RNA-Seq of recombinant Mumps virus
Cell culture supernatant was treated with micrococcal nuclease (New England Biolabs), prior to RNA extraction.  Viral RNA was extracted using the QIAamp MinElute Virus spin kit (Qiagen) and eluted in 25 l of DEPC treated water.  This RNA was processed using the NEBNext Ultra RNA Library Prep kit for Illumina (New England Biolabs). 50 ng of RNA extracted from virus r88+JL(M/F/SH/HN) shown by qRT-PCR to contain at least 7.68E6 molecules of full-length recombinant viral RNA was mixed with 1.79E6, molecules of in vitro transcribed DVG Urabe930 RNA in the presence of fragmentation buffer.  This RNA mixture was chemically fragmented and reverse-transcribed into cDNAs.  Double strand cDNAs were adenylated at the 3 ends and individually indexed, followed by 12 cycles of amplification and then purified using Agencourt AMPure magentic beads (Beckman Coulter).  cDNA libraries were checked for size and quality using a BioAnalyser (Agilent Technologies) and subjected to paired-end sequencing (250 x 2 cycles) on an Illumina MiSeq