title: MCF7C1
organism: Homo sapiens
description: Protocols: MCF-7/SP10+ and MCF-7/C breast cancer cells were routinely cultured in a humidified 95% air/5% CO2 incubator at 37 °C in complete medium Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) 1.0 mM sodium pyruvate, 2mM L-glutamine and 100 IU/mL penicillin. The low metastatic MCF-7 (ERα positive) epithelial breast cancer cells were transfected with transduction ready lentiviral particles containing short hairpin RNA (shRNA) against ERα (MCF-7/SP10+) and with control shRNA lentiviral particles that encode a scrambled shRNA sequence (MCF-7/C) Total RNA was isolated from cells using NucleoSpin® RNA plus Kit (Macherey-Nagel). The amount of isolated RNA was quantified by measuring its absorbance at 260 nm. Total RNA for AmpliSeq analysis was extracted in 3 biological replicates and 3 technical replicates as described above. 50 ng of RNA were reverse transcribed to cDNA using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit Preparation protocol (Revision A.0, Thermo Fisher). The acquired cDNA was amplified using Ion AmpliSeq™ Transcriptome Human Gene Expression core panel (Thermo Fisher) and the primer sequences were then partially digested. Then, DNA adaptors (Ion P1 Adaptor and Ion Xpress™ Barcode Adaptor, Thermo Fisher) were ligated to the amplicons. Adaptor ligated amplicons were purified using the Agencourt® AMPure® XP reagent (Beckman Coulter) and eluted in amplification mix (Platinum® PCR SuperMix High Fidelity and Library Amplification Primer Mix, Thermo Fisher) and amplified. Size-selection and purification was conducted using Agencourt® AMPure® XP reagent (Beckman Coulter). The amplicons were quantified using the Fragment AnalyzerTM instrument (Advanced Analytical Technologies, INC.) with DNF-474 High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies, INC.). Samples were then pooled, six or less per pool, followed by emulsion PCR on either the Ion OneTouch™ 2 System using the Ion PI™ Hi-Q™ OT2 Kit (Thermo Fisher), or on the Ion Chef™ System using the Ion PI Hi-Q Chef Kit (Thermo Fisher). The pooled samples were loaded on Ion PI™ v3 chips and sequenced on the Ion Proton™ System using the Ion PI™ Hi-Q Sequencing 200 Kit chemistry (200 bp read length, Thermo Fisher). Mean read length was 114 bp.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-10-22T17:03:04Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2017-03-01T11:02:45Z
attributes.tag: External Id
attributes.value: SAMEA102061168
attributes.tag: INSDC center name
attributes.value: Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University
attributes.tag: INSDC first public
attributes.value: 2018-10-22T17:03:04Z
attributes.tag: INSDC last update
attributes.value: 2017-03-01T11:02:45Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5537:MCF7C1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MCF-7
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: breast adenocarcinoma
attributes.tag: phenotype
attributes.value: estrogen receptor alpha positive
attributes.tag: sample name
attributes.value: E-MTAB-5537:MCF7C1
attributes.tag: scientific_name
attributes.value: Homo sapiens