GSM7092454: ribotag blue fio1 replicate 2; Arabidopsis thaliana; OTHER
TRAP of polysomes was performed as described previously (Mustroph et al., 2009). Briefly, 1 mL of pulverized tissue was added to a 15 mL tube containing 5 mL of polysome extraction buffer (PEB: 200 mM Tris, pH 9.0, 200 mM KCl, 25 mM EGTA, 35 mM MgCl2, 1% PTE, 1 mM DTT, 1 mM PMSF, 100 μg/mL cycloheximide, 50 μg/mL chloramphenicol) and 1% detergent mix [20% (w/v) polyoxyethylene(23), 20% (v/v) Triton X-100, 20% (v/v) Octylphenyl-polyethylene glycol, 20% (v/v) Polyoxyethylene sorbitan monolaurate 20] and incubated until thawed on ice, before homogenized with a glass homogenizer. The homogenized mixture was incubated on ice for ten minutes before being centrifuged at 16,000g at 4 degrees Celsius for fifteen minutes. After centrifugation, the supernatant was transferred to a fresh tube and filtered through sterile Miracloth (Millipore) to create an extract that had been clarified. 10% of the total cleared extract was preserved to be used as an input for isolating RNA. Anti-FLAG M2 Protein beads (1.5 mL) that were pre-washed twice with wash buffer [WB; 200 mM Tris (pH 9.0), 200 mM KCl, 25 mM EGTA, 35 mM MgCl2, 1 mM DTT, 1 mM PMSF, 100 g/mL cycloheximide, 50 g/mL chloramphenicol] were added to the clar The binding of ribosome-mRNA complexes with a FLAG epitope tag was achieved by incubating them at 4°C for two hours while gently shaking the tubes. Remove the supernatant and carefully resuspend the beads in 6 mL WB for 2 minutes at 4°C while rocking. This procedure was performed twice more before the beads were resuspended in 1 mL WB. The beads were washed twice more with 1 mL WB, and then the supernatant was extracted. Added 300μL of WB with 200ng/μL of FLAG3 peptide and 20U/μL RNase inhibitor (Cat. # N8080119 Themo Fisher) and incubated for 30 mins at 4°C with shaking. Centrifuged and collected the supernatant for RNA purification. For meRIP, WT and mutant seedlings grown in the dark or in blue light (30 μmol m−2 s−1) for 6 days were used for m6A-seq.Total RNA was isolated using Direct-zol RNA Miniprep Kits (Zymo, Cat no. R2052). MeRIP-seq was performed as described previously (Wang et al., 2021).The libraries from two biological repeats for each sample were sequenced on Illumina Novaseq6000 instruments in pair-end mode with 100 bp per reads. The RNA was used for preparation of RNA-seq libraries with TruSeq RNA library prep kit (Illumina). The libraries from 3 biological repeats for each sample were sequenced on the Illumina HiSeq 2500 sequencing systems.