Modern Approaches in Genetic Toxicology Research: Insights from Next-Generation-Based RNA Sequencing.

Transcriptomics-based approaches, particularly in human-derived cell systems, are emerging as key New Approach Methods (NAMs). These methods offer high-throughput, high-content, and human-relevant analyses of DNA damage. Transcriptomic profiling, which tracks gene expression changes resulting from chemical exposure, provides insights into both adverse and adaptive biological responses. This chapter gives an insight into the methodology (Wet Lab and Dry Lab) to be followed for the RNA sequencing and RNA data analysis, which can be used for identifying the biomarkers/signatures for the genotoxicity-based studies. RNA sequencing is a useful next-generation sequencing (NGS) technique, which has wide variety of applications which include studying altered gene expression, alternative spliced transcripts, gene fusion, post-transcriptional modifications, and mutations/single nucleotide polymorphisms (SNPs). Not only mRNA, RNA-Seq is used to study small RNAs as well as long non-coding RNA profiles in a cell or tissues. The basic steps for RNA-Seq include RNA isolation, cDNA synthesis, attachment of adapters, cDNA library preparation, followed by sequencing and bioinformatic data analysis workflow. The depth to which the library is sequenced depends on the usage of the output data. The sequencing usually follows either single-read or paired-end sequencing methods. Once the data is generated, it can be processed using R-programming, which is briefly discussed here as well.