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Old 11-28-2011, 06:21 AM   #6
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Location: Purdue University, West Lafayette, Indiana

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Okay, we are starting to get into the details of RNAseq here. But most commonly RNAseq library protocols follow these steps:

(1) Ribodepletion (removing most of the ribosomal RNA that composes the majority of all total RNA preps.)
(2) RNA fragmentation. For Illumina this fragmentation will generate a peak around a few hundred nucleotides.
(3) First and second strand cDNA synthesis. Because of the RNA fragmentation step, the majority of cDNAs will be in the target range.
(4) ds cDNA end polishing, adapter ligation

After you ligate the adapters to the cDNA, then you have your library of amplicons. These are usually subjected to other manipulations, but eventually end up being diluted to low titres and amplified by cluster or emulsion PCR to create ~ 1um splotches of area on a flow cell surface with thousands or millions of copies of the same amplicon. Sequencing proceeds from a priming site in one of the adapters.

Why a few hundred bases instead of something close to the polymerase size limit?

First, your read length is only 50-100 nt or so. Reading from the very ends of full length cDNAs will only interrogate a percentage of your entire transcriptome. To read the entirety you either need very long read lengths, or you need to fragment your transcripts prior to sequencing them. Second, PCR is much more efficient at replicating smaller amplicons than larger ones. So by going shorter you get a signal boost for your template clusters or beads.

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