title: RNA-seq, 4 h post VACV infection
design: Ribosome foot printing was carried out as described elsewhere with minor modification (16). Briefly, HeLa cells pretreated with translational inhibitors were lysed, treated with DNase (Ambion) and the lysate was clarified. A portion of the lysate was removed for mRNA isolation and subsequent mRNA-Seq analysis. For mRNA-Seq, the isolated mRNA was fragmented by digestion with RNase III (New England Biolab). The fragmented RNAs were resolved by denaturing polyacrylamide gel electrophoresis  in a 15% gel with urea and fragments between 50 and 80 nucleotides (nt) were extracted. The remainder of the lysate was digested with RNase I (Ambion) after which the ribosomes were pelleted by ultracentrifugation. The ribosome-protected RNA fragments were isolated and separated by electrophoresis as described above except that fragments between 28 and 34 nt were extracted. The purified RNA fragments were then subjected to a series of enzyme treatments and molecular manipulations as described (16) to generate libraries for deep sequencing. The final libraries were purified from primers and unligated adaptors by electrophoresis and size selection of 150-200 bp fragments in a 4% agarose gel