GSM9178872: TAg/Mettl3-depleted Liver rep2 (input); Mus musculus; RNA-Seq
Total RNA extraction was carried out using ISOGEN (NIPPON GENE, Tokyo, Japan), and the isolated RNA was fragmented to approximately 100 nt using ZnCl₂ (FUJIFILM Wako Pure Chemical, Osaka, Japan). RNA fragmentation was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Tokyo, Japan). About 1/20 of the fragmented RNA was reserved as the input control for subsequent RNA sequencing analysis. The remaining RNA was treated with an anti-m6A antibody (#202 003, Synaptic Systems GmbH, Göttingen, Germany) at 4 °C for 2 hours, followed by mixing with pre-washed Pierce™ Protein A/G Magnetic Beads (#10004D, Thermo-Fisher Scientific) in immunoprecipitation buffer at 4 °C for another 2 hours. The mixture underwent magnetic separation. m6A-containing RNA was eluted from the complex using 6.7 mM N6-methyladenosine 5'-monophosphate sodium salt (#M2780, Sigma-Aldrich) at 4 °C for 2 hours, and the methylated RNA was subsequently purified for MeRIP-seq analysis using the RNeasy Mini Kit (QIAGEN, Tokyo, Japan). RNA libraries were prepared following the manufacturer's protocol with the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA), with the exception of utilizing oligo-dT beads.
GEO
GSM9178872_r1