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.
Unliganded Estrogen Receptor Beta cooperates with Argonaute 2 in the modulation of transcription rate and splicing of target genes in breast cancer cells.
Sample 8
RNA was extracted from cells expressing or not ER using the standard RNA extraction method with TRIzol (Life Technologies) After Ago2 knock-down in C-TAP-ER and MCF7 cells starved for 5 days, nascent RNA was extracted from each sample following the procedure developed by Khodor et al.28. Nascent RNA isolation was also conducted on non-transfected and transfected with pLKO.1-puro Non-Target shRNA Control Plasmid DNA (Sigma-Aldrich) cells as control. TRIzol reagent (Life Techonolgies) was added, an incubation at 65C was performed to dissolve DNA-Histone-Pol II-RNA pellets28 and RNA was extracted following the manufacturers protocol.  For sequencing, indexed libraries were prepared using 1 g of Nascent RNA as starting material, with TruSeq Stranded Total RNA Sample Prep Kit (Illumina Inc.)