Editing BioMicroCenter:RNAseq (section)

== Additional Chemistries Available in the BioMicro Center ==
=== Strand Specific Sequencing ===
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 |For samples with high amounts of input, we can modify the chemistry of cDNA creation to allow detecting the strand of the RNA using the dUTP 2nd strand marking protocol that preformed best in J. Levin et al 2010. In this method, actinomycin D is added to the 1st strand synthesis to prevent reinitiation of the RT-PCR enzymes. Then, dUTP is substituted in for dTTP in the second strand, allowing a clear distinction between the forward and reverse strands. After library construction, but before amplification, the dUTP containing strand is degraded and only the reverse strand (template strand) remains. This method is highly efficient but requires significant amount of RNA as the actinomycin causes a significant reduction in 1st strand yield. 
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 |[[Image:BMC_strandSpecific.gif|thumb|600px|left|Sample Data from Strand Specific RNAseq]]
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=== [[BioMicroCenter:PippinPrep|Size Selection]] ===
For some applications of RNAseq, such as splice choice determination, having a precise knowledge of the insert size is critical. While the [[BioMicroCenter:SPRI-Works|SPRIworks]] does provide some size selection (typically restricting fragments to between 150 and 350bp), this can be too wide for some methodologies. In these cases, after libraries are amplified, they can be run on the [[BioMicroCenter:PippinPrep|Sage BluePippin]] (either singly or pooled). Here the size distribution can be much tighter, with most of the DNA fragments being within a 50nt range.