Dnazol

Genomic DNA was extracted from G-415 cells using DNAzol (Thermo Fisher Scientific) according to manufacturer protocol and specific DNA sequences were amplified for exons 2-11 of human TP53, according to protocols of IARC TP53 Database (https://p53.iarc.fr, accessed on 31 August 2018). PCR products were cloned on pGEM^®-T Easy Vector (Promega, Madison, WI, USA) for sequencing (Macrogen Corp., Seoul, Korea), according to manufacturer instructions.

Faecal samples were collected in the sterile laboratory before being sacrificed. The faecal samples were placed into sterile centrifuge tubes with PBS and mixed. All samples were centrifuged (4000 r/min, 4°C, 30 min), and precipitates were retained. After, total DNA was extracted according to the protocol of the kit (DNAzol, Thermo Fisher, USA). The DNA of bacillus coli was applied as a template for amplification. Clone databases of the 16S rDNA genes were constructed according to the DNA Sample Prep Kit (TruSeq™, Illumina, USA), and DNA sequences were detected (MiSeq, Illumina, USA). Finally, original sequences were spliced and filtered.
Principal component analysis (PCA) was applied to compare the bacterial diversity of the gut microbiome. The analysis of similarities (ANOSIM) was applied to analyze whether there was comparability between each group. In addition, the linear discriminant analysis (LDA) was applied to screen out the different species through LEfSe. Finally, the function of the gut microbiome was annotated according to the Kyoto Encyclopedia of Genes and Genomes database (KEGG database).

Forty fungal isolates were randomly picked to evaluate the fungal library’s diversity. The fungi were cultured with liquid malt extract medium at RT for one week, and mycelium was collected for DNA extraction using DNAzol (Thermo Fisher). Genomic DNA applied as PCR templates were isolated using DNAzol Reagent following the manual (Thermo Fisher Scientific). To identify the fungal species, nuclear ribosomal ITS regions were amplified by PCR with specific primers (Table 1) (Op De Beeck et al., 2014 (link)) using the following approach: 94 °C 2 min; 94 °C 30 s, 55 °C 30 s, 72 °C 1 min, 35 cycles; 72 °C 5 min. The amplified products were sequenced and blasted against the NCBI database to identify fungal species (Raja et al., 2017 (link)).

Plasmid pSRS_CM1 (Shames et al., 2017 (link)), also called pSB4807 (Fowler and Galan, 2018 (link)), a chloramphenicol-resistant derivative of pSAM_Bt (Goodman et al., 2009 (link)), was a gift of C. Fowler and J. Galan. This plasmid was transferred into donor strain E. coli β-2163Δnic35 (Demarre et al., 2005 (link)) and propagated at 37°C in LB containing DAP and carbenicillin. Conjugative-based transposon mutagenesis of recipient S. Typhimurium strains was performed by mixing donor and recipient strains in a 3:1 ratio. Bacterial conjugations were performed at room temperature for 24 h and transconjugants selected by plating on LB agar plates containing 15 mg/ml chloramphenicol and 40 mg/ml X-gal (MP Biomedicals) to identify blue colonies. Approximately 63,000 colonies were screened (28,000 14028s and 35,000 SL1344) and blue colonies purified by streaking to single colonies on X-gal/chloramphenicol plates. Genomic DNA was isolated with DNAzol (Thermo Fisher), partially digested with Sau3AI and ligated to a DNA adaptor digested with BamHI. Ligation-mediated PCR (Mueller and Wold, 1989 (link)), using primers that anneal to adaptor and transposon sequences, was used to amplify sites of transposon insertion from genomic DNA.

To isolate sperm DNA of adult rat of the F0 generation, cauda sperms of each father were washed twice with PBS, resuspended in 1.0 mL lysis buffer containing 20 mM Tris (pH 8), 10 mM dithiothreitol, 150 mM NaCl, 10 mM EDTA (pH 8), and 1% SDS, and incubated for 20 h at 37°C [44 (link)]. The DNA was extracted from the lysed tissue using DNAzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) following the manufacturer's instructions. Subsequently, 5mC DNA ELISA Kit (Zymo Research s.r.l., Irvine, CA, USA) was used to evaluate differences in global 5mC in sperm DNA from treated fathers with respect to controls.
SNpc and the striatum nuclei of each rat of F0 and F1 generations were also used to extract genomic DNA using DNAzol (Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer's instructions. For SNpc and the striatum nuclei DNA analysis, four subgroups for each treatment were set up, basing the grouping on quartiles of dopamine data distribution. 100 ng of DNA for each subgroup sample was then used to evaluate global 5mC and 5hmC levels using, respectively, the 5mC DNA ELISA Kit™ and the Quest 5hmC™ DNA ELISA Kit (Zymo Research s.r.l., Irvine, CA, USA). Results are presented as percentage of total CpG of rat genome. Each sample was analyzed in duplicate following the manufacturer's instructions.