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Sample GSM2433084

Query DataSets for GSM2433084

Status

Public on Jan 24, 2017

Title

TRAP-rep3

Sample type

SRA

Source name

epididymal WAT

Organism

Mus musculus

Characteristics

strain: C57BL/6J
tissue: adipose

Growth protocol

Mice were maintained on a standard chow diet (8664 Harlan Teklad, 6.4% w/w fat) under a regular 12h light/12h dark cycle at constant temperature (23°C).

Extracted molecule

total RNA

Extraction protocol

TRAP was performed as previously described (Long et al., Cell Metab 2014) with modifications. Adipose tissues were homogenized and processed to immunoprecipitation using anti-GFP antibody. Immunoprecipitates were washed and subjected to RNA extraction using Qiagen Micro RNeasy kit according to the manufacturer’s instructions. For input RNA, 5% of homogenates were mixed with TRIzol and processed according to the manufacturer’s instructions to extract total RNA. Isolated RNA was quantified by Qubit, and RNA integrity was analyzed by Agilent Bioanalyzer.
Extracted RNA (100ng) was processed for ribosomal RNA removal using the Epicentre rRNA depletion kit according to the manufacturer’s instructions. Ribosomal RNA-depleted RNA was subsequently used to generate paired-end sequencing libraries using the Illumina RNA TruSeq Library Kit according to the manufacturer’s instructions. Quantity and quality of RNA-Seq libraries were analyzed by Qubit and Agilent Bioanalyzer, respectively, and the libraries were pooled at a final concentration of 12pM and sequenced by HiSeq2500

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

RNA-seq data were aligned using TopHat2 (Trapnell et al., 2009) to the mm9 mouse genome.
Duplicates and low quality reads were removed by Picard (http://picard.sourceforge.net)
Reads were assigned to transcripts, normalized and quantified using featureCounts (Liao et al., 2014) and EdgeR (Robinson et al., 2010).
Low expressed genes (log2 CPM <1) were filtered out, and differentially expressed genes were defined at log2 fold-change (FC) ≥0.5 and log2 false discovery rate (FDR)≤0.25
Gene set enrichment analysis was performed using RDAVIDWebService (Fresno and Fernandez, 2013) and GOstats (Falcon and Gentleman, 2007).
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text file includes raw read counts for each sample

Submission date

Dec 19, 2016

Last update date

May 15, 2019

Contact name

Evan Rosen

E-mail(s)

erosen@bidmc.harvard.edu

Organization name

Beth Israel Deconess Medical Center

Department

Endocrinology