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| Status |
Public on Jun 08, 2020 |
| Title |
iAD mouse negative Dox |
| Sample type |
SRA |
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| Source name |
uterine tumor
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| Organism |
Mus musculus |
| Characteristics |
cell type: uterine tumor
genotype: iAD (Arid1aflox/flox)
treatment: No treatment
mouse id: 4822
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| Treatment protocol |
Uteri from 4 iPAD and 4 iPD mice were harvested two weeks after gene knockout (by feeding doxycycline). Hematoxylin-and-eosin stained tissue sections were prepared, and the diagnosis of endometrioid carcinoma from all iPAD mice and EIN from all iPD mice was confirmed by histopathology. Uteri from iAD mice were harvested with and without two weeks of feeding doxycycline.
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| Growth protocol |
Generation of Arid1aflox/flox mice on the C57BL/6 background was previously described (PMID: 24899687). Cre-mediated deletion of Arid1a removes exon 8 and introduces a frameshift mutation and a premature stop codon (p.Gly809Hisfs*6; wild type ARID1A protein has 2283 amino acids). Ptenflox/flox mice on the BALB/c background (Strain C;129S4-Ptentm1Hwu/J) were obtained from the Jackson Laboratory . Cre-mediated deletion of Pten removes exon 5 and introduces a frameshift mutation (p.Val85Glyfs*14). Arid1aflox/flox;Ptenflox/flox mice were generated by intercrossing these transgenic strains. In order to express Cre recombinase specifically in the mouse uterine epithelium for all subsequent genetic alterations described, we used Pax8-Cre mice which were generated by crossing mice expressing the reverse tetracycline-controlled transactivator (rtTA) under the control of the Pax8 promoter (Pax8-rtTA) with mice expressing Cre recombinase in a tetracycline-dependent manner (TetO-Cre) (PMID: 24332043). To generate mouse models with Arid1a and Pten individual or combined knockout in uterine epithelium, we crossed Pax8-Cre mice with Arid1aflox/flox mice, Ptenflox/flox mice, and Arid1aflox/flox;Ptenflox/flox mice. All experimental mice were maintained on a mixed genetic background (C57BL/6, BALB/c, and S129). The resultant genotype of each model was confirmed by genomic DNA PCR, using primer sequences described previously (PMID: 24899687). The wild-type, floxed, and recombined Arid1a generate 669, 845, and 298 bp PCR products, respectively. The predicted PCR products for wild-type, flox, and recombined Pten are 1018, 1100, and 400 bp, respectively. Knockout was initiated by treating mice with doxycycline either through oral gavage (2 mg/mouse/day) or subcutaneous implantation of doxycycline pellets (200 mg) when they reached puberty (6-8 weeks old). Hysterectomy (either unilateral or bilateral) was performed 2 weeks, 6 weeks, 10 weeks, 12 weeks, or 16 weeks later. All of the animal procedures were approved by the Johns Hopkins University Animal Care Committee.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from iPAD and iPD mouse uterine tissues using the Qiagen RNeasy Plus Mini Kit. Uteri from iAD mice were harvested and subjected to epithelial cells isolation using Multi Tissue Dissociation Kit 1, CD45 MicroBeads, and CD326 MicroBeads (all from Miltenyi Biotech) according to manufacturer protocols. Total RNA from the isolated iAD mice uterine epithelial cells were extracted using the Qiagen RNeasy Plus Micro Kit.
RNA quality was assessed using the Agilent 2100 Bioanalyzer RNA Nano Chip. RIN values ranged from 5.8 to 7.8 for iPD and iPAD mice uteri, and 7.0 to 9.4 for iAD mice uterine epithelial cells. RNA-sequencing was performed by GeneWiz, Inc. on the Illumina HiSeq2500 platform, in a 2x150 bp paired-end high output V4 chemistry configuration.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
iAD_plus_minus_Dox.tabular.txt
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| Data processing |
Bioinformatics analysis was performed using Galaxy, an open access web-based program that contains a variety of next-generation sequencing analysis tools. Reads were processed and aligned to Mus musculus reference genome build mm10 using TopHat gapped-read mapper (ver. 2.1.0). The aligned reads were processed with Cufflinks transcript assembly (ver. 2.2.1.0), and output of the resulting GTF files was amalgamated to UCSC hg19 or mm10 RefSeq genes annotation files using Cuffmerge (ver. 2.2.1.0). Differential expression analysis was performed using Cuffdiff (ver. 2.2.1.3) to produce a list of genes whose expression changes were significant (FDR < 0.05) between the groups tested.
Genome_build: mm10
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| Submission date |
Jan 02, 2019 |
| Last update date |
Jun 08, 2020 |
| Contact name |
Yohan Suryo Rahmanto |
| E-mail(s) |
suryoysr@gmail.com
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| Organization name |
Johns Hopkins School of Medicine
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| Department |
Department of Pathology
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| Street address |
CRB-2, Rm 376, 1550 Orleans St
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE106662 |
Inactivation of ARID1A-SWI/SNF Complex Alters Chromatin Compactness at Enhancer Regions and Affects Transcription of Key Tumor Signaling Circuitry [RNA-Seq, mouse] |
| GSE106665 |
Inactivation of ARID1A-SWI/SNF Complex Alters Chromatin Compactness at Enhancer Regions and Affects Transcription of Key Tumor Signaling Circuitry |
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| Relations |
| BioSample |
SAMN10686903 |
| SRA |
SRX5195761 |
| 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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