Transcriptome profiling of the cytarabine response in U2OS cells overexpressing DNMT3A R882C isoform (compared to DNMT3A wt and empty vector control)
Purpose:  The goal of this study was to investigate the effect of the cytarabine treatment on the transcriptional profiles of U2OS cells retrovirally expressing DNMT3A R882C isoform, compared to DNMT3A WT and empty vector controls Methods: U2OS DNMT3A WT, R882C, and empty vector cells were treated with 10uM cytarabine or vehicle control for 24 hours. Biological triplicates were generated, ie each replicate was performed on different day using a new batch of cells. Total RNA was collected and QC'd; mRNA was enriched using Poly-T oligo attached to magnetic beads and Illumina libraries were prepared and sequenced on Novaseq6000 (PE 150). Library construction and sequencing were performed by Novogene Corporation, Inc according to standard workflow. Results: Using a custom built pipeline in PartekFlow, the reads were aligned to human (build hg38) genome with the STAR aligner 2.6.1d and low quality alignments were filtered. About 25 million sequence reads per sample were mapped to the human genome. We identified 5714 transcripts/sample and low expression genes with counts less than 10 were filtered and the raw read counts were normalized using TMM model with an offset of 1 to avoid zero counts. Unsupervised hierarchical clustering was performed on the top 2000 genes with highest variance Conclusions: The study identifies gene networks involved in cytarabine response that are deregulated by presence of mutant DNMT3A. Overall design: RNA profiles of U2OS overexpression cell lines (DNMT3A WT, DNMT3A R882C, and MigR1 empty vector control), treated with 10uM cytarabine for 24 hours, or vehicle control