title: microbiome study of C57BL/6N and C57BL/6J_21N mice
design: 16S rRNA gene sequence analysis was performed to compare the community composition of cecal luminal samples of the two mouse substrains C57BL/6N (n = 11) and C57BL/6J (n = 8) after high-fat diet intervention. For PCR 16S rRNA gene forward primer 926F (5´-AAACTYAAAKGAATTGACGG_3´) attached to the Roche A adapter for 454-library construction and reverse primer 630R   (5´- CAKAAAGGAGGTGATCC - 3´) attached to the Roche B adapter were used. For multiplexing purposes each primer included a 10-nt barcode sequence. Three independent PCRs were performed for each sample with Fast Start High Fidelity PCR System (Roche, Mannheim, Germany) containing 20 ng of DNA with an optimal annealing temperature of 50°C and 22 cycles. PCR reactions were pooled together and purified using QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany). After quantification using a Quant-iT™ PicoGreen dsDNA quantification kit (Invitrogen, Paisley, UK) samples were pooled equally. The sequencing of the partial 16S-rRNA genes was performed on a Roche 454 GS FLX Titanium system Pyrosequencer (Roche, Mannheim, Germany). Total bacterial diversity was estimated analyzing reverse sequences only.