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Sample GSM3245016

Query DataSets for GSM3245016

Status

Public on Dec 31, 2019

Title

124G_HF_HFS: GDM High fat sucrose 1

Sample type

SRA

Source name

islets

Organism

Rattus norvegicus

Characteristics

strain: Sprague Dawley
tissue: islets
age: 15 weeks of age

Treatment protocol

Briefly, female Sprague-Dawley rats were obtained at 3 weeks of age from the University of Manitoba colony and n=6 were randomly assigned to a low-fat (LF) diet (10% fat, Research Diets D12450B) and n=6 were randomly assigned to a high-fat and sucrose (HFS) diet (45% fat, Research Diets D12451) for a period of 6 weeks. After 6 weeks of dietary intervention animals were mated with LF diet fed males and diets were continued in the same dams throughout pregnancy and suckling period. Food intake and body weight of the dams were measured weekly and a glucose test was performed at e15.5. Dams were allowed to deliver naturally and at birth, litters were reduced to eight pups to avoid competition for food

Growth protocol

Pancreatic islets were isolated from Rattus norvegicus (Sprague Dawley) as described in Li, et al. 2009, with modifications to accommodate the features of the rat anatomy. After euthanizing the animal, the pancreas was localized and perfused via the bile duct in situ with 10mL of ice-cold Collagenase V solution (Sigma, St. Louis MO, USA) to achieve distension of the pancreas (approximately 90%). After the pancreas was fully distended, pancreatectomy was performed and the excised pancreas was immersed into a 50-mL conical tube containing 2.5 mL ice-cold collagenase V solution. All animal protocols were conducted in accordance with guidelines set by the Canadian Council on Animal Care and were approved by the University of Manitoba animal care committee. The pancreas was digested in a water bath at 37 oC for 15 min with gentle manual shaking at 2-3 min intervals. Solution was then immediately topped up to 30-35 mL with islet media (RPMI (11.1 mM glucose, Invetrogen) + 10% FBS + 1% P/S + 1% L-glutamine) and placed on ice. Samples were inverted to create a homogenous solution and then centrifuged at 290 x g for 30 sec. Carefully (without disturbing the soft pellet) the supernatant was decanted and the pellet was washed with 20 mL ice-cold 1XPBS by centrifuging at 290 x g for 30 sec. After a 100 m cell strainer (VWR, Pennsylvania, USA) was pre-wet with 1-2 mL 1XPBS, tissue suspension was re-suspended to create a homogeneous solution and filtered by passing through the cell strainer over a 50-mL tube. Strainer was tapped to enhance liquid flow and subsequently washed with 3 mL 1XPBS. Captured islets on the strainer were washed with 10 mL 1XPBS. Cell strainer was inverted over a sterile 100 mm tissue culture plate and the strainer was rinsed with 15 mL of ice-cold media collecting the released islets in the cell culture dish. Using a dissection microscope (Olympus SZ61; NCL 150 Illuminator), individual clean, healthy looking islets were picked manually using a 20 L or 100 L pipettor. Approximately about 200-300 islets were picked, centrifuged at 290 x g for 30 sec. Supernatant was removed and pellets were re-suspended in 300 L RLT buffer (Qiagen, Valencia CA, USA) and stored at -80°C until needed.

Extracted molecule

total RNA

Extraction protocol

Total RNA from the islets was extracted using RNAeasy Micro kit (Qiagen, Valencia CA, USA) as per manufacturer instructions. Subsequently, ribosomal RNA was removed from two micrograms total RNA by using Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Illumina). Total RNA and mRNA purity and integrity was assessed using Bioanalyzer 2000 Total RNA Pico and mRNA Pico, respectively (Agilent Technologies, Santa Clara CA, USA).
The library was prepared according to NEBNext Ultra II Directional RNA Library Prep for Illumina (NEB, Ipswich, MA, USA). Ribosomal RNA depleted RNA (10-100 ng) was fragmented and random primed, converted to cDNA and ligated to sequencing adaptors. PCR enrichment of adaptor-ligated libraries was performed and libraries were analyzed and measured using Bioanalyzer 2000 2100 Expert DNA 1000 chip (Agilent Technologies, Santa Clara CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

The base calls are made by the RTA software on the computer that runs the instrument. The RTA for the Hiseq 4000 is v .2.7.7. Using default parameters.
After quality check the reads were aligned to Rattus norvegicus (Rnor_5) genome assembly using STAR v2.5.1b .
All the "chrUn" were not considered in the downstrem analysis
For differential analysis software HOMER (v4.9) was used. First using homer script makeTagDirectory, the tag directories were made, which contains basic configuration information, such as the total number of reads, the total number of unique positions with aligned reads. Then homer script analyzeRepeats.pl was used to generate raw read counts based on exons and the counts were not adjusted and were condensed to genes. Further, the raw reads were subjected to homer script getDiffExpression.pl where we used DESeq2 18 to obtain the differentially regulated genes. We used a cutoff of ≥1.5 fold and FDR of ≤0.05 to find the significant genes.
Genome_build: Rattus norvegicus (Rnor_5) genome assembly
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

Submission date

Jul 05, 2018

Last update date

Dec 31, 2019

Contact name

Dr. Vernon Dolinsky

E-mail(s)

Vernon.Dolinsky@umanitoba.ca

Phone

2047893559

Organization name

University of Manitoba