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| Status |
Public on Apr 15, 2024 |
| Title |
TC32, pLOC RFP, Rep 2 |
| Sample type |
SRA |
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| Source name |
Cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Cell line
cell line: TC-32
cell type: Ewing sarcoma
genotype: pLOC RFP
treatment: pLOC RFP
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| Treatment protocol |
To generate the ETS1-pLOC construct, we used a synthetic human ETS1 cDNA (NM_001143820) with custom flanking restriction enzyme sites (GENEWIZ, South Plainfield, NJ) cloned into the pLOC vector (OHS5832; Dharmacon/Horizon Discovery, Cambridge, UK). Lentivirus was produced in HEK-293T cells using either the Trans-Lentiviral ORF Packaging Kit (Dharmacon/Horizon Discovery). Viral supernatants were collected 72 hrs post-transfection and concentrated (PEG Virus Precipitation Kit, Abcam, Waltham, Boston). Concentrated virus was added dropwise to cells in the presence of media supplemented with 8 µg/ml polybrene and transduced cells selected using puromycin (2 µg/ml) and subjected to single cell sorting.
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| Growth protocol |
TC-32 cells were cultured in RPM1 1640 supplemented with 10% FBS and Plasmocin Prophylactic (Invivogen, San Diego, CA), and grown at 37°C, 5% CO2. We confirmed the identity of cell line using short tandem repeat (STR) analysis (ATCC) at intervals throughout experimentation, and we monitored for mycoplasma contamination using the MycoAlert Plus system (Lonza, Walkersville, MD)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Maxwell 16 LEV simply RNA kits (Promega)
RNA libraries for RNA-seq were prepared using Illumina® Stranded mRNA Prep Ligation kit following manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Data processing |
RNA-seq analysis were carried out using center for cancer research bioinfoirmatics resource (CCBR) pipeline (https://github.com/CCBR/Pipeliner/wiki/Pipeline-Documentation - rna-seq)
Sequence reads were triimmed for adaptor sequence using cutadapt v4.4
Trimmed reads were mapped to the GRCh38/hg38 human genome using STAR v.2.7.3 with standard parameters.
FeatureCounts in the Subread v.2.0.3 package (Liao et al., 2013) was used to summarize gene level reads by counting reads that overlapped the GRCh38/hg38 annotated gene exons.
Gene counts were normalized and used to quantify differential gene expression between control and experimental conditions using limma v.3.38.3.
Assembly: GRCh38/hg38
Supplementary files format and content: Tab-delimited text file includes raw counts for each samples
Supplementary files format and content: Tab-delimited text file includes FPKM
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| Submission date |
Sep 14, 2023 |
| Last update date |
Apr 15, 2024 |
| Contact name |
Natasha J Caplen |
| Organization name |
National Institutes of Health
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| Department |
National Cancer Institute
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| Lab |
Functional Genetics
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| Street address |
37 Convent Drive, Bldg. 37, Rm 6128
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| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE243184 |
ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 |
| GSE243279 |
EWSR1::FLI1 fusion oncoproteins’ regulation of ETS1 (RNA-seq) |
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| Relations |
| BioSample |
SAMN37398949 |
| SRA |
SRX21781316 |
| 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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