Milk microbiota, Sample 2425_HF_T1_qAR
For each quarter, 5 ml of milk sample were centrifuged at 500 g for 10 min at 4°C, the supernatant was discarded and the pellet was resuspended with 1 ml of saline solution (NaCl 0.9%) and centrifuged at 1500 rpm for 5 min at 4°C. The supernatant was again discarded and the bacterial DNA pellet was extracted from the samples as described previously (Cremonesi et al., 2006) and, then, stored at -20°C until use. Bacterial DNA was amplified using primers described which target the V3-V4 hypervariable regions of the 16S rRNA gene. All PCR amplifications were performed in 25-μl volumes per sample, using 0.2 μl of each primer (100 μM) and 2 μl of genomic DNA (5 ng/μl). A first amplification step was performed as follows: samples were denatured at 98 °C for 30 seconds, followed by 25 cycles with a denaturing step at 98 °C for 30 seconds, annealing at 56 °C for 1 minute and extension at 72 °C for 1 minute, with a final extension at 72 °C for 7 min. Amplicons were cleaned-up and libraries were prepared following the 16S Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA). The libraries obtained were quantified, pooled in equimolar proportion and sequenced in one Miseq (Illumina) run with 2×300 bp paired-end reads.