title: GSM4086609: VACV infected RPF replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: Cells were lysed in polysome buffer. One portion of the lysate was used to prepare RPF mRNA, while the other portion of lysate was used to purify poly(A)+ mRNA. Poly(A)+ was enriched from total mRNA using magnetic oligo-dT DynaBeads and subjected to alkaline fragmentation. Fragmented mRNAs were size-selected on a denaturing 10% polyacrylamide-urea gel and the bands corresponding to 30–50 nucleotides were excised, eluted, and precipitated with isopropanol. For isolation of RPF mRNA, lysates were subjected to digestion with Rnase I. Monosomes were pelleted by ultracentrifugation. Ribosome protected fragments of mRNA were size-selected via gel purification. Fragmented total mRNA or size-selected RPF mRNA was dephosphorylated, ligated, and reverse-transcribed. The resulting cDNA was circularized, rRNA contamination was reduced by subtractive hybridization, and the resulting library was PCR amplified using indexed primers.