|
Status |
Public on May 22, 2018 |
Title |
WT_28C-7 |
Sample type |
SRA |
|
|
Source name |
Brown adipose tissue
|
Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 tissue: Brown adipose tissue condition: housed at 28C
|
Treatment protocol |
BL6 mice were housed at 28C or exposed to 4C for 6h prior to brown adipose tissue isolation.
|
Extracted molecule |
total RNA |
Extraction protocol |
Brown adipose tissues were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent and Rneasy mini kit. The SOLiD SAGE technology (Serial Analysis of Gene Expression) was utilized to determine mRNA expression levels by generating the SOLiD SAGE libraries of unique 27-bp sequence tags for all mRNAs and sequencing unique sequence tags isolated from the 3’ ends of mRNAs using the Life Technologies 5500XL SOLiD system. Briefly, total RNA was bound to Dynabeads Oligo(dT) EcoP magnetic beads and transcribed into cDNA on the beads using SuperScript III Reverse Transcriptase and E.coli DNA polymerase. Double-stranded cDNA was digested with NlaIII restriction enzyme and ligated to Barcode adaptor A containing an EcoP15L restriction enzyme recognition site and a truncated internal adaptor sequence. An EcoP15I digestion was carried out to cleave the tag off from the magnetic beads. The tag consists of the adaptor sequence and 27 bp of unique sequence from the 3¢ end of a single mRNA. The 5¢ end of each tag was ligated to adaptor B that contains the P1 primer binding site for SOLiD emulsion PCR. Each library was amplified in a PCR reaction with a different barcoded SOLiD 3¢ Primer from the SOLiD RNA Barcoding kit and a SOLiD 5¢ Primer. SOLiD SAGE library sequencing was performed with next-generation sequencing using the Life Technologies 5500XL SOLiD system.
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
AB 5500xl Genetic Analyzer |
|
|
Description |
housed at 28°C
|
Data processing |
demultiplex, trimming, and mapping using Applied Biosystems SOLiD_SAGE Analysis software V1.10 allowing 0 mismatches differential expression analysis performed with DESeq V 1.12.1 and EdgeR V 3.2.4, R V 3.0.1 and Rstudio V0.97.551, P value cutoff of 0.05 and a relative fold change cutoff of +/- 1.5 Principal Component Analysis and additional statistical tests performed with JMP Genomics V 6.0 Unsupervised hierarchical clustering and heat maps generated using the Morpheus (https://software.broadinstitute.org/morpheus) Pathway enrichment analysis performed with Gene Set Enrichment Analysis (GSEA) (http://software.broadinstitute.org/gsea/index.jsp) Genome_build: GRC38/mm10 Supplementary_files_format_and_content: tab-delimited text, DESeq normalized counts
|
|
|
Submission date |
Feb 02, 2018 |
Last update date |
May 22, 2018 |
Contact name |
Ji Suk Chang |
Organization name |
Pennington Biomedical Research Center
|
Department |
Gene Regulation and Metabolism
|
Street address |
6400 Perkins Rd.
|
City |
Baton Rouge |
State/province |
LA |
ZIP/Postal code |
70808 |
Country |
USA |
|
|
Platform ID |
GPL15907 |
Series (2) |
GSE110055 |
A map of the PGC-1α- and NT-PGC-1α-regulated transcriptional network in brown adipose tissue [SAGE] |
GSE110056 |
A map of the PGC-1α- and NT-PGC-1α-regulated transcriptional network in brown adipose tissue |
|
Relations |
BioSample |
SAMN08457561 |
SRA |
SRX3643071 |