To enable the investigation of RNA by Next Generation Sequencing, the RNA starting material has to be processed to create a sequencing library. This is carried out using a number of molecular biology processes and, in this case, a library preparation kit by Illumina.
Once a library has been made it can then go on to be sequenced on one of the Illumina sequencing platforms (see DNA Sequencing – MiSeq)
RNA libraries can be used to study transcriptome sequence, gene expression and alternative splicing.
How does it work?
This particular library prep is for use with poly-adenylated RNA (although user-isolated mRNA can also be used to start the protocol after mRNA enrichment). Once the mRNA is isolated, it is fragmented and cDNA is made of these fragments using reverse transcription. The resulting double-stranded DNA fragments are ligated to DNA adaptors. The DNA adaptors are known DNA sequences that allow the library fragments to anneal to the sequencing flowcell. A sequencing run can then be performed.
Other RNA library prep protocols are available but are costed on a per project basis.
Each library requires 0.1-1ug of total RNA with a RIN ideally above 7.
Dr Ian Hancox, 024 76 150 380
email i dot hancox at warwick dot ac dot uk.
Mrs Jeanette Selby, 024 76 5 75172
email j dot m dot selby at warwick dot ac dot uk.
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