This repo is for learning purposes only!! ideally you would want to use a RNA analysis analysis like Nextflow rnaseq pipeline. Together they can be used to do full RNA-seq Analysis as seen as in Odor-evoked Transcriptomes
RNA sequencing (RNA-seq) is a high-throughput technique used to analyze the entire transcriptome, which is the complete set of RNA molecules in a cell. It involves isolating RNA, converting it into complementary DNA (cDNA), and sequencing it to obtain detailed data on gene expression levels, splicing patterns, and the presence of non-coding RNAs. RNA-seq enables the identification of novel transcripts, alternative splicing events, and gene mutations, providing insights into cellular processes and disease mechanisms. Unlike microarrays, RNA-seq does not require predefined probes, making it highly sensitive and capable of detecting low-abundance transcripts. It has become a powerful tool for comparative genomics, functional genomics, and personalized medicine.
Modulation of odorant receptors mRNA induced by prolonged odor exposure is highly correlated with ligand-receptor interactions in Drosophila as well as mammals of the Muridae family. If this response feature is conserved in other organisms, this presents an intriguing initial screening tool when searching for novel receptor-ligand interactions in species with predominantly orphan olfactory receptors. We demonstrate that mRNA modulation in response to 1-octen-3-ol odor exposure occurs in a time- and concentration-dependent manner in Aedes aegypti mosquitoes. To investigate gene expression patterns at a global level, we generated an odor-evoked transcriptome associated with 1-octen-3-ol odor exposure. Transcriptomic data revealed that ORs and OBPs were transcriptionally responsive whereas other chemosensory gene families showed little to no differential expression. Alongside chemosensory gene expression changes, transcriptomic analysis found that prolonged exposure to 1-octen-3-ol modulated xenobiotic response genes, primarily members of the cytochrome P450, insect cuticle proteins, and glucuronosyltransferases families. Together, these findings suggest that mRNA transcriptional modulation of olfactory receptors caused by prolonged odor exposure is pervasive across taxa and can be accompanied by the activation of xenobiotic responses.
RNA library preparation and sequencing were conducted at GENEWIZ, LLC (South Plainfield, NJ, USA). RNA sample integrity and quantification was assessed using TapeStation (Agilent Technologies, Palo Alto, CA, USA) and Qubit 2.0 Fluorometer respectfully (Invitrogen, Carlsbad, CA, USA). NEBNext Ultra RNA Library Prep Kit was then used to prepare sequencing libraries according to manufacturer’s instructions (NEB, Ipswich, MA, USA). Oligo(dT) beads were used to enrich mRNA from 200 ng total RNA prior to fragmenting for 15 minutes at 94°C. After fragmentation cDNA first and second strand were synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) as well as quantitative PCR (KAPA Biosystems, Wilmington, MA, USA) were used to validate and quantify the sequencing libraries. Libraries were sequenced across three Illumina HiSeq lanes with 2x150 bp Paired End chemistry according to manufacturer’s instructions. HiSeq Control Software (HCS) were used to conduct base calling and Image analysis and Illumina HiSeq Raw sequence data (.bcl files) were converted into fastq files and de-multiplexed using Illumina software. Index sequence identification allowed for one mismatch
