Editing Transcriptome (section)

===RNA sequencing===
{{Main|RNA-Seq}}
RNA sequencing is a [[next-generation sequencing]] technology; as such it requires only a small amount of RNA and no previous knowledge of the genome.<ref name="etymology" /> It allows for both qualitative and quantitative analysis of RNA transcripts, the former allowing discovery of new transcripts and the latter a measure of relative quantities for transcripts in a sample.<ref name="cellerino12" />

The three main steps of sequencing transcriptomes of any biological samples include RNA purification, the synthesis of an RNA or cDNA library and sequencing the library.<ref name="cellerino12">{{Harvnb|Cellerino|Sanguanini|2018|p=12}}</ref> The RNA purification process is different for short and long RNAs.<ref name="cellerino12" /> This step is usually followed by an assessment of RNA quality, with the purpose of avoiding contaminants such as DNA or technical contaminants related to sample processing. RNA quality is measured using UV spectrometry with an absorbance peak of 260&nbsp;nm.<ref name="cellerino13">{{Harvnb|Cellerino|Sanguanini|2018|p=13}}</ref> RNA integrity can also be analyzed quantitatively comparing the ratio and intensity of [[28S RNA]] to [[18S RNA]] reported in the RNA Integrity Number (RIN) score.<ref name="cellerino13" /> Since mRNA is the species of interest and it represents only 3% of its total content, the RNA sample should be treated to remove rRNA and tRNA and tissue-specific RNA transcripts.<ref name="cellerino13" />

The step of library preparation with the aim of producing short cDNA fragments, begins with RNA fragmentation to transcripts in length between 50 and 300 [[base pair]]s. Fragmentation can be enzymatic (RNA [[endonuclease]]s), chemical (trismagnesium salt buffer, [[Hydrolysis|chemical hydrolysis]]) or mechanical ([[sonication]], nebulisation).<ref name="cellerino18">{{Harvnb|Cellerino|Sanguanini|2018|p=18}}</ref> [[Reverse transcription]] is used to convert the RNA templates into cDNA and three priming methods can be used to achieve it, including oligo-DT, using random primers or ligating special adaptor oligos.