title: Nucleotide-Resolution Profiling of RNA Recombination in the Encapsidated Genome of a Eukaryotic RNA Virus by Next-Generation Sequencing
design: 0.4 µg of RNA was prepared for Next-Generation Sequencing using a modified version of the Illumina protocol (http://www.illumina.com/applications/sequencing/rna.ilmn#strand_specific_rna_seq) where 12 cycles of PCR were performed and standard Truseq adapters and Truseq barcoded primers were used. A final size selection was performed by native agarose gel electrophoresis to yield a library of inserts approximately 200 bases in length suitable for 100 base single read sequencing. The library was extracted from the agarose gel using standard oligo purification columns. The prepared library was then loaded onto an Illumina HiSeq v3 single-read flowcell, standard cluster generation was performed on a Cbot and sequencing was performed for 100 bases of the insert and 7 bases of the index read using standard HiSeq sequencing reagents on an Illumina HiSeq 2000 instrument. Reads were processed using CASAVA 1.8.2 and demultiplexed based on index sequences.