Generation of cDNA Libraries: Methods and Protocols

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What is RNA-seq?

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It analyzes the transcriptome of gene expression patterns encoded within our RNA. Here, we look at why RNA-seq is useful, how the technique works, and the basic protocol which is commonly used today 1. Understanding the transcriptome is key if we are to connect the information on our genome with its functional protein expression. RNA-seq can tell us which genes are turned on in a cell, what their level of expression is, and at what times they are activated or shut off 2.

This allows scientists to more deeply understand the biology of a cell and assess changes that may indicate disease. This can give researchers vital information about the function of genes.

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For example, the transcriptome can highlight all the tissues in which a gene of unknown function is expressed, which might indicate what its role is. It also captures information about alternative splicing events Figure 1 , which produce different transcripts from one single gene sequence. These events would not be picked up by DNA sequencing.

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  • These reads must then be aligned back to the reference genome. Early RNA-seq techniques used Sanger sequencing technology, a technique that although innovative at the time, was also low-throughput, costly, and inaccurate. Adapters are then added to each end of the fragments.

    These adapters contain functional elements which permit sequencing; for example, the amplification element and the primary sequencing site. The cDNA library is then analyzed by NGS, producing short sequences which correspond to either one or both ends of the fragment. The depth to which the library is sequenced varies depending on techniques which the output data will be used for. The sequencing often follows either single-read or paired-end sequencing methods.

    A further choice must be made between strand-specific and non-strand-specific protocols. The former method means the information about which DNA strand was transcribed is retained. The value of extra information obtained from strand-specific protocols make them the favorable option.

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      Advertisement Hide. Front Matter Pages i-xiii. Complementary DNA Libraries. Pages Mikhail V. Matz, Naila O. Alieva, Alex Chenchik, Sergey Lukyanov.