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Sample GSM1864933

Query DataSets for GSM1864933

Status

Public on Sep 01, 2015

Title

1wk Dox-Mphs_rep3

Sample type

SRA

Source name

CD11b+ islet-derived macrophages

Organism

Mus musculus

Characteristics

strain: RIP-rtTA; TetO-VEGF-A
treatment: 1 week Dox
cell type: CD11b+ islet-derived macrophages

Extracted molecule

total RNA

Extraction protocol

Isolated whole islets (100-300) or sorted islet-derived cells (8,000-400,000) were added to 200-400 μL lysis/binding solution in the RNAqueous small scale or micro scale phenol-free total RNA isolation kits (Ambion, Austin, TX). Trace contaminating DNA was removed with TURBO DNA-free kit (Ambion, Austin, TX). RNA quantification and quality analysis was performed using a Qubit Fluorometer and an Agilent 2100 Bioanalyzer.
RNA samples that had RNA integrity number (RIN) ≥7.0. RNA was amplified using the NuGEN Technologies Ovation RNA amplification kit (Part # 7102-08) using manufacturer's instructions. Approximately, 2.5 µg amplified DNA from each sample was sheared on a Covaris S200 focused- ultrasonicator (Woburn, MA, USA) with a target yield of an average 200bp fragment size. The fragmented DNA was taken into standard library preparation protocol using NEBNext® DNA Library Prep Master Mix Set for Illumina® (New England BioLabs Inc., Ipswich, MA, USA) with slight modifications. Briefly, end-repair was done followed by polyA addition and custom adapter ligation. Post-ligated materials were individually barcoded with unique in-house genomics service lab (GSL) primers and amplified through 8 cycles of PCR using KAPA HiFi HotStart Ready Mix (Kapa Biosystems, Inc., Woburn, MA, USA). The quality of the libraries were assessed by Qubit® 2.0 Fluorometer , and the concentration of the libraries was estimated by utilizing a DNA 1000 chip on an Agilent 2100 Bioanalyzer, respectively. Libraries were sequenced on the Illumina HiSeq2500 following the manufacturer's protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Raw reads were mapped to reference mouse genome mm9 using TopHat v2.0 (Trapnell et al., 2009). Aligned reads were imported onto the Avadis NGS data analysis platform (Strand NGS, San Fransisco, CA, USA). Briefly imported mapped reads were first filtered on their quality metrics, and then duplicate reads were removed. Normalized gene expression was quantified using the TMM (Trimmed Mean of M-values) method (Dillies et al., 2012; Robinson and Oshlack, 2010). The transcriptional profile from each sample group (whole islets at No Dox, 1wk Dox, 2wk WD and sorted islet-derived MΦs and ECs at 1wk Dox) was compared by principle component analysis (PCA) and hierarchal clustering analysis to determine the layout and spread of the samples. Differential expression between conditions was calculated on the basis of fold change (cutoff ≥2.0) and the p-value was estimated by z-score calculations (cutoff 0.05) as determined by the Benjamini Hochberg false discovery rate (FDR) method (Benjamini and Hochberg, 1995). Differentially expressed genes underwent gene set enrichment analysis (GSEA), gene ontology (GO) analysis, and pathway analysis using DAVID, and Ingenuity Pathway Analysis.
Genome_build: mm9
Supplementary_files_format_and_content: txt file contains normalized expressions for each sample

Submission date

Aug 31, 2015

Last update date

May 15, 2019

Contact name

Alvin C Powers

Organization name

Vanderbilt University Medical Center

Department

Medicine

Street address

2213 Garland Avenue