Gut mouse microbiota, Sample: 4,  Pathogen free
We investigated a naturally H. hepaticus-colonized B10.D2 mouse breeding, comparing the gut microbiota of 6  H. hepaticus-colonized mice to 6 Helicobacter-free rederived B10.D2 controls by high-throughput 16S rRNA gene sequencing. Controls were re-derived by early post-natal (<72 h after birth) transfer of B10.D2 pups born from naturally H. hepaticus-colonized mothers towards Helicobacter-free B10.D2 foster mothers and resulted  in the eradication of this colonization.  DNA was extracted from fecal pellets from individual mice and the V3-V4 region of the 16S rRNA gene was PCR-amplified in 25 μl volumes containing 12.5 ng of microbial DNA and 200 nM of S-D-Bact-0341-384 b-S-17/S-D-Bact-0785-a-A-21 primers (Klindworth A, et al. (2013)) carrying Illumina overhang adapter sequences.Thermal cycle consisted of an initial denaturation at 98°C for 3 min, 25 cycles of denaturation (95°C for 30 sec), annealing (55°C for 30 sec), and extension (72°C for 30 sec), plus a final extension step at 72°C for 5 min. Amplicons of about 460 bp were purified and sequenced on Illumina MiSeq platform using a 2×250 bp paired end protocol.