RNA structure is a primary determinant of its function, and methods that merge 
chemical probing with next generation sequencing have created breakthroughs in 
the throughput and scale of RNA structure characterization. However, little work 
has been done to examine the effects of library preparation and sequencing on 
the measured chemical probe reactivities that encode RNA structural information. 
Here, we present the first analysis and optimization of these effects for 
selective 2'-hydroxyl acylation analyzed by primer extension sequencing 
(SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly 
reproducible reactivity data over a wide range of RNA structural contexts with 
no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a 
'universal' method that can obtain reactivity information for every nucleotide 
of an RNA without having to use or introduce a specific reverse transcriptase 
priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible, 
with reactivity data that can be used as constraints in RNA folding algorithms 
to predict structures on par with those generated using data from other SHAPE 
methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding 
the RNA sequence-structure relationship at the heart of some of life's most 
fundamental processes.