characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-10-02T17:04:17Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T21:37:38Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2785585
characteristics.characteristic: External Id
characteristics.text: Systems Biology Ireland School of Medicine and Medical Sciences
characteristics.characteristic: INSDC center name
characteristics.text: 2014-10-02T17:04:17Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T21:37:38Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS555275
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2969:24h-1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: MCF 10A
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001200
characteristics.characteristic: cell line
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Monolayer cells were maintained in T75 (10ml) or T175 (20ml) culture flasks, with the following media per flask: 500ml DMEM/F12 (Hyclone), 5% horse serum (Sigma), 0.5ug/ml hydrocortisone (Sigma, stock 1mg/ml in ethanol), 100ng/ul cholera toxin (Sigma, stock 1mg/ml in water), 10ug/ml insulin (Sigma) and 5ml Penicillin/Streptomycin (Pen/Strep) (Gibco, Invitrogen). The media mixture was finally sterilised using a 0.2um filter (Stericup, Millipore). 20ng/ml epidermal growth factor (Sigma) was added freshly to growth media after each cell splitting procedure. For the work performed in MCF10A 3D culture, cells were cultured on a laminin-rich extracellular matrix (ECM) Matrigel (Corning) to form acini. Cold Matrigel was applied to dishes or slides with a sterile pipette and spread evenly using an inoculation loop. Dishes or slides were then placed at 37C for at least 30 minutes to allow the matrix solidify. Prior to seeding of MCF10A cells onto laminin-coated tissue culture plastic/glass slides, cells were treated with Trypsin and detached from plastic flasks as described above, then resuspended in assay medium (containing 2% horse serum). In order to perform cell counting, an aliquot of cells was taken and mixed at a 1:1 ratio with Trypan Blue Stain (Invitrogen). Cells were then counted using a Countess Automated Cell Counter (Invitrogen). For RNA or protein extraction, cells were counted and seeded at a density of 200,000cells/6cm plastic dish (Greiner) pre-coated with 150ul of Matrigel.For all assays performed in 3D, medium was supplemented with 2% Matrigel and 5ng/ml EGF. RNA was harvested from dishes using the RNeasy Kit (Qiagen) or TRI Reagent (Sigma) according to manufacturers protocol. DNA was digested with the DNA-free Kit (Applied Biosystems). RNA concentrations were measured using a Nanodrop2000 (Thermo Scientific) and RNA quality was verified on a 2100 Bioanalyser (Agilent) using a Eukaryote Total RNA Nano Chip (version 2.6). Sequencing libraries were generated from 2ug of total RNA per sample with TruSeq RNA sample preparation Kit v2 (Illumina) according to manufacturers protocol. Size and purity of the libraries were analysed on a Bioanalyser High Sensitivity DNA chip (Agilent).
characteristics.characteristic: description
characteristics.text: 3D in matrigel for 24h
characteristics.characteristic: growth condition
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: 24h-1
characteristics.characteristic: title