GSM8721730: liver.input_B12_2; Mus musculus; OTHER
After 14 days of treatment, the mice were euthanized, brain, liver, and testis tissues were collected for subsequent library construction experiments, while the remaining tissues were stored in a -80°C freezer. The m6A MeRIP-seq libraries were constructed using the m6A-seq2 protocol. Poly-A selected mRNA (1.8 μg per sample) was extracted using Oligo d(T)25 Magnetic Beads and fragmented to approximately 150 nucleotides (nt) using RNA fragmentation buffer. Fragmented RNA was DNase digestion, dephosphorylation, and purification using T4 PNK, TURBO DNase, FastAP (NEB, Thermo Scientific), and VAHTS RNA Clean Beads (Vazyme Biotech). A 3′ RNA barcode adapter was ligated using T4 RNA Ligase (NEB), and barcoded RNA samples were pooled, reserving 10% as input control. For m6A immunoprecipitation, RNA was heat-denatured and incubated with anti-m6A antibody (NEB)-coated Protein A/G magnetic beads (Thermo Scientific) in IPP buffer. Beads were washed with low- and high-salt IPP buffers to remove nonspecific binding. Enriched RNA was eluted with RLT buffer (Qiagen), purified, and reverse-transcribed to cDNA using SuperScript III (Thermo Scientific) with a specific rTd primer. cDNA synthesis was followed by RNA hydrolysis and Illumina 5′ adapter ligation. PCR enrichment was performed using the KAPA HiFi PCR Kit, with a universal forward primer and barcode-containing reverse primer. The amplified libraries were purified, quantified using a Qubit fluorometer, and assessed for quality using an Agilent Bioanalyzer 4200 TapeStation. RNA-seq and MeRIP-seq were conducted on the NovaSeq 6000 platform (Illumina) to generate paired-end 150 bp reads, producing ~10 GB of raw data per library.