title: 1stset_control_Mouse_5
design: The V3-V4 hypervariable region of the 16S rRNA gene was amplified by PCR using the following primers:  a forward primer 5-CTT TCC CTA CAC GAC GCT CTT CCG ATC TAC GGR AGG CAG CAG-3 (28-nt illumina adapter (in bold) followed the 14 nt broad range bacterial primer 343F) and a reverse primer 5-GGA GTT CAG ACG TGT GCT CTT CCG ATC TTA CCA GGG TAT CTA ATC CT-3 (28-nt illumina adapter (in bold) followed by the 19 nt broad range bacterial primer 784R). The PCR reaction mix consisted of 10 ng of fecal DNA template, 0.5 M primers, 0.2 mM dNTP, and 0.5 U of the DNA-free Taq-polymerase (MolTaq 16S, Molzym, Bremen, Germany). The PCR cycles were as follow: 1 cycle at 94C for 60 s, followed by 30 cycles at 94C for 60 s, 65C for 60 s, 72C for 60 s, and a final step at 72C for 10 min. The PCR reactions were sent to the @Bridge platform (INRAe, Jouy-en-Josas, france) for sequencing (Illumina Miseq technology). Single multiplexing was performed using home-made 6 bp index, which were added to R784 during a second PCR with 12 cycles using forward primer (AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC) and reverse primer (CAA GCA GAA GAC GGC ATA CGA GAT-index-GTG ACT GGA GTT CAG ACG TGT). The resulting PCR products were purified and loaded onto the Illumina MiSeq cartridge according to the manufacturer instructions. Run quality was checked internally using PhiX, and sequences assigned to the corresponding based on the 6 pb index.