title: Mouse fecal sample 18
design: For each mouse, a single fecal pellet was deposited into a Qiagen PowerBead glass 0.1 mm tube. 1 mL of Invitrogen TRIzol Reagent was added to each tube. The tubes were sealed and clipped to a horizontal microtube attachment on a Vortex Genie 2 and vortexed at high speed for 20 minutes. The tubes were centrifuged quickly, and 200 L of chloroform were added. The tubes were then vortexed for 1 minute, allowed to homogenate at room temperature for 3 minutes, and then centrifuged at 4C for 15 minutes. The upper aqueous layer was carefully transferred to a 1.5 mL Eppendorf tube. A 1:1 ratio of 70% ethanol was added to each tube. The tubes were then sealed and vortexed for 1 minute followed by centrifugation at room temperature for 1 minute. The samples were then passed through Qiagen RNeasy Mini Kit spin columns, 600 L at a time, and the flow-through was discarded. RNA was extracted as per the Qiagen RNeasy Mini Kit protocol, including the on-column DNase digestion step. 80% ethanol was used in place of RPE for the second RPE wash step. RNA was eluted by adding 50 L of RNAse-free water to the center of each spin column membrane and incubating at room temperature for 5 minutes. The tubes were centrifuged at maximum speed at room temperature for 30 seconds. The captured eluate was then transferred to the center of each spin column membrane, and the spin columns were centrifuged at maximum speed for 2 minutes. The extracted RNA was quantified using the Invitrogen Qubit RNA HS Assay Kit, and the quality of the RNA was assessed using an Agilent RNA 6000 Pico Kit.  50 l of Beckman Coulter RNAClean XP beads were added to the 50 l of extracted RNA ofr each sample. Samples were washed per Beckman Coulters wash protocol and eluted in 20 l. Bacterial ribosomal RNA was removed using the Invitrogen RiboMinus Transcriptome Isolation Bacteria Kit, and the ribo-depleted RNA was eluted in 10l. Sequencing libraries were generated using the Illumina TruSeq Stranded Total RNA Library Prep Gold kit to ensure host ribosomal RNA depletion and mitochondrial RNA depletion. TruSeq RNA indexes were used to barcode samples.  cDNA libraries were washed using Beckman Coulter AMPure XP magnetic beads. Library quality and size verification were performed using the PerkinElmer LabChip GXII instrument with the DNA 1K Reagent Kit. Library concentrations were quantified using the Quant-iT dsDNA High Sensitivity Assay Kit using a Promega GloMax plate reader. Library molarity was calculated based on library peak size and concentration. Libraries were normalized to 2nM using the PerkinElmer Zephyr G3 NGS Workstation and pooled together using the same volume across all normalized libraries into a 1.5ml Eppendorf DNA tube. Pooled libraries were sequenced on an Illumina NextSeq500 High Output 75bp Single Read Sequencing run.