16S of gut digestive tract metagenome: stomach  of rabbit 6C
Bacterial DNA was extracted from each sample of intestinal contents by using the commercial QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany), and amplified using primers which target the V3-V4 hypervariable regions of the 16S rRNA gene. All the PCR amplifications were performed in 25 L volumes per sample. A total of 12.5 L of KAPA HIFI Master Mix 2 (Kapa Biosystems, Inc., Wilmington, MA, USA) and 0.2 L of each primer (100 M) were added to 2 L of genomic DNA (5 ng/L). The blank controls (no DNA template added to the reaction) were also performed. A first amplification step was performed in an Applied Biosystem 2700 thermal cycler (ThermoFisher Scientific, Waltham, MA USA). The samples were denatured at 95 C for 3 min, followed by 25 cycles with a denaturing step at 98 C for 30 s, annealing at 56 C for 1 min, and extension at 72 C for 1 min, with a final extension at 72 C for 7 min. Sequencing libraries were prepared following the 16S Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA), quantified by using the Real Time PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc., Wilmington, MA, USA), pooled in equimolar proportion, and then sequenced in one MiSeq (Illumina, San Diego, CA, USA) run with 2  250-base paired-end reads.