RNA-Seq of mus musculus: EAE Gut Metagenome
Bacterial DNA was isolated from feces using MoBio PowerLyzer PowerSoil Kit (Qiagen). DNA concentration was quantified using the Quant-iT dsDNA Assay Kit, broad range (Life Technologies) and normalized to 2 ng/ul on a Beckman handling robot. Amplicons spanning variable region 4 (V4) of the bacterial 16S rRNA gene were generated as previously described. Bacterial 16S rDNA PCR including no template controls were setup in a separate PCR workstation using dual-indexed primers. PCR reactions contained 1 M for each primer, 4 ng DNA, and Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific). Reactions were held at 98C for 30 s, proceeding to 50 cycles at 98C for 10 s, 45C for 30 s, and 72C for 30 s and a final extension of 2 min at 72C. Amplicons were evaluated by gel electrophoresis. The sequencing library was prepared by combining equivolume amounts of each amplicon, size-selected and concentrated using AMPure XP beads (0.8X, Beckman). Library concentration was quantified by Qubit and qPCR, mixed with 15% PhiX, diluted to 4 pM and subjected to paired-end sequencing (Reagent Kit V2, 2x150bp) on an Illumina MiSeq sequencer at the Icahn School of Medicine Microbiome Core Facility.