title: 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 2
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 2
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 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 libraries were generated from 120ng of the same pooled RNA using TruSeq Small RNA Sample Prep Kits (Illumina Inc.)
attributes.tag: Experimental Factor: estrogen receptor Beta
attributes.value: immunoprecipitate