m6A RNA Immunoprecipitation sequencing (MeRIP-seq) and data analysis

MeRIP-seq was performed as described before[22] with modifications. Briefly, total RNA was extracted from 14 day-old Arabidopsis thaliana seedlings using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) and treated with DNAase I (RapidOut DNA Removal Kit, Thermo Scientific). 300 μg of total RNA was mixed with 10×Fragmentation buffer (1 M Tris-HCl pH = 7.0, 1 M ZnCl2) and placed at 94°C for 5 min then snap cooled on ice for 5 minutes. The volume of fragmented RNA was then adjusted to 755 μl with RNase-free water. Next, 10 μL RNasin Plus RNase inhibitor (Promega, cat. no. N2611), 10 μL Ribonucleoside vanadyl complexes (RVC; 200 mM; Sigma-Aldrich, cat. no. R3380), 200 μL 5×IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% (vol/vol) Igepal CA-630), and 25 μL of m6A antibody (Synaptic Systems, cat. no. 202 003) were added to samples and samples were rotated at 4°C for 2 hours. After 2 hours, pre-blocked Protein A Dynabeads (Thermo Fisher, 1001D) was added to the RNA samples and rotated for an additional 2 hours at 4°C. After 2 hours, Dynabeads were pelleted using a magnetic stand and washed three times with 1 mL 1×IP buffer. RNA was eluted from Dynabeads by adding 98 μL elution buffer (20 mM Tris-HCl pH 7.5, 300 mM sodium acetate, 2 mM EDTA, 0.25% SDS), 2 μL of proteinase K (Thermo Fisher, AM2546) and then shaking for 1 hour at 37°C. All samples were precipitated using 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol and kept at -80°C overnight. Libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs, E7300S) according to the manufacturer’s instructions. Novogene (Beijing) performed sequencing on an Illumina HiSeq4000 instrument. About 3.0 Gb high-quality 150-bp paired-end reads were generated from each library. FastQC (Galaxy Version 0.72 + galaxy1) was initially run to assess the overall quality of all sample reads. Poor quality bases and adapters were filtered out using Trim Galore (Galaxy Version 0.6.3). The quality-filtered reads were aligned to the A. thaliana reference genome using HISAT2 (Version 2.1.0 + Galaxy4) with default parameters. To identify regions in which m6A modifications occurred, MACS [23] was used to call peaks on aligned files. The peaks showing an absolute value of log2 FC (fold change; fio1 mutant / WT) ≥ 0.5 and RPM ≥ 5 were considered as differentially modified peaks. MeRIPseq data generated in this study has been deposited in NCBI’s Gene Expression Omnibus under GEO Series accession no. {"type":"entrez-geo","attrs":{"text":"GSE171928","term_id":"171928"}}GSE171928.