title: 16S metagenome of mus musculus: colon and ileum lipid diets
design: Frozen ileum and colon tissues were homogenized (60Hz for 1 minute) using stainless steel beads in Mixer Mill MM 400 (Retsch). Bacterial genomic DNA was extracted with QIAamp_DNA Stool Mini Kit (Qiagen) according to the manufacturer_ instructions. DNA concentration and purity were checked with Nanodrop 2000 (Thermo Scientific).  Primers were used to amplify the 16S rRNA gene. The same linker primer (CCTACGGGNGGCWGCAG) and reverse primer (CTATGCGCCTTGCCAGCCCGCTAGGACTACHVGGGTATCTAATCC) were used for all samples while specific barcode sequences (TACTCTCGTG, TAGAGACGAG, TCGTCGCTCG, ACATACGCGT, TGTACTACTC, ACGACTACAG, CGTAGACTAG, TACGAGTATG, TCTCTATGCG, TGATACGTCT, CATAGTAGTG, CGAGAGATAC, ATACGACGTA, TCACGTACTA, CGTCTAGTAC, TCTACGTAGC, ACGAGTGCGT, ACGCTCGACA, AGACGCACTC, AGCACTGTAG, ATCAGACACG, ATATCGCGAG, CGTGTCTCTA, CTCGCGTGTC) were used to identify each sample.   EconoTaq_PLUS 2X Master Mix (Lucigen Corporation, Middleton, USA) was used for the amplification: the 25ul PCR mixture contained 1X EconoTaq master mix, 1uM forward and reverse primers and 50-200ng template genomic DNA isolated. The samples were amplified with a PCR protocol of initial denaturation at 94_ for 2 min, 30 cycles of denaturation, annealing and extension at 94_ for 30s, 58_ for 30s and 72_ for 60s, then a final extension at 72_ for 10min. The PCR product was run on a 1% agarose gel with the band at 464bp gen purified with QIAquick_Gel Extraction Kit (Qiagen). PCR amplicons concentration was normalized with SequalPrepTM Normalization Plate (96) Kit (Invitrogen). Library preparation, emPCR amplification and picotitre plate pyrosequencing using titanium chemistry was carried out by Vancouver Prostate Centre, UBC and Vancouver General Hospital Centre of Excellence in accordance with Roche/454 Life Sciences protocol on the 454 GS FLX+ System
identifiers.namespace: SUB3195524
identifiers.id: MaleColon