Illumina Genome Analyzer IIx sequencing; miRNA expression profile in breast cancer cells
miRNA expression profile in breast cancer cells
Extract_MCF7_N-Tap_ER-beta
Human breast cancer MCF-7 wt were used to produce stable clones expressing ER-beta tagged with TAP-tag at the N-term(Nt-ER-beta ) as previously described. 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 compound. 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).