triton
DNA was extracted using the QIAamp DNA Micro Kit, (Qiagen) selecting the protocol for isolation of genomic DNA from tissues and proceeding according to manufacturers instructions. DNA was quantified using the Quant-iTTM Picogreen dsDNA reagent and kit (Invitrogen) according to the manufacturer's instructions. The 2-step PCR amplification targeted the V3V4 region of the 16S rRNA gene with specific primers which also contained an adaptor and barcode sequence. PCR products were purified using the Nucleospin Gel and PCR clean up kit (Machery-Nagel), according to manufacturer instructions, followed by quantification using the QubitTM dsDNA high sensitivity assay kit (Invitrogen) and pooled equimolarly. The 16S amplicon library pool was cleaned and concentrated using the AMPure XP magnetic beads (Beckman Coulter) and re-quantified using the mentioned Qubit assay. Sequencing was performed using the MiSeq sequencing technology (Illumina Inc., LA Jolla, CA) generating paired-end sequences with 300 base pairs per read.