Ribo-seq of MH63: panicle
For the Ribo-seq, all samples were first treated with specific lysis buffer with cycloheximide (50mg/mL) to acquire the lysate, and concentration of the lysate was measured by the NanoDrop? 2000 Spectrophotometer. To digest the RNA other than RPFs, sample lysate was treated with unspecific endoribonuclease RNase I. Isolation of monosomes was performed by size-exclusion chromatography with MicroSpin S-400 HR columns. The RNA samples were then treated with rRNA depletion kit to deplete the samples of as much rRNA contamination as possible before PAGE purification of the relatively short (20~38 nt) RPFs. Following PAGE purification, the both ends of RPF were phosphorylated and ligated with 5 and 3 adapters respectively. Then the fragments were reversely transcribed to the cDNAs and amplified by PCR.