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===Future=== *try experiment where adding in Rnase H, NPA primer and polymerase into the reaction at once. Then incubate at 37C for 5 minutes, and then incuvbate the reactions at suggested temperatures for the polymerases for the suggested amounts of time. Currently some protocols use this methodto reduce the number of steps. *'''UPDATE AUG 7, 2013''' *Stumbled upon a paper in genome research [http://genome.cshlp.org/content/22/1/134.long Transposase mediated construction of RNA-seq libraries] that describes generating sequencing libraries though using nextera transposons, for polyA selected RNA. some considerations can be taken from the paper. *1. Random hexamer single strand DNA synthesis is basically the same. *2. Second strand synthesis: can use this protocol instead, but seems negligible since for my method a RNA is wanted to be highly degraded, primer is added, and extension is wanted from that primer (sss_NPA_prmr) 20 uL sample (all sample) 16 uL water 5 uL 10X synthesis strand buffer ((500 mM Tris-HCl at pH 7.8, 50 mM MgCl2, 10 mM DTT)) 3 uL 10 mM dNTPs 1 uL Rnase H 5 uL DNA polymerase I (10 u/Ul) Reactions were incubated at 16C for 2.5 hours. (Rnase H nicks, and DNAP extends DNA from the nicks) *3. Mention in methods that using 75 ng or less of double-stranded cDNA in the transposition reaction is important and larger amounts of cDNA results in large >1000 bp insert sizes. Can take this into consideration when running assay again. Can titrate amounts of RNA used for transposition and see what gives better sequencing results.
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