alpha diversity was calculated using Mothur v1.42.1, beta diversity were calculated using the ‘vegan’ package in R and pathway enrichment was calculated using the PICRUSt2 software package (https:// github.com/picrust/picrust2). <br> <strong>2. FMT reverses the imbalance of gut microbiota induced by MCAO/R in rats</strong> We next characterized the microbial composition of the rectal fecal samples collected from rats 10 days after treatment using 16S rRNA gene sequencing analyses. Microbial community barplots show that the microbiota composition of the genus and phylum was altered significantly (Figure 3A and 3B) after cerebral infarct. In terms of the microbial composition on a genus level, MCAO/R decreased the relative abundance of <em>Ligilactobacillus</em> from 56.21 to 14%, <em>Romboutsia</em> from 10.74 to 1.01% and HT002 from 22.75 to 5.68%, whilst increasing the relative abundance of <em>Escherichia-Shigella</em> from 0.43 to 16.49%, <em>Staphylococcus</em> from 0.01 to 2.15%, <em>Lactobacillus</em> from 6.29 to 26.38%, <em>Streptococcus</em> from 0.09 to 2.64%, <em>Akkermansia</em> from 0.17 to 18.74%, <em>Corynebacterium</em> from 0.26 to 2.00%, <em>Bacteroides</em> from 0.01 to 0.64%, <em>Bilophila</em> from 0.04 to 1.4% and <em>Firmicutes</em>_unclassified from 0.02 to 0.29% compared with the sham control rats. Compared with the MCAO/R model group, FMT appeared to have partially reversed the imbalance (Figure 3A). On the phylum level, we analyzed the components and found that the relative abundance of Proteobacteria (0.59 to 17.14%), <em>Verrucomicrobiota</em> (0.17 to 18.74%), <em>Actinobacteriota</em> (0.34 to 2.06%), Bacteroidota (0.19 to 1.16%) and <em>Desulfobacterota</em> (0.14 to 1.47%) were significantly increased after MCAO/R compared with those in the sham control rats, whilst the relative abundance of <em>Firmicutes</em> (98.51 to 59.40%) was significantly decreased in the MCAO/R group (Figure 3B). FMT prevented these changes at the phylum level induced by MCAO/R injury. <br> <br>