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| Status |
Public on Jan 01, 2016 |
| Title |
DEN treated WT RNAseq_replicate 3 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/CBA
age: 8.5 months
genotype/variation: WT
tissue: liver
agent: diethylnitrosamine (DEN)
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| Treatment protocol |
Mice were left untreated or treated by a single intraperitoneal injection of 25mg/kg diethylnitrosamine (N0756 Sigma) and sacrificed at 8.5 months of age. DEN (diethylnitrosamine) is a hepatotoxic agent whose administration triggers late onset hepatocellular carcinoma (HCC). DEN treatment is commonly used to model HCC in mice. Livers were dissected and divided for the preparation of RNA and crosslinked chromatin.
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| Growth protocol |
C57Bl6 mice were maintained in grouped cages in a temperature controlled, pathogen-free facility on a 12-h light/dark cycle and fed by a standard chow diet (Altromin 1324; 19% protein, 5% fat) and water ad libitum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 20 µg of total RNAs were used for mRNA isolation using the Dynabeads® mRNA DIRECT™ Micro Kit (Life Technologies, Carlsbad, CA, USA)
For RNA-Seq experiments, the isolated mRNA was digested with RNaseIII, hybridized and ligated to Ion Adaptors, reverse transcribed, barcoded and amplified, using the Ion Total RNA-Seq Kit v2 (Life Technologies, Carlsbad, CA, USA). Samples were processed on an OneTouch 2 instrument and enriched on a One Touch ES station. Templating was performed using the Ion PI™ Template OT2 200 Kit (Life Technologies, Carlsbad, CA, USA) and sequencing with the Ion PI™ Sequencing 200 Kit on Ion Proton PI™ chips (Life Technologies, Carlsbad, CA, USA) according to commercially available protocols. For ChIP-Seq experiments, chromatin immunoprecipitation was performed as described in (Schmidt et al., 2009) http://www.ncbi.nlm.nih.gov/pubmed/19275939 except the first step of crosslinking, which was performed as follows. Liver tissue was minced to small pieces in PBS and after addition of formaldehyde to a 1% final concentration immediately was subjected to 10 strokes of dounce homogenization. Cross-linking was continued for 10 min and stopped by the addition of glycine at 0.125 M final concentration. Immunoprecipitations were performed with antibodies against RNA Pol-II (Euromedex 1PB-7G5 LOT number 072105), H3K4Me3 (Abcam ab8580 - LOT number GR164534-1) and Smyd3 antiserum raised in-house in rabbits injected with full length recombinant Smyd3 protein.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
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| Description |
mRNA from liver extract from 8.5mo C57BL6/CBA DEN treated WT mouse 3
WT_DEN-WT_Smyd3KO_DEN-Smyd3KO_norm_counts.txt.gz
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| Data processing |
Base calling was performed using software provided with the Ion Torrent Proton sequencer.
Sequenced reads were trimmed for adaptor sequence using cutadapt software and mapped to mm9 whole genome using: for RNA-Seq tophat 2.0.9 with default settings and using additional transcript annotation data for the mm9 genome from Illumina iGenomes (http://cufflinks.cbcb.umd.edu/igenomes.html), for ChIP-Seq, using Bowtie2 with default parameters.
Tags overlapping the Ensembl build 67 mouse exons were counted and filtered for artifacts as follows: if an annotated gene had up to five exons, tag presence was required in at least two exons. If an annotated gene had E >5 exons, tag presence was required in at least E/5 exons. The final gene counts were calculated as the sums of their exon tags. The counts table was normalized using EDASeq and analyzed for differential expression using DESeq. The final list of differentially expressed genes was derived by the genes demonstrating a binomial test p-value less than 0.05.
Genome_build: mm9
Supplementary_files_format_and_content: [RNA-Seq] Tab-delimited files containing DESeq normalized counts for each Ensembl gene. [ChIP-Seq] bigWig and bed files.
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| Submission date |
Apr 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Iannis Talianidis |
| E-mail(s) |
talianid@imbb.forth.gr
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| Organization name |
Foundation for Research and Technology - Hellas
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| Department |
Institute of Molecular Biology and Biotechnology
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| Street address |
100 N. Plastira Str
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| City |
Vassilika Vouton, Herakleion |
| State/province |
Crete |
| ZIP/Postal code |
70013 |
| Country |
Greece |
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| Platform ID |
GPL18635 |
| Series (1) |
| GSE67790 |
Smyd3 is a transcriptional potentiator of multiple cancer-promoting genes and required for liver or colon cancer development |
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| Relations |
| BioSample |
SAMN03481088 |
| SRA |
SRX992463 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1656150_DEN_WT_RNA_BR3.txt.gz |
167.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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