Nuclear RNA immunoprecipitation of ERα in MCF-7 BC cell line
Nuclear RNA immunoprecipitation of ERα in MCF-7 BC cell line
Cells were washed twice with cold PBS supplemented with 0.1% of EDTA (500 mM), harvested by scraping and collected in a tube. After a centrifugation at 3000 rpm for 5 min at 4°C, the nuclear fraction was extracted by resuspending the pellet in nuclear isolation Buffer (NIB) (1.28 M Sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2 and 4% Triton X-100) supplemented with 100 U/ml RNAse inhibitor (RiboLock RNase Inhibitor, Invitrogen) and proteinase inhibitors (1mM PMSF and 1x PIC). Samples were incubated on ice for 20 min and then centrifuged at 2500 x g at 4°C for 15 min. Once removed the supernatant, nuclear pellets were resuspended in 400 µl of RIP Buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT e 0.5% NP40) supplemented with 100 U/ml RNAse and proteinase (1mM PMSF and 1x PIC) inhibitors. Cell nuclei were sonicated for 10 cycles (15 ON and 15 OFF) using Bioruptor (Diagenode, Denville, New Jersey, USA) and subsequently centrifuged at 13000 rpm for 10 min at 4°C. 1 ml of TRIzol (Life Technologies, Thermo Fisher) was added directly to the beads and RNA extraction was performed according to the manufacturer's guidelines TruSeq Stranded Total RNA Library Prep Gold (Cat. 20020599, Illumina, San Diego, California, USA)