title: Sample 81
organism: mouse gut metagenome
description: Protocols: C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). All mice were male and 4-6 weeks old. Thirty-two male C57BL/6J mice, were first randomly divided into 4 groups (WT group, and T2D group). Mice in the T2D group were given an intraperitoneal injection of sterile citrate buffer containing streptozotocin (25 mg/kg, Sigma Aldrich, St. Louis, MO, USA), while mice in the WT group were injected intraperitoneally with the same volume of citrate buffer. After continuous administration of streptozotocin for 5 days, the T2D group was fed a high-fat diet (42% Cal from fat, TD.88137, ENVIGO) for 4 weeks to establish a model of type 2 diabetes. The WT group was given adjusted control diet (4% Cal from fat, TD. 08405, ENVIGO). Fecal samples were collected in different time point to understand the effect of several sequential treatment in gut microbiota. Fecal samples collection was performed at 1, 4, 6 and 10th weeks' after 1st injection of streptozotocin. Samples were collected in a sterile Eppendorf tube inside a biosafety cabinet with sterile forceps. Each group consisting eight animals were randomized (group wise) to nullify cage-effect in microbiome studies among the groups. After 10 weeks, all animals were sacrificed according to protocols approved by University of Miami Animal Care Committee. Thirty-two male C57BL/6J mice, were first randomly divided into 4 groups (WT group, and T2D group). Mice in the T2D group were given an intraperitoneal injection of sterile citrate buffer containing streptozotocin (25 mg/kg, Sigma Aldrich, St. Louis, MO, USA), while mice in the WT group were injected intraperitoneally with the same volume of citrate buffer. After continuous administration of streptozotocin for 5 days, the T2D group was fed a high-fat diet (42% Cal from fat, TD.88137, ENVIGO) for 4 weeks to establish a model of type 2 diabetes. The WT group was given adjusted control diet (4% Cal from fat, TD. 08405, ENVIGO). In tumor groups, After 2 weeks of tumor implantation when subcutaneous pancreatic cancer model was established, the two groups of mice were each further randomly divided into two subgroups (WT and WT + GP; T2D and T2D + GP). The WT + GP and T2D + GP groups received intraperitoneal injections of 100 mg/kg of gemcitabine and 10 mg/kg of paclitaxel twice in a week for consecutive 4 weeks, while the WT and T2D groups were only receive equal volume of saline. 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).