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| Status |
Public on Feb 14, 2020 |
| Title |
Ewing Sarcoma PDX-111: A540U403 |
| Sample type |
SRA |
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| Source name |
Ewing Sarcoma PDX-111
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Ewing Sarcoma
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| Treatment protocol |
No treatment
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| Growth protocol |
Ewing Sarcoma Patient Derived Xenografts (PDX) were established in the laboratory by subcutaneous implantation of tumor samples in SCID mice. Patients consented preoperatively to take part in the study which received agreement by the Institutional Review Board Protocol.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Ewing PDX were dissected from mice and mechanically dissociated. The finely minced tissue was transferred to a digestion mix consisting of C02 independent medium (Gibco) containing 1 mg/mL collagenase D (Roche), 2 mg/mL hyaluronidase (Sigma) and 25 µg/mL DNAse (Sigma), incubated for 45 min at 37 °C and gently resuspended every 10 min. Cell suspension was then filtered using 70 µm and 30 µm cell strainers (Miltenyi Biotec).
Single-cell RNA-seq was performed using the Single Cell 3’GEM Code Single-Cell instrument (10x Genomics, Pleasanton, CA, USA), according to the manufacturer’s protocol. Cellular suspension (5,300 cells) was loaded on a 10x Chromium instrument to generate 3,000 single-cell GEMs, using the Chromium™ Single Cell 3' Library & Gel Bead Kit v2. The initial step consisted in performing an emulsion where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10x cell barcodes, UMI (Unique Molecular Identifiers) and poly(dT) sequences. GEM-RT was performed to generate barcoded full-length cDNA (53°C for 45 min, 85°C for 5 min, held at 4°C). After RT, GEMs were broken using the recovery agent and the single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific).
Bulk cDNA was amplified (98 °C for 3 min; 12 cycles : 98 °C for 15 s, 67 °C for 20 s, 72 °C for 1 min; 72 °C for 1 min; held at 4 °C) and then cleaned up with the SPRIselect Reagent Kit (Beckman Coulter). A qualitative analysis on the amplified cDNA was performed using Agilent Bioanalyzer high sensitivity chip.
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)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Raw data not submitted due to patient privacy concerns.
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| 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
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| Submission date |
Apr 18, 2019 |
| Last update date |
Feb 14, 2020 |
| Contact name |
Olivier Delattre |
| Organization name |
Institut Curie
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| Street address |
26 rue d'Ulm
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| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE130023 |
Single cell RNA-Seq of Ewing Sarcoma PDX data (PDX_C1) |
| GSE130025 |
Transcriptional programs define intratumoral heterogeneity of Ewing sarcoma at single cell resolution |
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| Relations |
| BioSample |
SAMN11462863 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3730278_A540U403.mapped.counts.txt.gz |
161.8 Kb |
(ftp)(http) |
TXT |
| Raw data not provided for this record |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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