title: 16S amplicon sequencing of alimentary microbiota in CAST/EiJ mouse: Mouse 1617, adult female inoculated with pilTL201C Neisseria musculi, unstable colonization, medial small instestine  lumen contents.
design: Snap frozen tissue was divided in rostral-caudal thirds. DNA then was extracted from lumen contents using a modified bead beating protocol in lysis buffer and a 25:24:1 mixture of phenol, chloroform and isoamyl alcohol, followed by ethanol precipitation. Samples were RNAse treated and re-purified by phenol-chloroform extraction and ethanol precipitation. Purified samples were next amplified with Platinum HiFi polymerase using V4 and V5 fusion primers to incorporate the Truseq adaptor, barcode and index.   Amplicon cleanup and sizing was conducted using Ampure beads, and multiplexed pools were quantitated by KapaBiosystems qPCR prior to clustering on the flowcell and sequencing as 250 nt PE reads.