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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 05, 2024 |
| Title |
pdox_shh_T3_3 |
| Sample type |
SRA |
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| Source name |
MB tumor cells treated with T3
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| Organism |
Homo sapiens |
| Characteristics |
tissue: MB tumor cells treated with T3
cell line: ICb-5610MB
cell type: human SHH PDOX medulloblastoma cells
treatment: T3
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| Treatment protocol |
For in vitro experiments, 200nM of T3 was added to the culture medium for 48 hours. For EZH2i conditions, 1mM of GSK126 was added to culture. Controls were treated with PBS only.
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| Growth protocol |
Medulloblastoma cells were suspended in NB-B22 culture medium (Neurobasal, 1mmol/L sodium pyruvate, 2mml/L L-glutamine, B22 supplement, and 1% penicillin/streptomycin).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Primary cells were freshly isolated from tumor-bearing mice at 6 to 8 weeks of age. Briefly, tumor tissues were isolated from mouse brains and digested in a papain solution to obtain a single cell suspension and then centrifuged through a 35% and 65% Percoll gradient. Cells from the 35% to 65% interface were suspended in Dulbecco's PBS (DPBS) plus 0.5% BSA. Cells were then suspended in NB-B22 (Neurobasal medium with B22 supplement. Cell suspension was plated on poly-D-lysine (PDL)-coated coverslips (BD Biosciences).
Total RNA was extracted from tumor cells treated with T3 or PBS. Strand-specific mRNA-seq libraries for the Illumina platform were generated and sequenced following the manufacturer’s protocol. High-quality total RNA was used as input for the so-called dUTP library preparation method. Briefly, the mRNA fraction was purified from total RNA by polyA capture, fragmented and subjected to first-strand cDNA synthesis with random hexamers in the presence of Actinomycin D. The second-strand synthesis was performed incorporating dUTP instead of dTTP. Barcoded DNA adapters were ligated to both ends of the double-stranded cDNA and subjected to PCR amplification. The resultant library was checked on a Bioanalyzer (Agilent) and quantified. The libraries were multiplexed, clustered, and sequenced on an Illumina NovaSeq X Plus. The sequencing run was analyzed with the Illumina BCL-Convert, with demultiplexing based on sample- specific barcodes.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq X Plus |
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| Description |
bulk transcriptome
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| Data processing |
Paired-end RNA sequencing fastq files were processed for adapter and low quality base call removal. Each paired-end file was split into equal read numbers across all conditions to account for library depths. Files were then mapped to either the mm39 or hg38 gencode genome index with the light-weight transcript abundance quantifier, Salmon. Variance-mean dispersions of raw counts were estimated using the negative binomial generalized linear modeling Wald test.
Assembly: mouse:mm39, human:hg38
Supplementary files format and content: Comma-separated values text file of raw counts for each sample.
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| Submission date |
Jul 03, 2024 |
| Last update date |
Jul 05, 2024 |
| Contact name |
Duancheng Guo |
| E-mail(s) |
[email protected]
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| Phone |
2679382222
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| Organization name |
Fox Chase Cancer Center
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| Department |
W345
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| Lab |
Zeng-jie Yang
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| Street address |
333 Cottman AVE
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19111 |
| Country |
USA |
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| Platform ID |
GPL34284 |
| Series (1) |
| GSE224974 |
Thyroid Hormones Promotes Terminal Differentiation of Medulloblastoma Tumor Cells |
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| Relations |
| BioSample |
SAMN42286536 |
| SRA |
SRX25199788 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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