title: 16S of mouse feces: Bmp2fl fl Rorc cre Mouse 3
design: A single fecal pellet is 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 is added to the PowerBead tube containing the sample. The sample/buffer is 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 is removed and clipped to a horizontal microtube attachment on a Vortex Genie2 (SI-H524) and vortexed at high-speed for 20 minutes. The sample is removed from the Vortex and centrifuged on an Eppendorf Centrifuge 5430R at 40*C, 12700 rpm for 10 minutes. Upon completion, the sample is centrifuged again for an ADDITIONAL 10 minutes to eliminate foam. Sample tube cap is removed, and the sample is checked for foam and particulates (they will clog the robotic instrument tips). If foam or particulates are found in the sample, they are carefully removed using P1000 pipette. The opened tube is 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 is 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 is loaded into Promega Maxwell RSC 48 for DNA extraction & elution. DNA is eluted in 100l and transferred to a standard 96-well plate. DNA is quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087).16S Library GenerationLibrary generation follows the protocol from Earth Microbiome Project http://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/.Library Verification, Quality Check, PoolingAmplicon libraries are washed using Beckman Coulter AMPure XP magnetic beads. Library quality & size verification is performed using PerkinElmer LabChip GXII instrument with DNA 1K Reagent Kit (CLS760673). Library concentrations are quantified using Quant-iT dsDNA High Sensitivity Assay Kit using Promega GloMax plate reader on a microplate (655087). Library molarity is calculated based on library peak size & concentration. Libraries are 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).