Polyattract mrna isolation system 3

Total RNA of adult earthworms was extracted using TRIzol reagent^{94 (link)}, while mRNA was isolated using the kit PolyATtract mRNA Isolation System III (Promega, Madison, WI, USA). A total of 1.5 μg RNA per sample was used as the input material for preparation of individual RNA libraries. Libraries were constructed using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (NEB, Beverly, MA, USA) following the manufacturer’s recommendations. Polymerase chain reaction (PCR) products with insert sizes of 150–200 bp were purified using the AMPure XP system and library quality was assessed with the Agilent Bioanalyzer 2100 system. Finally, the library was clustered using the cBot Cluster Generation System and sequenced on the Illumina HiSeq 2000 sequencing platform to generate paired-end reads.

Total RNA was extracted from the cells using Trizol reagent (TAKARA). mRNA was isolated and purified using Poly Attract mRNA Isolation System III with Magenetic Stand (Promega) following the manufacturer’s instructions. For m⁶A dot blot, mRNA was hybridized onto the Hybond-N + membrane (GE Healthcare). After crosslinking at 80 °C for 30 min, the membrane was blocked with 5% non-fat milk (Biorad) for 1 h, incubated with rabbit anti-m⁶A antibody (1:1000, Synaptic Systems, cat. No. 202003) at 4 °C overnight. Then the membrane was incubated with HRP-conjugated mouse anti-rabbit IgG (1:3000, Santa,sc-2357) at room temperature for 2 h. After being incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore), the immunocomplex was photographed using the ECL imaging system (Bio-Rad). Finally, the membrane was stained with 0.02% methylene blue to eliminate the difference in mRNA amount. Relative m⁶A level was quantified via gray intensity analysis using Image J.

For m⁶A immunoprecipitation, the procedure was modified from the previously reported methods [15 (link)]. In brief, total RNA was extracted using TRIzol reagent followed by purification using PolyATtract^® mRNA Isolation System III (Promega). Subsequently, purified mRNAs were digested using DNase I and then fragmented into roughly 100-nt fragments by incubation for 15 min at 70°C in fragmentation buffer (10 mM Tris-HCl, pH 7.0, 10 mM ZnCl₂). 500 ng mRNA was saved as input control for RNA-seq. Five μg fragmented mRNA was incubated with 12 μg anti-m⁶A antibody (Synaptic Systems) in 1 × IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Igepal CA-630) for 2 h at 4°C. At the same time, recombinant protein A bead (Invitrogen) was washed twice followed by incubation in 1 × IP buffer with 0.5 mg/ml BSA on a rotating wheel for 2 h at 4°C. The m⁶A-IP mixture was then incubated with protein A beads for additional 2 h at 4°C on a rotating wheel. After washing three times with IP buffer, bound mRNA was eluted using 100 μl elution buffer (6.7 mM N⁶-Methyladenosine-5’-monophosphate sodium salt in IP buffer) followed by ethanol and sodium acetate precipitation. Immunoprecipitated RNA fragments and comparable amounts of input were subjected to first-strand cDNA synthesis. Sequencing was performed on Illumina HiSeq2500 according to the manufacuture’s instructions.