title: Illumina HiSeq 2500 sequencing; Microbial changes in early pancreatic cancer progression
design: Microbial changes in early pancreatic cancer progression
library_construction_protocol: Spontaneous pancreatic animal model KRASG12D TP53R172HPdx-Cre (KPC) animals from both genders were enrolled in the study at 1 month of age. PDX-cre (Cre) animals that were age matched with the KPC animals were used as control. Fecal samples were collected at 2 months; 3 months and 4 months of age. Initially, 8-9 animals were kept in each group. At 4 months, all animals were sacrificed according to protocols approved by University of Miami Animal Care Committee. DNA from the mouse 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).
attributes.tag: Experimental Factor: disease
attributes.value: pancreatic neoplasm