MetNeo_16S_day4_7
DNA was extracted from mouse fecal samples using the PureLink Microbiome DNA Purification Kit (Invitrogen) according to the manufacturers protocol and following a 2 minute bead-beating step. The V4 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the 515F (AATGATACGGCGACCACCGAGATCTACACGCT) barcoded and 806R (TATGGTAATTGTGTGYCAGCMGCCGCGGTAA) primers67. A reaction containing a final concentration of 0.04% of each primer and 0.5% of PrimeSTAR Max DNA Polymerase (TaKaRa) 50l total volume. PCR reactions were carried out by 30-35 cycles of denaturation (95C), annealing (55C) and extension (72C), with final elongation at 72C. PCR products were purified using AMPure magnetic beads (Beckman Coulter) and quantified using a Denovix spectrophotometer. Samples were then pooled at equal amounts, loaded on 2% agarose E-Gel (Thermo Fisher), purified and sent for sequencing using the Illumina MiSeq platform (Genomic Center, Azrieli Faculty of Medicine, BIU, Israel).