Hiseq novaseq instrument

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.

Total RNA was extracted from the cochleae using TRIzol Reagent/RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantified with an Agilent NanoDrop (ThermoFisher Scientific). A total of 1 μg RNA from each sample was used for the library preparations for next generation sequencing, and these were constructed according to the manufacturer’s protocol. The cDNA was synthesized by ProtoScript II Reverse Transcriptase and Strand Synthesis Enzyme Mix, and then treated with End Prep Enzyme Mix to repair both ends and to add tail-A, which followed by a ligation to add adaptors. Each sample was then amplified by PCR using P5 and P7 primers, with both primers carrying sequences that can anneal with the flow cell to perform bridge PCR and carrying indexes that allow for multiplexing. The libraries were then multiplexed and loaded onto an Illumina HiSeq/Novaseq instrument according to the manufacturer’s instructions (Illumina, San Diego, CA). The expression levels of all mapped genes were estimated by stringtie (v2.0), and the gene expression levels were determined by fragments per kilobase of transcript per million fragments mapped. Differential expression analysis was based on the DESeq2 Bioconductor package, and p values of less than 0.05 indicated DEGs. The analysis of pathways was based on the Kyoto Encyclopedia of Genes and Genomes database.