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| Status |
Public on May 22, 2024 |
| Title |
JN-DSRCT-1, siCTRL, R3, RNA-seq |
| Sample type |
SRA |
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| Source name |
JN-DSRCT-1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: JN-DSRCT-1
cell type: Desmoplastic Small Round Cell Tumor
treatment: control non-targeting siRNA
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| Treatment protocol |
JN-DSRCT-1 cell line was transfected with a custom siRNA targeting EWSR1::WT1 (3’ GAT CTT GAT CTA GGT GAG A 5’) or non-targeting siRNA (Horizon Discovery «on»-TARGETplus Non-targeting siRNA#1, reference D-001810-01-05), according to manufacturer’s instructions. After cell seeding and obtention of 50% confluency, transfection was performed using LipofectamineTM RNAiMAX (InvitrogenTM reference 13778150), and the medium was replaced the day after. A 48-hour silencing time point was used for each described experiment. The experiment was performed in triplicate.
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| Growth protocol |
JN-DSRCT-1 cell line was maintained in 2D adherent culture within DMEM/F-12 (GibcoTM) supplemented with 10% FBS, 1% Penicillin-Streptomycin (GibcoTM), 1% Sodium Pyruvate (GibcoTM), 1% Sodium Bicarbonate (GibcoTM), 1% Non-Essential Amino Acids (GibcoTM) and 1% HEPES (GibcoTM). Cell passaging was performed at 1/10 twice a week. Used cells were controlled for mycoplasma-free status.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were collected at 4°C in PBS after 48-hour silencing. Cell pellets were lyzed and RNA extracted using Qiagen RNEasy kit according to manufacturer's instructions. RNA quality control was performed before library construction based on RNA Integrity Number (RIN) evaluated on Agilent 2100 Bioanalyzer.
After the QC procedure, rRNA was removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs(dUTP, dATP, dGTP and dCTP), RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Then followed by purification by AMPure XP beads, terminal repair, polyadenylation, sequencing adapter ligation, size selection and degradation of second-strand U-contained cDNA by the USER enzyme. The strand-specific cDNA library was generated after the final PCR enrichment.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
column H48_siCTRL_R3
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| Data processing |
RNA-seq data were analyzed according to nf-core/rnaseq pipeline
Raw reads quality control (QC) was performed using FastQC
Adapter trimming was done with Trim Galore!
Pseudoalignment to the reference genome and transcript quatification were performed using Salmon
Assembly: hg19 (GENCODE version 19)
Supplementary files format and content: Count matrix in transcript per million
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| Submission date |
May 18, 2024 |
| Last update date |
May 24, 2024 |
| Contact name |
Clémence Henon |
| E-mail(s) |
clemence.henon@gustaveroussy.fr
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| Organization name |
Gustave Roussy
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| Street address |
114 rue Edouard Vaillant
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| City |
Villejuif |
| ZIP/Postal code |
94800 |
| Country |
France |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE263523 |
Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in Desmoplastic Small Round Cell Tumors |
| GSE267804 |
Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in Desmoplastic Small Round Cell Tumors [Cell_line_RNA-seq] |
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| Relations |
| BioSample |
SAMN41447696 |
| SRA |
SRX24681656 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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