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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2014 |
| Title |
TT4 |
| Sample type |
SRA |
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| Source name |
patient-derived non-small cell lung cancer xenograft total tumour
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| Organism |
Homo sapiens |
| Characteristics |
tissue: patient-derived non-small cell lung cancer xenograft total tumour
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| Growth protocol |
Patient derived xenografts (PDX) were established from lung cancer primary tumors and expanded as previously described (Moro et al. 2012). Briefly, after approval from the Internal Review and the Ethics Boards of the Fondazione IRCCS Istituto Nazionale Tumori, samples of primary non-small cell lung cancer (NSCLC) were obtained from patients undergoing surgical resection. Each sample was cut in small pieces (25-30 mm3) and implanted subcutaneously using a trocar gauge in the flank of female SCID mice. PDXs were then expanded in vivo through successive rounds of transplantation from donor to recipient mice.
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| Extracted molecule |
total RNA |
| Extraction protocol |
To obtain single cell suspension, PDXs were mechanically and then enzymatically digested in a solution of collagenase IV (5mg/ml) and DNAse (100U/ml) (Sigma-Aldrich) in DMEM/F12 (Lonza) for 1h at 37 °C. Partially digested tissue was filtered through a 100µm cell strainer (BD Falcon) and red blood cells were removed by Lysing Buffer 1X (BD Bioscience). Single cell suspensions from dissociated NSCLC-PDXs were washed and incubated in staining solution 1% BSA and 2mM EDTA with specific antibodies at appropriate dilutions for 30 min at 4 °C: PE anti-human CD133/1, FITC anti-human CD326 (EpCAM) (Miltenyi Biotech), APC anti-human CD187 (CXCR4) (BD Pharmingen), Alexa Fluor® 488 anti-human HLA-ABC (BD Pharmingen) , PerCP-eFluor 710 anti-mouse MHC class I (e-Bioscience). Prior to sorting, cells were resuspended to a final concentration of 10x106 cells/ml in Hepes Buffered Saline (HBS) (Lonza) + 0.1% B27 Supplements (Gibco) and 7-AAD viability staining solution (1:10) (e-Bioscience) for dead cells exclusion. PDX-cells were sorted with FACSAria (Becton Dickinson) into a chilled 96-well plate (Corning Incorporated). For sorting of different CSC fractions, an initial gate excluding doublets, dead cells and mouse MHC class I+ cells was set. Then, within the gate of human viable CD133+ cells, the fraction of EpCAM+ and CXCR4+EpCAM- cells were identified and sorted. Total tumor population was isolated based on human HLA-ABC+ expression. For each cell fraction, 300 cells were directly sorted in 30 μl of CLS (Complete Lysis Solution) per well and within 1h approximately 100 cells/10 μl were transferred into 0.5 ml tubes and stored to -80 0C. To ensure all cDNA generated using the Epistem RNA Amp kit was double stranded one cycle of re-amplification was performed.
RNA (1ng-25pg) or cell lysates in CLS were adjusted to 6.75ml volume and RNA-amplified using the EpiStem RNA-Amp Kit according to the manufacturers protocols (EpiStem, Manchester, UK). Briefly the samples underwent oligo-dT priming and 5’ capping prior to x35 cycles of PCR amplification using the conditions 90ºC 30 sec, 42ºC 2 min and 72ºC 6 min. Following amplification all samples were purified using a MoBio UltraClean® PCR Clean-Up Kit (Carlsbad, CA, USA) and quantified on a NanoDrop spectrophotometer.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB 5500xl Genetic Analyzer |
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| Description |
Amplified cDNA from volume equivalent to 10 cells
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| Data processing |
50mer single-ended strand specific RNA-seq data were generated using a SOLID 5500 sequencing machine and aligned to human genome hg19 using SHRIMP2.
Filtering; reads that aligned to multiple loci were discarded
Normalisation; For each sample, reads were positioned relative to annotated genes from ENSEMBL version 70, using the annmap database and Bioconductor package(4), and the number of reads hitting exonic regions was counted for each gene. These data were then used to identify differentially expressed (DE) genes using the Bioconductor package EdgeR(10) (FDR < 0.05; absolute fold change > 2; exact test method(11)). Three gene lists were produced: TT vs. MCIC, TT vs. RCIC, and MCIC vs. RCIC samples.
Genome_build: hg19
Supplementary_files_format_and_content: RPKM in exonic regions of all protein coding genes which has at least one read in one of 10 samples (Ensembl HS70)
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| Submission date |
Nov 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ged Brady |
| Organization name |
Caner Research UK Manchester Institute
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| Department |
Clinical and Experimental Pharmacology
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| Street address |
Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Rd
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| City |
Manchester |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
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| Platform ID |
GPL16288 |
| Series (2) |
| GSE52715 |
Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells: RNA-Seq CIC data |
| GSE52717 |
Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells |
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| Relations |
| BioSample |
SAMN02420391 |
| SRA |
SRX383197 |
| 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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