Illumina HiSeq 1000 sequencing; miRNA expression profile in breast cancer cells
miRNA expression profile in breast cancer cells
Extract_ZR75.1_ER-beta
Human breast cancer ZR75.1 cells were used to produce stable clones expressing ER-beta tagged with 3xFlag-tag respectively at the N-term (ZR-flag-ER-beta). All were grown in Dulbeccos modified Eagles medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (both from Invitrogen SpA, San Giuliano, Milanese, Italy), 100 U/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml Amfotericin-B, 50 ug/ml G418 (normal growing condition). For G0-G1 synchronization cells were plated at 40% confluence in steroid-free medium (phenol red-free DMEM medium, with 5% fetal bovine serum pre-treated with dextran-coated charcoal and antibiotics) and maintained for 5 days without any additional scompound Cells were harvested in absence of estrogen stimuli Total RNA was extracted using TRIZOL reagent  (Invitrogen) according to the manufacturer instructions. 1ug of total RNA was used in a library preparation according to the Illumina TruSeq small RNA sample preparation protocol rev. E (Illumina, USA).