Illumina HiSeq 2500 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 5
Unliganded Estrogen Receptor Beta cooperates with Argonaute 2 in the modulation of transcription rate and splicing of target genes in breast cancer cells. Part 5
Sample 6_s
C-TAP-ERβ and MCF7 control cells were hormone-deprived for 5 days For each assay, a total of 5 × 106 cells were fixed, lysed, sonicated and diluted as earlier described29. An aliquot of whole-cell extract was taken as input. For ERβ immunoprecipitation, chromatin samples were incubated at 4°C for 3 hours with 30µl of IgG Sepharose 6 fast Flow (GE Healthcare Bio-Science AB) properly equilibrated in a Poly-Prep chromatography columns (0.84 cm, Bio-Rad), according to the manufacturer’s instructions. For Ago2 immunoprecipitation, chromatin samples were incubated at 4°C overnight with 1µg of mouse monoclonal anti-Ago2/eIF2C2 (ab57113, Abcam) and then at 4°C for 1 hour with 40 µl of equilibrated slurry Protein A/G Plus-Agarose (sc-2003, Santa Cruz Biotechnology). As negative control of Ago2 immunoprecipitation, chromatin samples were also incubated overnight with 1 µg of mouse monoclonal anti-Flag M2 affinity purified (F1804, Sigma-Aldrich). Beads washing, elution, reverse crosslinking and DNA extraction were then performed as described29.  Size distribution of each ChIP DNA sample was assessed by running a 1µl aliquot on Agilent High Sensitivity DNA chip using an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). The concentration of each DNA sample was determined by using Quant-IT DNA Assay Kit-High Sensitivity and a Qubit Fluorometer (Life Technologies). 10 ng of purified ChIP DNA were used as starting material for sequencing libraries preparation, by pooling three independent ChIP experiments. Indexed libraries were prepared with TruSeq ChIP Sample Prep Kit (Illumina Inc.).