title: 16S rRNA seq of mouse cecal DNA
design: The cecal DNA was extracted using a ZymoBIOMICS-96 MagBead DNA kit (Zymo Research, Irvine, CA, USA), and the DNA samples were prepared for targeted sequencing with the Quick-16S NGS Library Prep kit using Primer Set V4 (Zymo Research, Irvine, CA). The ZymoBIOMICS Microbial Community Standard (Zymo Research, Irvine, CA) was used as a positive control for each DNA extraction. The sequencing library was prepared using a library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with the Select-a-Size DNA Clean & Concentrator (Zymo Research, Irvine, CA), then quantified with TapeStation (Agilent Technologies, Santa Clara, CA) and Qubit (Thermo Fisher Scientific, Waltham, WA). The ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e., blank extraction control and blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.