title: RNA-seq of influenza virus infection in Cavia porcellus and Mustela furo
design: Respiratory tract tissues were homogenized in Qiagen RNeasy RLT buffer, and RNA was isolated with the RNeasy kit according to the manufacturers protocol.  Whole blood was collected in PAXgene blood RNA tubes and RNA isolated with the PAXgene Blood RNA kit as per protocol. On-column DNA digestion was performed during RNA isolation.  RNA quality was assessed by Agilent Bioanalyzer, and RNA quantity by Qubit Fluorometer. Using the NEBNext Poly(A) mRNA kit with magnetic isolation module (New England Biolabs), poly(A) mRNA was selected from 1 g of total RNA and fragmented for 20 min at 94C.  First- and second-strand cDNA synthesis were performed with the NEBNext RNA First Strand and Ultra Directional RNA Second Strand Synthesis Modules (New England Biolabs), with second-strand reaction mix containing dUTP, according to the protocols.  cDNA fragments of ~300 nt were selected and quality assessed by Agilent Bioanalyzer.  Fragmented cDNA ends were repaired by addition of adenosine base at 3 ends, and adapters were ligated according to protocol with the TruSeq DNA PCR-Free Low Throughput Library Prep Kit (Illumina).  End-product size was verified by Agilent Bioanalyzer DNA nano chip. The adapter-ligated cDNA was amplified by PCR for 15 cycles with the NEBNext High Fidelity 2X PCR master mix (New England Biolabs), cleaned and sent for size analysis, and then cDNA libraries were multiplexed and sequenced on Illumina HiSeq 2500 and HiSeq 4000 sequencers.