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| Status |
Public on Jun 14, 2017 |
| Title |
GRO-seq, BAT HDAC3, HDAC3 floxed, 22C/5pm |
| Sample type |
SRA |
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| Source name |
Brown adipose tissue
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: HDAC3 floxed
time: 5pm
temperature: 22C
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| Treatment protocol |
Male mice for thermoneutrality (29C) cohorts were allowed to acclimate for 2 weeks prior to tissue harvest. Mice were acclimated to 29C in environmental rodent incubator maintained on the same 12:12 hour dark/light cycle. Room temperature (22C) cohorts were maintained in standard housing room.
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| Growth protocol |
Male wild-type and HDAC3 KO mice maintained on a C57BL/6 background maintained on a 12 hour dark /light cycle and were sacrificed at 5PM (ZT10) at 12 weeks of age for the collection of interscapular brown adipose tissue.
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Nuclear Run-On (NRO): Nuclei were mixed with an equal volume of run-on buffer (10 mM Tris pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 200 U/ml Superase-In, 1% Sarkosyl, 500 μM ATP, GTP and Br-UTP, 2 μM CTP) and incubated for 5 min at 30°C. Nuclear RNA was extracted with Trizol (Invitrogen) and treated with DNase (Ambion). RNA was hydrolyzed using fragmentation reagents (Ambion) for 13 min at 70°C and purified through a Micro Bio-Spin Column P-30 (Bio-Rad). RNA was treated with T4 polynucleotide kinase (New England Biolabs) for 1h at 37°C, then with an additional T4 polynucleotide kinase for 1h more, and inactivated for 5 min at 75°C. NRO-RNA immuno-precipitation: Anti-BrdU agarose beads (Santa Cruz) were rotated for 1h in blocking buffer (0.25x SSPE, 38 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.1% PVP, and 0.1% BSA). Then NRO-RNA was rotated with beads for 1h, followed by washes twice in binding buffer (0.25x SSPE, 38 mM NaCl, 1 mM EDTA, 0.05% Tween-20), twice in low salt buffer (0.2x SSPE, 1 mM EDTA, 0.05% Tween-20), once in high salt buffer (0.2x SSPE, 1 mM EDTA, 0.05% Tween-20, 138 mM NaCl), and twice in TET buffer (TE pH 7.4, 0.05% Tween-20). BrdU-labeled RNA was eluted from the beads four times for 10 min with 100 μl elution buffer pre-heated to 42°C. RNA was precipitated by adding 300 mM NaCl, 1 µl of glycogen and 2.5 volumes of ethanol and incubated overnight at -20°C. RNA was resuspended in water (with 1 U/µl Superase-In and 0.05% Tween-20), denatured for 3 min at 65°C, and treated with poly(A)-polymerase (New England Biolabs) for 30 min at 37°C.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
nacent RNA
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| Data processing |
Library strategy: GRO-seq
GRO-seq: GRO-seq libraries were sequenced twice for better coverage, and sequencing replicates were pooled for each condition. Before alignment, sequencing reads were cleaned by trimming off low quality base, adapter and tailing poly-A sequences using cutadapt16. Trimmed reads of at least 25bp were selected and aligned to UCSC mm9 using bowtie with an option ‘--best --strata -m 1 -v 3’. Aligned reads within extremely high signal regions such as rRNA, snoRNA, snRNA and tRNA were removed before downstream analysis to minimize read-depth bias. To visualize transcriptional activity in genome browser, bigwig files were generated for (+) and (−) strands separately, where all the reads were extended to 150bp for smooth profile and (−) strand signal were presented as negative values.
Genome_build: mm9
Supplementary_files_format_and_content: Read-per-million (RPM) normalized stack-height profile in bigWig format
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| Submission date |
Jun 30, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hee-Woong Lim |
| Organization name |
Cincinnati Children's Hospital Medical Center
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| Department |
Division of Biomedical Informatics
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| Street address |
3333 Burnet Ave. MLC 7024
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| City |
Cincinnati |
| State/province |
Ohio |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE83926 |
Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge [ChIP-Seq, GRO-Seq] |
| GSE83928 |
Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge |
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| Relations |
| BioSample |
SAMN05334770 |
| SRA |
SRX1890331 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2221751_GROseq.BAT_WT_22C_MJE12_pool.minus.bw |
285.9 Mb |
(ftp)(http) |
BW |
| GSM2221751_GROseq.BAT_WT_22C_MJE12_pool.plus.bw |
296.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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