Hiseq2000 2500 machine

RNA extraction used enrichment with magnetic beads coated with oligo (dT) primer. The extracted mRNAs were broken randomly into short fragments using fragmentation buffer, and the first strand cDNA was synthesized as random hexamers using a fragment of mRNA as a template. Subsequently, buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand cDNA. Then we used AMPure XP beads to double-purify products, T4 DNA polymerase and Klenow DNA polymerase to fix the sticky end of the DNA to the flat end. 3′-end added base A and the joint, then used AMPure XP beads (Agencourt Bioscience, Beverly, MA, USA) for fragment selection. Finally, qPCR amplification was used to obtain the final sequencing library. Sequencing was done by LC-Bio Technologies Co., Ltd. (Hangzhou, P. R. China) (http://www.lc-bio.com) using an Illumina HiSeq2000/2500 machine (Illumina, San Diego, CA, USA) for paired-end sequencing.

RNA samples were sequenced at the Centro Nacional de Análisis Genómico (Barcelona, Spain). Briefly, total RNA was assayed for quantity and quality using Qubit RNA HS Assay (Thermofisher Scientific, Waltham, MA, USA) and RNA 6000 Nano Assay on a Bioanalyzer 2100. The experimental protocol to construct stranded mRNA RNASeq libraries starting from the total RNA employed the TruSeqStranded mRNA LT Sample Prep Kit (Illumina Inc., Rev.E, October 2013, San Diego, CA, USA). The initial input was 0.5 ug of total RNA for each sample. The size and quality of each final library were validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay (Agilent, Santa Clara, CA, USA). Libraries were sequenced using TruSeq SBS Kit v3-HS in paired-end mode with read length 2 × 76 bp. Each sample was sequenced in a fraction of a sequencing lane on a HiSeq2000/2500 machine (Illumina, San Diego, CA, USA) following the manufacturer’s protocol, generating between 59 and 87 million paired-end reads per sample. Images analysis, base calling, and quality scoring of the run were performed using the manufacturer’s software Real-Time Analysis (RTA 1.13.48) and were followed by generation of FASTQ sequence files with CASAVA 1.8. For this analysis, the two biological replicates available for each MDV strain (RB-1B and CVI-988) were pooled together before analysis, to increase the overall read coverage.