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Status |
Public on Mar 31, 2017 |
Title |
PBS-treated Gcgr-/- 3 |
Sample type |
SRA |
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Source name |
liver
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Organism |
Mus musculus |
Characteristics |
strain: C57Bl6 tissue: liver age: 16-20 weeks genotype: Gcgr-/- treatment: PBS
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Treatment protocol |
Treated intraperitoneally once a week for 10 days with either PBS or 10mg/kg of a humanized monoclonal antibody against the Glucagon receptor (Amgen, Inc)
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Growth protocol |
Adult (16-20 weeks) male mice after 6 hr fast
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Extracted molecule |
total RNA |
Extraction protocol |
Qiagen Rneasy Mini The concentration and integrity of the extracted total RNA were estimated using the Qubit® 2.0 Fluorometer (Invitrogen)and Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA, USA), respectively. Five hundred nanograms of total RNA was required for downstream RNA-seq applications. Polyadenylated RNAs were isolated using NEBNext Magnetic Oligo d(T)25 Beads. The NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs Inc., Ipswich, MA, USA) was then used to prepare individually bar-coded next-generation sequencing expression libraries as per manufacturer's recommended protocol. Library quality was assessed using the Qubit 2.0 Fluorometer, and the library concentration was estimated by utilizing a DNA 1000 Chip on an Agilent 2100 Bioanalyzer. Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification Kit. Each library was diluted to a final concentration of 12.5 nM and pooled in an equimolar ratio prior to clustering. Paired-end sequencing (50 million, 50-bp, paired-end reads) was performed using a 200 Cycle TruSeq SBS HS v4 Kit on an Illumina HiSeq2500 sequencer (Illumina, Inc., San Diego, CA, USA).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
processed_data_column_header: 2552-ACP-0006 Normalized expression values_all_2552ACP
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Data processing |
Post-processing of the sequencing reads from RNA-seq experiments for each sample was performed using HudsonAlpha’s unique in-house RNA-seq data analysis pipeline. Briefly, quality control checks on raw sequence data for each sample were performed using FastQC (Babraham Bioinformatics, Cambridge, UK). Raw reads were mapped to the reference mm9 using TopHat v2.0. The alignment metrics of the mapped reads were estimated using SAMtools. Aligned reads were imported to the commercial data analysis platform AvadisNGS (Strand Scientifics, CA, USA). After quality inspection, the aligned reads were filtered on the basis of read quality metrics; reads with a base quality score of less than 30, alignment score of less than 95, and mapping quality of less than 40 were removed. Remaining reads were then filtered on the basis of their read statistics; missing mates, translocated, unaligned, and flipped reads were removed. The reads list was then filtered to remove duplicates. Samples were grouped and transcript abundance was quantified for this final read list using Trimmed Means of M-values (TMM) as the normalization method. Output data utilized for all subsequent comparisons were summarized as normalized signal values generated by AvadisNGS. Differential expression of genes was calculated on the basis of fold changes (using the default cut-off ≥ ±2.0) observed in comparisons between defined conditions, and the p-value of the differentially expressed gene list was estimated by ANOVA using Benjamini Hochberg corrections of 0.05 for false-discovery rate. Genome_build: mm9 Supplementary_files_format_and_content: Excel files include RPKM values for each Sample
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Submission date |
Oct 21, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Danielle Dean |
E-mail(s) |
danielle.dean@vanderbilt.edu
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Organization name |
Vanderbilt University Medical Center
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Department |
Division of Diabetes, Endocrinology, and Metabolism
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Street address |
7465 MRBIV, 2215 Garland Avenue
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City |
Nashville |
State/province |
TN |
ZIP/Postal code |
37232 |
Country |
USA |
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Platform ID |
GPL17021 |
Series (1) |
GSE89035 |
Interruption of Hepatic Glucagon Signaling Reveals a Hepatic-α-Islet Cell Axis Where L-Glutamine Stimulates α-Cell Proliferation |
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Relations |
BioSample |
SAMN05933962 |
SRA |
SRX2263490 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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