title: GSM5049413: m6A-seq FTO mutant root input rep1; Oryza sativa; OTHER
library_construction_protocol: For RNA-seq, 15-day-old seedlings of the Nipp and FTO plant were used for sequencing. Before purified non-ribosomal RNA with Ribo-Zero rRNA Removal Kit (Illumina), ERCC RNA spike-in control (Ambion) was added to each sample (0.1 μl per 5 ug total RNA). Library preparation was performed by using the NEBNext® Ultra™ I Directional RNA Library Prep Kit for Illumina® (NEB #E7420S) according to the manufacturer's protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China). For m6A-seq, 1 g of 15-day-old seedlings of the Nipp, FTOmut and FTO plants from hydroponic experiments were used to isolate poly(A) RNA for m6A sequencing. 5 pg spike-ins for m6A seq mixture containing 3 diverse 20% m6A modified and 3 diverse non-m6A modified poly(A) spike-ins (see Supplementary Table 6) which synthesized in vitro used the MEGAscript ® Kit (AM1333, Thermo Fisher Scientific, USA) was added to the poly(A) RNA, followed by fragmented into ~100 nt using RNA Fragmentation Reagents (Ambion, USA) according to the manufacturer's protocol.  m6A-IP was performed using EpiMark N6-Methyladenosine Enrichment Kit following the manufacturer's protocol. Library preparation was performed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer's protocol. Sequencing was performed on an Illumina HiSeq 4000 machine in pair-end mode with 150 bp per read (Genewiz, China). For RNA-seq, library preparation was performed by using the NEBNext® Ultra™ I Directional RNA Library Prep Kit for Illumina® (NEB #E7420S) according to the manufacturer's protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China). For m6A-seq, library preparation was performed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB #E7760S) according to the manufacturer's protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China).