Illumina HiSeq 2500 paired end sequencing; Unliganded Estrogen Receptor Beta cooperates with Argonaute 2 in the modulation of transcription rate and splicing of target genes in breast cancer cells. Part 4
Unliganded Estrogen Receptor Beta cooperates with Argonaute 2 in the modulation of transcription rate and splicing of target genes in breast cancer cells. Part 4
Sample 6_p
Stable clones expressing ERβ tagged with TAP-tag at either C-term (Ct -ERβ) or N-term (Nt-ERβ) were obtained from human breast cancer MCF-7 cells (ER-alpha positive; ATCC, Cat No. HTB-22) .Concerning the generation of ERβ tagged inducible clones, the human full-length cDNA clone pCMV6-ESR2 (RC218519) encoding human ESR2 was purchased from Origene (Rockville, MD). ESR2 sequence, including the Myc and Flag Tags, was then subcloned into the BamHI and EcoRI sites of the pTRE-Tight expression vector (Clontech, Mountain View, CA). All cell lines were propagated in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS (HyClone) and antibiotics: 100U/ml penicillin, 100mg/ml streptomycin, 250ng/ml Amfotericin-B. Steroid deprivation (starvation) was performed by culturing in DMEM w/o phenol red and 5% Dextran Coated Charcoal stripped serum (DCC-FBS) for 5 days. Cell lines were authenticated by short tandem repeat (STR) profiling and routinely tested for Mycoplasma contamination with MycoAlert mycoplasma detection kit (Lonza). Total RNA was extracted from cells expressing or not ERβ using the standard RNA extraction method with TRIzol (Life Technologies). Before use, RNA concentration in each sample was assayed with a ND-2000c spectrophotometer (Thermoscientific) and its quality and integrity assessed with the Agilent 2100 Bioanalyzer with Agilent RNA 6000 nano kit (Agilent Technologies).  For RNA sequencing experiments, indexed libraries were prepared using 1 µg of total RNA as starting material, with TruSeq Stranded Total RNA Sample Prep Kit (Illumina Inc.). Libraries were sequenced (paired-end, 2x100 cycles) at a concentration of 8 pM/lane on HiSeq 2500 platform (Illumina Inc.)