16S of mouse feces: RORgt-Cre+ Sting1 f/f  Mouse 5
200uL of homogenized liquid feces was deposited into a Qiagen PowerBead glass 0.1 mm tube (13118-50). Using a Promega Maxwell RSC PureFood GMO and Authentication Kit (AS1600), 1mL of CTAB buffer & 20l of RNAse A Solution was added to the PowerBead tube containing the sample. The sample/buffer was mixed for 10 seconds on a Vortex Genie2 and then incubated at 95C for 5 minutes on an Eppendorf ThermoMixer F2.0, shaking at 1500 rpm. The tube was removed and clipped to a horizontal microtube attachment on a Vortex Genie2 (SI-H524) and vortexed at high-speed for 20 minutes. The sample was removed from the Vortex and centrifuged on an Eppendorf Centrifuge 5430R at 40*C, 12700 rpm for 10 minutes. Upon completion, the sample was centrifuged again for an ADDITIONAL 10 minutes to eliminate foam. Sample tube cap was removed, and the sample was checked for foam and particulates (they will clog the robotic instrument tips). If foam or particulates were found in the sample, they were carefully removed using P1000 pipette. The opened tube was then added to a Promega MaxPrep Liquid Handler tube rack. The Liquid Handler instrument is loaded with proteinase K tubes, lysis buffer, elution buffer, 1000mL tips, 50mL tips, 96-sample deep-well plate, and Promega Maxwell RSC 48 plunger tips. The Promega MaxPrep Liquid Handler instrument was programed to use 300l of sample and transfer all sample lysate into Promega Maxwell RSC 48 extraction cartridge for DNA extraction. Upon completion, the extraction cartridge was loaded into Promega Maxwell RSC 48 for DNA extraction & elution. DNA was eluted in 100l and transferred to a standard 96-well plate. DNA was quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087). Library generation follows the protocol from Earth Microbiome Project (http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/). Amplicon libraries  were washed using Beckman Coulter AMPure XP magnetic beads. Library quality and size verification was performed using PerkinElmer LabChip GXII instrument with DNA 1K Reagent Kit (CLS760673). Library concentrations were quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087). Library molarity was calculated based on library peak size and concentration. Libraries were normalized to 2nM using the PerkinElmer Zephyr G3 NGS Workstation (133750) and pooled together using the same volume across all normalized libraries into a 1.5ml Eppendorf DNA tube (022431021). Pooled libraries were sequenced on the Illumina MiSeq instrument at loading concentration of 7pM with 15% PhiX, paired-end 250 using MiSeq Reagent Kit v2, 500-cycles (MS-102-2003).