Ribo-seq of A. thaliana: whole leaf RCNMV infected 5dpi Rep 8
Plants were grown for 1 month in a growth chamber. At 1 month old, plants were mock innoculated with sap from N. benthamiana. After 5 days post innoculation, 2-4 of the youngest leaves were cut and flash frozen in liquid nitrogen. Leaves from ~18 plants were pooled together to form this sample. Samples were ground and lysed with a buffer containing cycloheximide. Ribo-seq samples were treated with RNase1 and ultracentrifuged over a sucrose cushion to isolate monosomes. Monosome pellets were treated with proteinase K, DNase, and Ribo-zero prior to size selection via gel extraction. Ends were repaired with T4 PNK enzyme and 3' and 5' 4N adenylated adapters were ligated on with the NEXTflex kit. Bead cleanup was performed after  adpater cleanup followed by cDNA amplification and gel size selection. Fragment sizes were verified on a Bioanalyzer, samples were pooled, then sequenced on a NovaSeq 6000 S1 flow cell for 100 cycles.