Identification of Potential NR3C2 Targets by RNA-Seq in HK2 Cells
To identify potential targets of NR3C2 overexpression, RNA sequencing was performed to compare the transcriptomes of control and NR3C2-overexpressing (NR3C2-OE) HK2 cells. Overall design: We report the application of sequencing technology for high-throughput profiling of mRNA expressions in NR3C2-OE and Control cells. Total RNA was extracted from both control and NR3C2-OE HK2 cells (1×106 cells per sample) using Trizol reagent(Takara, Cat# 9109). RNA concentration and purity were meas assessed by agarose gel electrophoresis and the Agilent 2100 Bioanalyzer system for RIN determination. All RNA samples used for library preparation met the following quality control thresholds: total amount =1 µg, concentration =35 ng/µL, OD260/280 =1.8, OD260/230 =1.0, and RIN >8.0. 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 HK2 cells. This study provides abundance of mRNA that may predict kidney diseases.