Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNAs were isolated from embryos at the indicated stages using TRIzol reagent (Thermo Fisher). The mRNAs were purified using a NEBNext Poly(A) mRNA Magnetic Isolation kit (NEB) according to the user manual. The MeRIP-Seq was performed by Cloudseq Biotech Inc. (Shanghai, China) . Briefly, 5 ug of fragmented mRNAs were incubated with 5 ug of anti m6A polyclonal antibody (millipore) in IP buffer (150 mM NaCl, 10 mM Tris–HCl, 0.1% NP-40, pH 7.4) for 2 h at 4°C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4◦C for an additional 2 h. Beads with captured RNA fragments were then immediately washed 3 times with 500 μl of ice-cold IP buffer. Bound mRNAs were eluted from the beads with N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IP buffer and then extracted with Trizol reagent (Thermo Fisher). To RNA-seq and RIP-seq, Illumina Truseq libraries were constructed according to the manufacturer's instructions and sequenced using NovaSeq platform. with paired end read length of 150 bp. RNA-seq library generation with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA). MeRIP-Seq Library sequencing was performed on an illumina Hiseq instrument with 150bp paired-end reads.