title: NextSeq 500 sequencing; Age associated differences in microbiome in Rhesus Macaque
design: Age associated differences in microbiome in Rhesus Macaque
library_construction_protocol: We enrolled 12 Indian Rhesus Macaques (RM), consisting of 8 old animals (age > 18 years) and 4 young animals (age 3-6 years). Animals were housed and blood/tissue/stool samples were collected at the New Iberia Research Center (NIRC) at the University of Louisiana at Lafayette. Fresh fecal samples were collected from anesthetized animals by insertion of a fecal loop into the rectum and swiping several times and stored at -80°C. No treatments were done. The only variable is age. DNA from the macaque fecal samples was isolated using the Power Soil DNA Isolation Kit (Qiagen) according to manufacturer's instructions. All samples were quantified using the Qubit® Quant-iT dsDNA High-Sensitivity Kit (Invitrogen, Life Technologies, Grand Island, NY) to ensure that they met minimum concentration and mass of DNA, and were submitted to University of Minnesota Genomics Center for further analysis by Whole Genome Shotgun sequencing (WGS). Shotgun metagenomic library was constructed with the Nextera DNA sample preparation kit (Illumina, San Diego, CA), as per manufacturer's specification. Barcoding indices were inserted using Nextera indexing kit (illumina). Products were purified using Agencourt AMpure XP kit (Beckman Coulter, Brea, CA) and pooled for sequencing. Samples were sequenced using MiSeq reagent kit V2 (Illumina).Raw sequences were sorted using assigned barcodes and cleaned up before analysis (barcodes removed and sequences above a quality score, Q>30 taken forward for analyses).