title: Sample 2
organism: Homo sapiens
description: Protocols: ER-alpha positive BC cells: MCF-7 (ATCC HTB-22) were propagated in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS (HyClone) and antibiotics: 100 U/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin-B. MCF-10A (ATCC CRL-10317) was cultured in Mammary Epithelial Cell Growth Medium (MEGM BulletKit, Lonza, Catalog No. CC-3150) with added 100 ng/ml cholera toxin. None ER-alpha positive BC cells: MCF-7 (ATCC HTB-22) were propagated in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS (HyClone) and antibiotics: 100 U/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin-B. Cells were treated with ICI 182,780 10-7 M for 3 days and with EPZ004777 6.4 µM or DMSO for 3 and 6 days. Conversion from BCL to fastq was perfromed using BCL2Fastq 104cells/well were incubated with compounds for 3 or 6 days with media containing compounds or vehicle and in particular MCF-7 cells treated with EPZ004777 6.4µM or DMSO for for 3 and 6 days or with 4-hydroxytamoxifen 10-7 M (4-OHT) or fulvestrant 10-7 M (ICI 182,780) or DMSO for 3 days. LCC2 cells were treated with 4-hydroxytamoxifen 10-7 M (4-OHT) or DMSO for 3 days and medium replaced every 2 days. Medium was replaced every 2 days. Total RNA was extracted using standard RNA extraction method with TRIzol (Invitrogen). 0.3-0.4 mL of TRIzol™ Reagent per 1 × 105-107cells was added directly to the culture dish to lyse the cells pipetting several times to homogenize. Then was added 0.1 ml of 1-Bromo-3-chloropropane (Sigma- Aldrich) and after incubation of 2-3 minutes, the samples was centrifugated for 15 minutes at 12,000 × g at 4°C. Then, the aqueous phase, containing the RNA, was collected into a new tube and precipitate with isopropanol and resuspended in RNAse/DNase free water. Nascent RNA was extracted from each sample as described by Khodor et al. In brief, following TRIzol (Life Techonolgies) addition, samples were incubated at 65°C to dissolve DNA-Histone-Pol II-RNA pellets and RNA was extracted following the manufacturer's protocol. For sequencing, indexed libraries were prepared using 1 µg of Nascent RNA as starting material, with TruSeq Stranded Total RNA Sample Prep Kit (Illumina Inc.).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-02-08T17:04:04Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-06-07T16:31:53Z
attributes.tag: External Id
attributes.value: SAMEA4716188
attributes.tag: INSDC center name
attributes.value: Laboratory of Molecular Medicine and Genomics
attributes.tag: INSDC first public
attributes.value: 2019-02-08T17:04:04Z
attributes.tag: INSDC last update
attributes.value: 2018-06-07T16:31:53Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6871:Sample 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MCF-7
attributes.tag: cell type
attributes.value: mammary epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: invasive ductal carcinoma
attributes.tag: metastatic site
attributes.value: pleural effusion
attributes.tag: organism part
attributes.value: mammary gland
attributes.tag: sample name
attributes.value: E-MTAB-6871:Sample 2
attributes.tag: scientific_name
attributes.value: Homo sapiens