In general, the first step in library preparation of mRNA is fragmentation. Fragments shorter than the desired size are then removed using ethanol precipitation or spin columns. Random hexamer primers are then added and will hybridize to complementary RNA sequences. In the presence of a reverse transcriptase and deoxynucleotide triphosphates (dNTPs) (A, T, G, C), the mRNA becomes the template for the synthesis of the first complementary strand of DNA (first strand synthesis). After digestion of the mRNA template, the single-stranded cDNA (sscDNA) is the template for the second strand synthesis. When hydrophobic nucleic acids in the sscDNA are no longer hybridized to the RNA template, the cDNA strand tends to loop around itself to avoid interaction with the aqueous environment. This hairpin loop at the 3’ end generally acts as the primer site for the DNA polymerase. DNA polymerase moves along the sscDNA to transcribe the complementary sequence for the sscDNA template.
cDNA libraries synthesized from fragmented mRNA require end repair to ensure that each molecule is free of overhangs and has 5’ phosphates and 3’ hydroxyls. Libraries to be used for blunt end cloning or blunt-ended adaptor ligation (including libraries for Ion Torrent™ or SOLiD™ sequencing) can be directly incorporated into a cDNA-adaptor construct. For all Illumina® libraries and some libraries intended for the 454 platform, incorporation of a non-templated deoxyadenosine 5’-monophosphate (dAMP) onto the 3’ end of blunted DNA fragments, known as dA-tailing, is necessary. dA-tails prevent concatamer formation during downstream ligation steps, and enables DNA fragments to be ligated to adaptors or cloning vectors with complementary dT-overhangs. The desired adaptor ligated DNA size for Illumia and SOLiD platforms can be selected via gel electrophoresis before amplification with polymerase chain reaction (PCR).
mRNA Library Construction Workflow for Illumina
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