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Status |
Public on Apr 30, 2019 |
Title |
BAT-Mcu-KO RT #3 |
Sample type |
SRA |
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Source name |
Brown adipose tissue
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 tissue: Brown adipose tissue age: 8-12 weeks Sex: Male genotype: Mcu[fl/fl];Ucp1-Cre temperature: RT
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Treatment protocol |
Age and sex-matched animals were individually housed at 4C for up to six hours in pre-cooled cages without bedding, with ad libitum access to pre-cooled water. Animals at 4C did not have access to food unless otherwise indicated; if food was provided, it was pre-cooled to 4C overnight. Body temperature was measured rectally at indicated time points using a Physitemp BAT-12 thermometer outfitted with a RET-3 probe. All animals used for cold tolerance and RNA-seq experiments were males at 8-12 weeks of age.
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Extracted molecule |
total RNA |
Extraction protocol |
Animals were sacrificed by CO2 asphyxiation followed by cervical dislocation, and tissues were immediately harvested and snap frozen in liquid N2. Frozen BAT samples were homogenized in 1mL Qiazol per 100mg tissue using the Qiagen TissueRuptor II. The homogenate was mixed thoroughly with chloroform (1:5 chloroform:homogenate), incubated for 3 mins at room temperature, and centrifuged at 12,000g for 15 mins at 4C, and 100uL of the resulting aqueous phase was added to 350uL Qiagen buffer RLT plus 250uL of 100% ethanol. The resulting mixture was transferred to a column from the Qiagen RNeasy Mini Kit, and RNA was purified according to the manufacturer’s protocol. RNA-sequencing libraries were prepared by the Broad Technology Labs at the Broad Institute based on the Smart-seq2 protocol (S. Picelli, O. R. Faridani, Å. K. Björklund, G. Winberg, S. Sagasser, and R. Sandberg, “Full-length RNA-seq from single cells using Smart-seq2,” Nat. Protoc., vol. 9, no. 1, pp. 171–181, 2014.)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
2x25bp paired-end reads were aligned to the mouse genome (mm10, with gencode M7 annotations) using STAR 2.4.0j (default parameters). Counts of reads uniquely mapping within exonic regions of annotated genes (irrespective of strand) were collated using HTSeq. 14,651 genes with at least 8 reads mapping to them in at least 6 of the samples were retained for differential expression analysis. This was performed in R using DESeq2 [115] and the design formula ~Genotype + Temperature + Genotype:Temperature. Where gene expression levels are reported, they represent normalized read counts following application of the estimateSizeFactors function, which implements the median ratio method. Where log2 fold-changes and p-values are reported, they represent the result of the Wald test. P-values are adjusted with the method of Benjamini-Hochberg. GSEA was performed according to the following parameters. 1000 phenotype permutations, signal to noise ranking metric, weighted enrichment statistic, real sorting mode, descending ordering mode, gene set max 15 and min 5. Genome_build: mm10 Supplementary_files_format_and_content: flicker_normcounts.csv contains normalized counts for every transcript measured in each sample. flicker_foldchange.csv contains fold change, nominal p-value, and adjusted p-value for each gene in the following comparisons: control RT vs. BAT-Mcu-KO RT, control 4C vs. BAT-Mcu-KO 4C, control RT vs. control 4C, BAT-Mcu-KO RT vs. BAT-Mcu-KO 4C, (control RT vs. control 4C) vs. (BAT-Mcu-KO RT vs. BAT-Mcu-KO 4C).
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Submission date |
Sep 14, 2018 |
Last update date |
Apr 30, 2019 |
Contact name |
Daniel Flicker |
E-mail(s) |
flicker@fas.harvard.edu
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Organization name |
Massachusetts General Hospital
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Department |
Molecular Biology
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Lab |
Vamsi Mootha
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Street address |
Department of Molecular Biology, 185 Cambridge Street, 6th floor
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City |
Boston |
State/province |
MA |
ZIP/Postal code |
02114 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (1) |
GSE119964 |
Exploring the in vivo role of the mitochondrial calcium uniporter in brown fat bioenergetics |
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Relations |
BioSample |
SAMN10060510 |
SRA |
SRX4680526 |
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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