title: 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 1
design: Unliganded Estrogen Receptor Beta cooperates with Argonaute 2 in the modulation of transcription rate and splicing of target genes in breast cancer cells. Part 1
library_construction_protocol: C-TAP-ERβ and MCF7 Tet-Off control cells were hormone-deprived for 5 days as previously described, collected and lysed with Polysome lysis buffer following the procedure reported in Keene et al (Keene et al., 2006).  An aliquot of whole-cell extract (10% of total) was taken as input while each assay of immunoprecipitation was performed starting from 2mg of total protein lysate. For ERβ immunoprecipitation, samples were incubated at 4°C for 3 hours with 50μl of IgG Sepharose 6 fast Flow (GE Healthcare Bio-Science AB) pre-treated with NT2 buffer supplemented with 5% BSA. After bound, the isolation of RNA immunoprecipitated with ERβ was carried out by adding TRIzol (Life Technologies) directly to the washed beads, following the manufacturer’s instructions indexed sequencing libraries were preparated starting from 1μg of RNA input and 300 ng of RNA immunoprecipitated, pooling three indipendent experiments (biological replicates) and using TruSeq Stranded Total RNA
attributes.tag: Experimental Factor: Input RNA
attributes.value: immunoprecipitate
attributes.tag: Experimental Factor: expression of estrogen receptor  fused to C terminus tandem affinity purification tag
attributes.value: genotype