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Sample GSM5526512

Query DataSets for GSM5526512

Status

Public on Jun 29, 2022

Title

114

Sample type

SRA

Source name

pancreatic islet

Organism

Mus musculus

Characteristics

cell type: Pancreatic islets
Sex: male
strain background: C57BL6/J
diet: Western diet
genotype: Zfp148floxed/floxed
age (wks): 23

Treatment protocol

Mice were kept on a high-fat/high-sucrose Western-style diet (Envigo Teklad TD.08811, IACUC protocol A005821-R01) for 20 weeks.

Growth protocol

β-Zfp148KO and control littermate mice of both sexes were used.

Extracted molecule

total RNA

Extraction protocol

Mouse islets were isolated as described (Rabaglia et al. American Journal of Physiology-Endocrinology and Metabolism 289, E218-E224 (2005). PMID: 15741243). After isolation, islets were placed in 350 uL of QIAzol Lysis Reagent (Qiagen) and stored at -80˚C before purifying RNA using Mini RNeasy purification kit. Purified RNA was reverse-transcribed and sequenced by the UW Madison Biotechnology Center.
RNA libraries were prepared for sequencing at the UW Madison Biotechnology Center. RNA integrity was determined using assayed on Agilent 2100 BioAnalyzer. Libraries were prepared using Illumina® TruSeq® Stranded Total RNA Sample Prep Gold kit (Illumina Inc., San Diego, California, USA). Complementary DNA (cDNA) was purified using Agencourt AMPure XP bead purification (Beckman-Coulter) following rRNA reduction. Libraries were quantified using Qubit DNA HS kit (ThermoFisher) and assayed on Agilent HS DNA Chips. Sequencing was done on Illumina HiSeq 2500 units.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

For generation of the mice, see Keller et al. JCI 2019.
Islet RNA sequencing

Data processing

Mouse islet RNA-sequencing reads were mapped via bowtie against the RefSeq mm10 mouse reference genome.
Gene expression values were estimated via RSEM.
Expected transcripts per million were normalized using median-by-ratio normalization.
Normalized gene abundance was analyzed for differential expression by EBSeq (Leng, N., Dawson, JA., Stewart, RM., Ruotti, V., Rissman, AI., Smits, BMG., Haag, JD., Gould, MN., Thomson, JA., and Kendziorski, C. (2013). EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics 29.8 1035-1043). Significantly different genes were determined as having posterior probability of differential expression > 0.95 (equivalent to FDR < 0.05). For coding genes, PPDE was determine for three tests: Zfp148floxed/floxed vs beta-ZFP148KO males, Zfp148floxed/floxed vs beta-ZFP148KO females, Zfp148floxed/floxed vs beta-ZFP148KO with both sexes included. Only genes intersecting all of these tests with PPDE > 0.95 were included for further analysis. This was repeated for ncRNAs, and the resulting genes meeting PPDE threshold for coding and non-coding genes were pooled into the final list for GO and subsequent analyses.
Genome_build: mm10/GRCm38
Supplementary_files_format_and_content: Emfinger_et_al_RNA_seq_PPDE_0.95.xlsx: This is an Excel (.xlsx) file containing tabs for the PPDE results for the three independent tests for coding and non-coding sequences, and the final intersected list. Transcript abundance is indicated for each sample in transcript reads per million (TPM).

Submission date

Aug 17, 2021

Last update date

Jun 29, 2022

Contact name

Alan Attie

E-mail(s)

adattie@wisc.edu, attie@biochem.wisc.edu

Organization name

University of Wisconsin-Madison

Department

Biochemistry