|
| Status |
Public on Dec 31, 2014 |
| Title |
MCF10A_1ng_R5 |
| Sample type |
RNA |
| |
|
| Source name |
MCF10A cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10A
sample type: Amplified cDNA from 1ng RNA
|
| 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.
|
| 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.
|
| Label |
Biotin
|
| Label protocol |
Following amplification the cDNA was Biotin-labeled using the EpiStem RNA-Amp labeling kit (EpiStem, Manchester, UK)
|
| |
|
| Hybridization protocol |
The hybridisation cocktail containing 5mg of biotin cDNA was heated to 99°C for 5 mins. The hybridisation cocktail was then transferred to 45°C for 5 mins and centrifuged at maximum speed for 5 mins to remove insoluble material. Samples were then hybridised to Affymetrix Human HGU133 Plus 2 arrays for 16 hours overnight and then stained with SAPE using a biotin targeted antibody step and washed according to the EukGE_Ws2v4_450 fluidics protocol from Affymetrix.
|
| Scan protocol |
Samples were scanned in an Affymetrix 3000 scanner.
|
| Data processing |
Data were normalised and summarised using RMA, as implemented by Affymetrix Power Tools (APT) software package (http://www.affymetrix.com/estore/partners_programs/programs/developer/tools/powertools.affx) using the 'apt-probeset-summarize' default parameter setting. In subsequent analysis, gene level summaries (not included here) were computed as the geometric mean of all probesets mapping to a gene, as defined by annmap, using ENSEMBL version 70 as the source of underlying genome annotation. The empirical Bayes statistics implemented in the Bioconductor package LIMMA were used to identify protein-coding genes showing differential levels in MCF7 and MCF10A samples (FDR < 0.05; absolute fold change > 2)
|
| |
|
| Submission date |
Nov 25, 2013 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Ged Brady |
| Organization name |
Caner Research UK Manchester Institute
|
| Department |
Clinical and Experimental Pharmacology
|
| Street address |
Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Rd
|
| City |
Manchester |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE52712 |
Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells: Affymetrix array data |
| GSE52717 |
Flexible multiplatform RNA profiling at the single cell level applied to enriched cancer initiating cells |
|
