title: Human gut experiment
design: An aliquot (~25mg) of each gut tissue was suspended while frozen in a solution containing 200 µl buffer ATL and 200 µl of a slurry of 0.1-mm-diameter zirconia/silica beads (BioSpec Products, Bartlesville, OK). The mixed sample was then lysed by mechanical disruption with a bead beater (BioSpec Products) set on high for 2 min (20?), followed by extraction with the QIAamp® DNA Mini Kit (Qiagen, Inc., Valencia CA). Lysed mixture was completely lysed with 30 min incubation at 56? after adding 20 ul proteinase K. The standard tissue protocol was modified to extend the 70? incubation step from 10 minutes to 30 minutes. DNA from tissue was eluted in a final volume of 200 µl elution buffer and stored at – 20?. Tubes containing only the QIAamp® DNA Mini Kit extraction controls were included throughout the lysis and PCR steps to serve as negative controls.  Triplicate PCR reactions were performed for each sample using a primer set described by Costello et. al. (2009) and Turnbauggh et. al. (2009) respectively. The forward primer (5’-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG- 3’) contained the 454 Life Sciences primer B sequence, the broadly conserved bacterial primer 27F, and a two-base linker sequence (‘TC’). The reverse primer (5’- GCCTCCCTCGCGCCATCAGNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3’) contained the 454 Life Sciences primer A sequence, a unique 10-nt barcode used to tag each PCR product (designated by NNNNNNNNNN and provided by Roche), the broad-range bacterial primer 338R, and a ‘CA’ linker sequence inserted between the barcode and the rRNA primer. Each 25 µl reaction contained 0.2 µM forward and reverse primers, 3 µl template DNA, and 2.5 ul 10X PCR buffer plus Mg2+ (TaKaRa), 2.0 ul dNTP (2.5 mM each) (TaKaRa), 0.75 ul DMSO (100%), 0.25 ul TaKaRa TaqTM (5 U/µl). All dilutions were carried out using sterile ddH2O. PCR reactions were assembled within a PCR hood in which all surfaces and pipettes had been decontaminated with 30 min autoclaving at 120? and UV irradiated for 30 minutes. Thermal cycling was 95°C for 10 minutes, followed by 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, with a final extension of 10 minutes at 72°C. Replicate amplicons were pooled and visualized on 1.0% agarose gels using EB stain in 0.5X TE. Amplicons were cleaned using PCR Products Purification Kit (Spin-column) (BioTeke Corporation, Beijing, China) according to the manufacturer’s instructions. Amplicon DNA concentrations were determined using the Quant-iT PicoGreen dsDNA reagent and kit (Invitrogen).