title: Ribosome-sequencing-mock-replicate2
design: Arabidopsis seedlings were grown on 1/2 Murashige and Skoog (MS) plates containing 0.8% agar and 1% sucrose at 22 C under 12/12 h light/dark cycles with 55% relative humidity. For elf18 treatment, Arabidopsis seedlings were grown on plates for 7 days and transferred to liquid 1/2 MS solution and grown for one more day (8-day-old) before treated with 10 mM elf18 or water for one hour. Arabidopsis seedlings treated with elf18 or water were collected, frozen in liquid nitrogen, and ground using the Genogrinder (SPEX SamplePrep). The ground tissue was homogenized in the polysome extraction buffer and subject to centrifuging to remove cell debris. The supernatant was then layered on top of a sucrose cushion and the ribosome pellet was collected after ultracentrifuge. The pellet was then washed with cold water and subject to RNase I (Ambion) digestion. The reaction was quenched by adding SUPERase-In (Invitrogen). Ribosome-bound RNA was purified using the RNA Clean & Concentrator kit (Zymo) and subject to PNK treatment (NEB) and size selection through gel (Invitrogen) extraction. The recovered RNA was then subject to library preparation using NEBNext Multiplex Small RNA Library Prep Kit with slight modifications. Specifically, after the reverse transcription, rRNA depletion was performed. This rDNA-depleted product was used as the template for PCR amplification and library preparation steps. Agilent 2100 Bioanalyzer was used for the sample quality control. The six libraries for Ribo-seq were pooled at equal amount of DNA and subject to the next-generation sequencing using Illumina NovaSeq (S2, full flow cell) with pair-end 50 bp.