|
| Status |
Public on Feb 14, 2020 |
| Title |
ASP14_day22: A296U171 |
| Sample type |
SRA |
| |
|
| Source name |
ASP14_day22
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A673
passages: <10
tissue: Ewing sarcoma
|
| Treatment protocol |
EWSR1-FLI1 specific small hairpin RNA was induced in A673/TR/shEF cells by adding DOX at 1 ug/mL. After 7 days, DOX was removed and cells were washed three times to allow silencing of the shRNA and induction of EWSR1-FLI1. For ASP14 xenograft, 20 millions cells were resuspended in 200 µL of PBS and subcutaneously injected into severe combined immunodeficiency (SCID) mice. When tumor volume reached 1,000 mm3, DOX was added in the drinking water of a subset of mice (+ DOX group) for 7 days.
|
| Growth protocol |
A673/TR/shEF are cultured in DMEM (Gibco) 10% FBS (Eurobio), with 50 μg/mL Zeocin (Invitrogen), 2 μg/mL Blasticidin (Invitrogen) added ex-tempo.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Dissociated cells were captured and processed with the C1 Single-Cell Auto Prep System (FluidigmTM) following the manufacturer’s protocol
After harvest from the C1 device, each cDNA library was tagmented using the Nextera XT DNA Sample Preparation Kit (Illumina). Libraries were quantified by Qubit fluorometric assay (Invitrogen) with dsDNA HS (High Sensitivity) Assay Kit and qualified using LabChip (LabChip® GX Touch™ PerkinElmer)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
A673TRshEF cells, after 15 days of doxycycline removal in media of cells that were previously subjected to 7 days of doxycycline inducing downregulation of EWS-FLI1 (“d7+15”)
|
| Data processing |
Reads obtained from sequencing of cells were aligned on the human genome (v. hg19) using TopHat (version 2.0.6). Reads mapping more than once (parameter -x 1) or having edit distances of more than 3 (-N 3) were discarded.
Counting of reads on annotated genes from the GRCh37 gene build was done using htseq-count (v. HTSeq-0.5.3p9) with the following parameters: reads with a quality score less than 10 (-a 10) were discarded and reads partially overlapping with the annotated gene transcript were included in the counts unless they overlapped with another read. In all experiments analysed the STRANDED=no option was used.
Sample-to-sample normalization was performed by rescaling using DESeq-type size factors. For all data analyses the number of reads was log10(x+1) transformed.
Genome_build: hg19
Supplementary_files_format_and_content: GeneXCell tab-delimited text file with read counts
|
| |
|
| Submission date |
Apr 18, 2019 |
| Last update date |
Feb 14, 2020 |
| Contact name |
Olivier Delattre |
| Organization name |
Institut Curie
|
| Street address |
26 rue d'Ulm
|
| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE130019 |
Single cell RNA-Seq of A673/TR/shEF cell line - time course |
| GSE130025 |
Transcriptional programs define intratumoral heterogeneity of Ewing sarcoma at single cell resolution |
|
| Relations |
| BioSample |
SAMN11462592 |
| SRA |
SRX5708482 |
