Agencourt ampure rnaclean xp beads

All RNA present in samples EBW034 and EBW436 was sequenced using Illumina RNA-seq next-generation sequencing (28 (link)). This method was chosen because (i) the sample volume was too limited to generate whole genomes using primer walking, and (ii) it avoids using HCV-specific primers that might bias which sequences are amplified. Sample processing was performed as described in reference 29 (link). Briefly, sequencing libraries were constructed from 100 ng of total RNA using the NEBNext mRNA sample prep kit 1 (New England BioLabs), according to the manufacturer's guidelines, with minor modifications. This procedure breaks the mRNA into fragments, generates cDNA from the mRNA with random primers, repairs the ends of the cDNA library and appends a d(A) tail, ligates adapters to the ends of the cDNA fragments, and uses PCR to enrich the adapter-ligated cDNA library, resulting in inserts with a median length of 200 nt. The remaining adapter dimers and mRNA were removed using Agencourt AMPure RNAClean XP beads (Beckman Coulter). Amplicons were quantified and assessed for quality using the Quant-IT Qubit double-stranded DNA (dsDNA) high-sensitivity assay (Invitrogen) and 1% E-gel (Invitrogen), respectively, and then sequenced on an Illumina HiSeq 2000, according to standard Illumina protocols, creating 100-nt paired-end reads.