title: Illumina MiSeq paired end sequencing; 16S rRNA sequencing of cecal microbiota from neonatal mice pre- and post-weaning
library_construction_protocol: Cecal contents were collected for genomic DNA isolation and subsequent 16s rRNA sequencing. For pups aged 1, 2, or 3 weeks, the intact cecum (host tissue and cecal contents) was dissected out and placed into a tared cryotube, weighed, snap frozen in liquid nitrogen, and stored at -80C until nucleic acid extraction. This procedure was used to avoid loss of any cecal content material, as the ceca were very small at these ages. For the 6-week old pups, the cecum was much larger and so the cecum was dissected out and cut open along the greater curvature with to expose the luminal surface; cecal contents were collected using a sterilized spatula and placed into a tared cryotube weighed, snap frozen in liquid nitrogen, and stored at -80C until nucleic acid extraction. Six pregnant BALB/c dams with litter sizes of 10 pups each were obtained from Envigo. Dams and the resulting pups were maintained in a specific pathogen-free barrier facility with a strict 12-hr light cycle and ad libitum access to food and water. Mice were housed in complete autoclaved cage assemblies containing chow (Envigo NIH-31 Irradiated Modified Open Formula Mouse/Rat Diet 7913) and bedding (Envigo Teklad 7097 1/4\" Corncob bedding). Weaning was performed as usual at 3 weeks of age, with pups of the same sex housed only with littermates in fresh autoclaved cage assemblies. To minimize experimental variation that could potentially arise from single cages or dams, 2 pups were randomly selected from each litter per timepoint (total of n=12 per timepoint) at 1-week, 2 weeks, 3 weeks, and 6 weeks of age. All procedures were approved by the Institutional Animal Care and Use Committee at Washington University School of Medicine. 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. 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/L 5 U/L FastStart High Fidelity Enzyme Blend (Roche), 1X Access Array Loading Reagent (Fluidigm), 1ul DNA (5 ng/uL), 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.