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 4
C-TAP-ER and MCF7 control cells were starved for 5 days and then Ago2 knock-down was performed using a combination of three pLKO.1 plasmid vectors expressing shRNAs (Sigma Aldrich: TRCN0000007864; TRCN0000007867; TRCN0000011203) targeting Ago2 transcript (GenBankTM accession number NM_012154 ) in different regions. Ago2 silencing was conducted by co-transfecting C-TAP-ER and control cells with shRNA vectors, using Lipofectamine 2000 (Life Technologies), for 48 hours. The transfection medium was replaced with fresh culturing medium 6 h after treatment. Non-transfected and transfected cells with pLKO.1-puro Non-Target shRNA Control Plasmid DNA (Sigma-Aldrich) were used as control. Western blotting and qPCR were performed to verify the depletion of our target protein and mRNA, respectively.   cDNA synthesis and qPCR  Using 1 g of RNA as starting material, retro-transcription was performed employing AffinityScript cDNA Synthesis Kit (Agilent). qPCRs was performed in duplicate on Stratagene Mx3005PTM instrument (Agilent Technologies) using SensiFASTTM SYBR Lo-ROX kit (Bioline). 40 cycles of amplification were performed. Primers used for qPCR are: for GAPDH: Forward primer: GAGAAGGCTGGGGCTCATTT Reverse primer: GCAGGAGGCATTGCTGATGA for AGO2: Forward primer: AAGTCCTTCACAGAGCAGCT Reverse primer: CTGCTCCTGTCCGACTTGTA 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.)