Nebnext ultra 2 directional rna library prep kit

For D. mel, frog, zebrafish, H1, and H9 RNA samples, RNA BS-seq library construction was performed as we previously described^{29 (link)}. In brief, total RNA was isolated with TRIzol reagent and Direct-zol RNA MiniPrep kit. Polyadenylated RNA was separated from total RNA using Oligo dT Magnetic Beads (Vazyme). 100-1000 ng of polyadenylated RNA was converted using the EZ RNA methylation kit (Zymo Research) with a modified high-stringency conversion condition. The converted RNA was fragmented into 150–200 nt fragments by incubation at 94 °C for 8 min in fragmentation buffer (NEBNext Ultra II Directional RNA Library Prep Kit, NEB). The fragmented RNA was then used for library construction, following the manufacturer’s protocol (NEBNext Ultra II Directional RNA Library Prep Kit, NEB).
For human and mouse oocyte and embryo samples, due to the limited amount of total RNA obtained, RNA BS-seq was performed using rRNA-depleted RNA. rRNA was depleted by Ribominus Eukaryote kit v2 (Thermo, A15020) and concentrated by ethanol precipitation from 100 ng total RNA. Next, the RNA was converted and used for library construction as described above.
Libraries were sequenced on Hiseq X10 to produce paired-end 150 bp reads. All libraries were summarized in Supplementary Data 1.

MeRIP-seq maps m⁶A-methylated RNA. In this method, an m⁶A-specific antibody was used to immunoprecipitate RNA. Total RNAs were extracted from LECs. The RNAs were reverse-transcribed to cDNA and sequenced. Deep sequencing provided high-resolution reads of m⁶A-methylated RNA. The rRNAs were removed from the total RNA with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Ipswich, MA, USA). RNA libraries were constructed by using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). M⁶A RNA-Seq service was provided by Cloudseq Biotech Inc. (Shanghai, China). Briefly, m⁶A RNA immunoprecipitation was performed with the GenSeqTM m⁶A-MeRIP Kit (GenSeq Inc., Cyberjaya, Malaysia) by following the manufacturer's instructions. Both the input samples without immunoprecipitation and the m⁶A IP samples were used for RNA-seq library generation with NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). The library quality was evaluated with BioAnalyzer 2100 system (Agilent Technologies, Inc., Santa Clara, CA, USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired-end reads.

m⁶A RNA-Seq service was provided by Cloudseq Biotech Inc. (Shanghai, China). Briefly, m⁶A RNA immunoprecipitation was performed with the GenSeq m⁶A-MeRIP Kit (GenSeq Inc., China) by following the manufacturer’s instructions. Both the input samples without immunoprecipitation and the m⁶A IP samples were used for RNA-seq library generation with NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., United States). The library quality was evaluated with BioAnalyzer 2100 system (Agilent Technologies, Inc., United States). Library sequencing was performed on an Illumina Hiseq instrument with 150 bp paired-end reads. RNA high-throughput sequencing was performed by Cloud-Seq Biotech (Shanghai, China). Briefly, total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., MA, United States) following the manufacturer’s instructions. RNA libraries were constructed by using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., United States). Library sequencing was performed on an Illumina Hiseq instrument with 150 bp paired-end reads. Raw data of RNA-seq and m⁶A-seq have been uploaded to GEO database (accession number GSE181540)¹.