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Sample GSM1119527

Query DataSets for GSM1119527

Status

Public on Jun 10, 2014

Title

M145_2

Sample type

SRA

Source name

Liver

Organism

Mus musculus

Characteristics

sample id: M145
strain: Diversity Outbred heterogeneous stock
age (weeks): 26
genotyped sex: Male
diet: High Fat
block: 2
flowcell: 2
lane: L007
barcode: GTTTCG
tissue: liver

Growth protocol

Male and female Diversity Outbred mice (J:DO Stock# 009376) were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were received at 3 weeks of age, housed at The Jackson Laboratory, and given free access to either standard rodent chow containing 6% fat by weight (LabDiet 5K52, LabDiet, Scott Distributing, Hudson, NH) or high-fat chow containing 22% fat by weight (TD.08811, Harlan Laboratories, Madison, WI) from wean age throughout the study. Animals were phenotyped for multiple metabolic and hematological parameters as described in Svenson et al. (2012), and then sacrificed at 26 weeks of age. Liver samples were collected from each animal and stored in RNAlater® solution (Life Technologies) at -80°C.
Male mice from each of the eight DO founder strains (n= 16 for each strain, n= 128 total) were obtained from The Jackson Laboratory. Mice were received at ~3 weeks of age, housed at the University of Wisconsin Madison, and beginning at 4 weeks of age given free access to either a control diet containing 17% kcal fat (TD.8010) or a high-fat, high-sucrose diet containing 45% kcal from fat and 34% (by weight) sucrose (TD.08811). Both diets were from standard rodent chow containing 6% fat by weight (LabDiet 5K52, LabDiet, Scott Distributing, Hudson, NH) or high-fat chow containing 22% fat by weight (TD.08811, Harlan Laboratories, Madison, WI) from wean age throughout the study. With the exception of NZO mice, animals were sacrificed at 26 weeks of age, and liver samples were collected, snap frozen, and shipped on dry ice to The Jackson Laboratory for RNA-seq analysis. Due to a high level of lethality of NZO mice that were maintained on the high-fat/high-sucrose diet, all NZO mice were sacrificed at 20 weeks of age.

Extracted molecule

total RNA

Extraction protocol

Livers were dissected and stored in RNAlater at -80C (DO samples), or snap frozen and stored on dry ice (founder strain samples). Total RNA was isolated using the Trizol Plus RNA extraction kit (Life Technologies) with on-column DNase digestion.
Following the Illumina TruSeq standard protocol, indexed mRNA-seq libraries were generated from 1μg total RNA followed by QC and quantitation on the Agilent Bioanalyzer and the Kapa Biosystems qPCR library quantitation method.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Basecalls were performed with CASAVA version 1.8.0
Fragmented fastq files from CASAVA were concatenated together, and then reads that did not pass quality filtering were removed with fastq_illumina_filter (http://cancan.cshl.edu/labmembers/gordon/fastq_illumina_filter/)
Read Alignment - Individualized transcriptomes (isoform sequences) derived from the genome fasta file and corresponding gene annotation gtf files were used to construct Bowtie-compatible indexes with the rsem-prepare-reference command in RSEM software (Li and Dewey, 2010; RSEM version 1.2.1). For inbred genomes (CAST, NCBIM37), each Ensembl transcript identifier corresponds to a single sequence. The Bowtie aligner (version 0.12.8) was used to align reads to transcript sequences. Alignments with up to three mismatches (-v 3) across the 100bp read were considered valid, but only the best “strata” of alignments were reported. For a given read, the best stratum is the alignment(s) with the least mismatches.
Transcript Abundance Quantitation - Estimates of isoform and gene-level abundance were obtained with RSEM (Li and Dewey 2010). Bowtie alignment files in .bam format were input into the rsem-calculate-expression command. Based on the library fragment size distributions observed on the Agilent Bioanalyzer, fragment length mean was specified as 280bp with a standard deviation of 50bp (RSEM parameters --fragment-length-mean 280 –fragment-length-sd 50). For reads with multiple valid isoform alignments, RSEM implements an expectation-maximization (EM) algorithm that assigns all or a proportion of each multimapping read to one or more isoforms based on the number of uniquely-aligning reads to those transcripts. RSEM estimates isoform expression (counts), and gene-level counts are calculated as the sum of the corresponding isoforms.
Normalization - For each sample, RSEM count estimates were normalized to the upper quartile value (after trimming zeros).
Genome_build: Mouse NCBIM37/mm9. Individualized diploid DO genomes were based on NCBIM37 coordinates.
Supplementary_files_format_and_content: All processed data files are tab-deliminited txt files. Each file contains a data matrix with rows representing Ensembl gene id's and columns representing samples. Only genes with non-zero values in ≥85% of samples were included in the final analysis.

Submission date

Apr 09, 2013

Last update date

May 15, 2019

Contact name

Steven C Munger

E-mail(s)

steven.munger@jax.org

Organization name

The Jackson Laboratory

Lab

Steven Munger