title: Illumina HiSeq 2500 paired end sequencing; NascentSeq of human breast cancer cell MCF7 treated with EPZ, ICI and DMSO at 3 or 6 days
design: NascentSeq of human breast cancer cell MCF7 treated with EPZ, ICI and DMSO at 3 or 6 days
library_construction_protocol: 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: Experimental Factor: compound
attributes.value: dimethyl sulfoxide
attributes.tag: Experimental Factor: dose
attributes.value: 0.01
attributes.tag: Experimental Factor: time
attributes.value: 6