NR4A1 Inhibition Synergizes with Ibrutinib in Killing Mantle Cell Lymphoma Cells

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to identify NR4A1 targets by RNA-seq and high-throughput data analysis and verify these genes by quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods. Methods: Jeko/Rec-1 Cas9 control and NR4A1 sgRNA stable cell lines were generated with tet-on system vector, sg RNAs were induced for 48 hours after doxycycline addition, mRNA was extracted and used for RNA sequencing. After TopHat analysis followed by Cufflinks, down/up regulated genes list was generated. qRT–PCR validation was then performed using SYBR Green assays. Results: Using an optimized data analysis workflow and with a fold change =1.3 and p value <0.05, thousands of genes were changed. Altered expression of 6 genes was then confirmed with qRT–PCR, demonstrating the high qulity of the RNA-seq method. Overall design: Jeko/Rec-1 Cas9 control and NR4A1 sgRNA stable cell lines were generated with tet-on system vector, sgRNAs were induced for 48 hours after doxycycline addition, and total RNA from these cells was then used for RNA-seq analysis.