RNA immunoprecipitation sequencing (RIP seq) using anti-Argonaute2 (Ago2) antibody to identify the potential targets of kshv-miR-K12-1-5p in AC16 cells

RNA immunoprecipitation sequencing (RIP seq) using anti-Argonaute2 (Ago2) antibody to identify the potential targets of kshv-miR-K12-1-5p in AC16 cells. We report the application of sequencing technology for high-throughput profiling of mRNA expressions in AC16 cells. AC16 cells were lysed and then incubated with anti-Ago2 antibody (Abnova Corporation, Taiwan, China) after transfected with kshv-miR-K12-1-5p mimics or miR-con for 24 h. After incubating at 4 °C overnight, protein A/G magnetic beads (Thermo Scientific, Shanghai, China) were added into the solution and incubated for another 2 h at 4 °C. Then, the beads were washed five times with PBST and the remaining products were extracted with TRIzol agent. In this project, rRNA was removed from the total RNA by ribosomal RNA removal kit, and the way of ion breaking were used to break the RNA into 200-300bp fragments. By using RNA as template, the first strand of cDNA was synthesized with 6 base random primers and reverse transcriptase. The first strand cDNA was used as the template to synthesize the second strand cDNA. When the second strand cDNA is synthesized, the base T is replaced by U to achieve the goal of a chain specific library. All of our RNA-seq projects used strand specific kits to construct the libraries. Finally, we show that mRNA different expression in AC16 cells. This study provides abundance of mRNA that may predict heart diseases.