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Transcriptome sequencing

New sequencing technology has made the study of the transcriptome (the complete set of transcripts present in a biological sample) an integral part of functional genomic approaches. RNA-Seq makes it possible not only to generate a catalogue of expressed genes but also to quantify gene expression and determine the structure of each transcript. RNA-Seq data is also used for annotation of de nova genomes. Transcriptome analysis, due to its high resolution, allows identification of translation start sites and alternative splice sites.

Currently, a wide variety of kits are available for the preparation of RNA-Seq samples, thus allowing transcriptome analysis of eukaryote and prokaryote organisms. To obtain reliable high quality information from an RNA-Seq experiment, it is crucial that to obtain a high enough coverage rate to allow detection of the greatest number of transcripts, including those that are less well-expressed. This may necessitate a preliminary step of transcript enrichment, except for tRNA and rRNA transcripts. This is the case, for example, with the TruSeq Illumina kit protocol which is based on heat fragmentation of the RNA prior to synthesising the cDNA by random priming. In this case, polyadenylated RNA selection or ribosomal RNA depletion is performed prior to this step, depending on whether the study being undertaken involves eukaryote or prokaryote species. Other kits on the market are, in contrast, based on synthesis by oligo(d)T priming (e.g. SMARTer from Clontech, which is limited to the study of polyadenylated RNA), or are based on a set of selective primers used for synthesis that promotes messenger RNA (Nugen). Following synthesis, the cDNAs are used to prepare a standard Illumina library for single strand or paired-end sequencing (Figure 1)..

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ARN total : Total RNA
Enrichissement en ARNm ou déplétion en ARNr : mRNA enrichment or rRNA depletion
Fragmentation ARNm (chaleur et cations) : mRNA fragmentation (heat and cations)
Synthese d’ADNc par random primming : cDNA synthesis by random priming
Ligation des adaptateurs : Adapter ligation
ARNm (avec ou sans polyA) : mRNA (with or without polyA)
ARNm : mRNA
Random hexamers : Random hexamers
ARNm avec polyA : mRNA with polyA
Oligo(d)T : Oligo(d)T 
Synthèse d’ADNc par oligo(d)T primming : cDNA synthesis using oligo(d)T priming 
Fragmentation ADNc (mécanique) : Mechanical fragmentation of cDNA
Ligation des adaptateurs : Adapter ligation
Fig.1 Schéma de l’obtention des librairies non orientées d’ARN :
Fig 1 : Workflow diagram depicting non-directional RNA library preparation

Some kits allow preparation of libraries for directional RNA-Seq, which enables identification of the mRNA coding strand. This information is very valuable for transcriptome annotation and for identification of different isoforms.

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