title: 16S rRNA seq of rainbow Oncorhynchus mykiss intestine
design: The DNA from MI and MIC samples was extracted using the FastDNA SPIN kit for soil (MP Biomedicals, Solon, OH, USA) with some modifications (Mannelli et al., 2018) in comparison to the manufacturers protocol and further adapted to fish samples as follows: each sample was transferred to a lysis tube and thawed on ice, then 90 L of MT buffer and 724 L of phosphate buffer were added, and the mixture was homogenised with a Retsch MM300 disrupter (90s at 30 cycles/s) twice, with a break of 1 minute in ice bath for cooling down the samples, when necessary. The samples were incubated for 20min at 70C and inverted 5 times every 5 minutes, then centrifuged at 14,000g at 4C for 5 minutes. The supernatant was transferred to a sterile 1.5 mL tube, 185 L of PPS were added, and the tubes were inverted for 8 minutes, then centrifuged at 14,000g at 4C for 5 minutes. The supernatant was recovered and processed according to the manufacturers specification, with a double washing step with SEWS-M solution. The samples were eluted in 100 distilled water provided in the kit. Two extraction blanks (EB1, EB2) were extracted along the samples to assess the presence of contaminant sequences in the kit and in the environment. The extracted DNA quantified using Qubit (Life Technologies, Carlsbad, CA, USA).The extracted DNA was diluted to 1 ng/L and then sent to BMR Genomics (Padova, Italy) and processed following the Illumina protocol (Illumina, 2013). The V3-V4 region of the 16S rRNA gene was amplified with primer pair Pro341F (5-CCTACGGGNBGCASCAG-3) and Pro805R (5-GACTACNVGGGTATCTAATCC-3) (Takahashi et al., 2014) with 35 PCR cycles, added with Illumina adapters (Nextera XT index kit). An Illumina MiSeq platform with paired-end 2  300 bp sequencing chemistry was used.