Ribo-seq of Arabidopsis: leaf

Ribosomal profiling analysis: Polysomes were isolated and treated with RNase I (NEB, USA) and DNase I (NEB, USA) to degrade DNA and unprotected RNA. Digested RNAs were separated by size exclusion columns (illustra MicroSpin S-400 HR Columns; GE Healthcare) and SDS was added to denature RNA-associated proteins. RNAs with a size greater than 17 nt was isolated with RNA Clean and Concentrator-25 kit (Zymo Research, USA). rRNA was removed and RNAs were further purified using magnetic beads (Vazyme, China). Ribo-seq libraries were prepared with NEBNext Multiple Small RNA Library Prep Set for Illumina (NEB, USA) and PCR products with DNA fragments at 140-160 bp (representing insert sizes of 18-35 bp) were enriched to generate a cDNA library and sequenced with Illumina HiSeqTM .10 (Gene Denovo Biotechnology Co., China). Reads counts in the open reading frame (ORF) of coding genes was calculated by software RSEM, and gene expression level was normalized by using RPKM method.