Illumina MiSeq paired end sequencing; Administration of Probiotic Blend in Pancreatic Cancer Xenografted mice
Administration of Probiotic Blend in Pancreatic Cancer Xenografted mice
fresh fecal samples were collected from the cages of each group at the end of treatment DPT). Samples were immediately frozen and stored at − 80 °C until DNA extraction. Mouse fecal DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen) according to the manufacturer's instructions to increase the ratio of microbial to host DNA.  The Illumina 16S Metagenomic Sequencing Library Preparation instructions were followed for high-throughput sequencing. Firstly, 12.5 ng of each DNA extract was employed for the amplification of the V3–V4 hypervariable regions of the bacterial 16S ribosomal RNA (rRNA) gene, using the following primers with Illumina adapters (underlined): forward primer: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, reverse primer: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC, selected from Klindworth et al. [43]. The amplification reaction was carried out in the presence of the 2× KAPA HiFi HotStart Ready Mix (Roche, Milan, Italy) under the following conditions: initial denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 30 s, extension at 72 °C for 30 s, with a final elongation at 72 °C for 5 min. PCR amplicons were then purified by means of Agencourt AMPure XP beads (Beckman Coulter, Milan, Italy). The purified DNA products were then subjected to a further PCR to attach dual Illumina indices (Nextera XT Index Kit, Illumina Inc., San Diego, CA, USA) necessary for multiplexing. The reaction was performed under the following conditions: initial denaturation at 95 °C for 3 min, followed by eight cycles of denaturation at 95 °C for 30 s, primer annealing at 55 °C for 30 s, extension at 72 °C for 30 s, with a final elongation at 72 °C for 5 min. Following a further PCR purification, the eluted DNA products were quantified using the Qubit dsDNA BR Kit assay, diluted to a concentration of 4 nM, and pooled in equal proportion into a single library.