Gut metagenome of Canis lupus familiaris: Control_10
Fecal samples (1-3 g) were collected directly from the rectal ampoule with sterile gloves both in epileptic dogs before treatment (E, n=10) and healthy controls (C, n= 12), and immediately frozen al -80C to fix bacterial growth and preserve DNA content. In the case of nine epileptic dogs, samples were also collected after one month on a stable dose of a single-AED treatment (Ed, n=9). Of these, four were being treated with phenobarbital and five with imepitoin. Bacterial DNA was extracted from faecal samples using the NZY Soil gDNA Isolation kit (NZYTech, Lisboa, Portugal). 16S rRNA gene of V4 region was amplified using specific primer (515F-806R) (Caporaso et al., 2011) with a barcode. All PCR reactions were carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs, UK). Sequencing libraries were generated using NEBNext Ultra DNA Library Pre Kit for Illumina. Later, the library was sequenced on an Illumina MiSeq platform and 250 bp paired-end reads were generated.