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| Status |
Public on Dec 31, 2014 |
| Title |
MCF7_RS_SC_R4 |
| Sample type |
SRA |
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| Source name |
MCF7 single cell
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
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| Growth protocol |
The human epithelial cell lines MCF7 and MCF10A were grown in DMEM (Gibco, Paisley, Scotland) supplemented with 10% heat-inactivated FCS, 2.5% HEPES buffer (pH 7.2), 0.1% β-mercaptoethanol and 2mM L-glutamine (all from Sigma, UK). Both cell lines were maintained in a 5% CO2 humidified incubator at 37°C and were routinely tested for the presence of mycoplasma.
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| Extracted molecule |
total RNA |
| Extraction protocol |
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. To ensure all cDNA generated using the Epistem RNA Amp kit was double stranded one cycle of re-amplification was performed.
A library was then prepared using 1ug of the dsDNA in the Life Technologies 5500 SOLiD Fragment Library Core Kit (Life Tech, Paisley, UK) according to the manufacturer's instructions. The libraries were quantified using the Life Technologies SOLiD Library Taqman Quantitation kit (Life Tech, Paisley, UK) and Emulsion PCR was performed using the Life Technologies SOLiD EZ Bead System (Life Tech, Paisley, UK). 50bp single read sequencing was carried out on the Life Technologies 5500XL SOLiD System (Life Tech, Paisley, UK).
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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 single cell
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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)). A MCF10A vs. MCF7 gene list was produced.
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) |
| GSE52716 |
Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells: RNA-Seq MCF7 and MCF10A single cell data |
| GSE52717 |
Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells |
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| Relations |
| BioSample |
SAMN02420402 |
| SRA |
SRX383214 |
| 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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