title: Illumina MiSeq paired end sequencing; 16S rRNA sequencing of fecal samples from Atg16l1 T300A and wildtype littermate mice that were either exposed or not to mainstream cigarette smoking for 6 weeks
design: 16S rRNA sequencing of fecal samples from Atg16l1 T300A and wildtype littermate mice that were either exposed or not to mainstream cigarette smoking for 6 weeks
library_construction_protocol: Mice were housed in an enhanced barrier specific pathogen-free facility under a strict 12-hour light cycle and fed irradiated chow diet (PicoLab Rodent Chow 20, Purina Mills). Mice were maintained on a C57BL/6 background. A heterozygous x heterozygous breeding scheme was used to generate Atg16l1 T300A and wildtype littermate control mice. All study protocols were approved by the Washington University Animal Studies Committee. Four- to six-week-old mice were exposed to mainstream cigarette smoking 4 cigarettes/day, 5 days/week, for 6 weeks, using Kentucky research cigarette 3R4F in a smoking chamber. All mice were sacrificed after completing 6 weeks of smoking exposure, and fecal samples were frozen down at the time of collection. DNA was isolated using Qiagen QIAmp DNA Stool Kit, according to the kit protocol. Total DNA concentration and purity was obtained from an absorbance ratio at 260nm and 280nm.  14 PCR amplicons, representing all 9 16S variable regions, were constructed using the Fluidigm Access Array System. 5ng/ul of DNA were input into each reaction.  The sample inlets consisted of 1X High Fidelity FastStart Reaction Buffer without MgCl2 (Roche), 4.5nM MgCl2 (Roche), 5% DMSO (Roche), 200uM PCR Grade Nucleotide Mix (Roche), 0.05 U/μL 5 U/μL FastStart High Fidelity Enzyme Blend (Roche), 1X Access Array Loading Reagent (Fluidigm), 1ul DNA, and water.  The primers were added to the assay inlets at 200nM forward and reverse primers with 1X Access Array Loading Reagent.  The samples were harvested after PCR amplification was performed on the BioMark HD system from Fluidigm.  Each sample was harvested and indexed using unique 10 base pair sequences with 14 rounds of PCR to incorporate each index sequence.  All samples were pooled into 48 sample libraries and cleaned using bead purification.