Mrna seq library prep kit

OCI-AML3 cells were incubated with either 5 μmol/L HEL or 0.1% DMSO in triplicate for 48 h. Total RNA was isolated using an RNeasy kit (#74004; Qiagen, Hilden, Germany). RNA libraries were prepared from 1 μg of total RNA using an mRNA-Seq Library Prep Kit (#001.24; Lexogen, Wien, Austria). Total RNA and library quality were analyzed using a Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Samples were sequenced using an Illumina HiSeq/Novaseq instrument according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). RNA-seq reads were aligned to the human genome using Hisat2 (v2.0.1) and gene expression was determined using HTSeq (v0.6.1). Differential expression was analyzed using the DESeq2 Bioconductor package (https://bioconductor.org/packages/release/bioc/html/DESeq2.html) with the parameters set to default. GOSeq (v1.34.1) was used to identify gene ontology (GO) terms from an annotated list of enriched genes with a significance of P < 0.05. Gene set enrichment analysis (GSEA) was performed using the Java GSEA Desktop Application with default parameters and the C5 ontology gene sets, which are provided as part of MSigDB V6.2 (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp) as default screening gene sets.