RNF5 Regulation of RBBP4 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

To investigate a potential role for RNF5 in AML, we monitored changes in gene expression profiles in multiple AML cell lines after RNF5 knock-down. PolyA RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module, and bar-coded libraries were constructed using the NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA). Libraries were pooled and single end-sequenced (1×75) on the Illumina NextSeq 500 using the High output V2 kit (Illumina, San Diego, CA). Quality control was performed using Fastqc (v0.11.5, Andrews S. 2010)), reads were trimmed for adapters, low quality 5′ bases, and minimum length of 20 using CUTADAPT (v1.1). The number of reads per sample and the number of aligned reads suggested that read quality and number were good and that the data were valid for analysis. High-quality data were then mapped to human reference genome (hg19) using STAR mapping algorithm (version 2.5.2a). FeatureCounts implemented in Subread (v1.50) was used to count the sequencing reads from mapped BAM files. Analyses of differentially expressed genes (DEGs) were subsequently performed using a negative binomial test method (edgeR) implemented using SARTools R Package. A list of the DEGs was observed and pathway analysis was performed using IPA (http://www.ingenuity.com) based on the following criteria: |log2(fold change)| >0.4 and p value < 0.05. RNA was prepared 5 days following RNF5 knockdown, and triplicates were used for RNAseq analysis.