ASALV small RNA sequencing in U4.4 cells

U4.4 cells were cultured as described previously [21 (link)], and were seeded at a density of 2×10⁶ cells per well in six-well plates and infected the next day with ASALV at an m.o.i. of 0.1. The cells were harvested for total RNA isolation at 72 hpi in RNA-Solv reagent (Omega Biotek R630-02). Small RNA libraries were prepared from 1 µg of total RNA using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina (NEB E7560S). The amplified libraries were size selected on a 6 % acrylamide/1× TBE gel and quantified using the Agilent 2100 Bioanalyzer System, and pooled libraries were sequenced on an Illumina HiSeq4000 machine by Plateforme GenomEast. Small RNA reads were mapped to the ASALV genome (ASALV-PP) using Bowtie (Galaxy Tool Version 1.1.2 [22 (link)]) allowing for one mismatch and the genome distribution was obtained by plotting the 5′ end position of the ASALV mapping reads on the viral genome. Reads were normalized to total library size (reads per million).