GSM8208846: zhx13; Sus scrofa; OTHER
The experiment was carried out according to the procedure previously reported. mRNA was isolated from total RNA using the Dynabeads™ mRNA DIRECT™ kit (Ambion, Cat NO. 61012). The purified mRNA was then fragmented by 30 cycles of ultrasound, with each cycle comprising 30 s on and 30 s off. The concentration of fragmented mRNA was determined using a QubitTM RNA HS assay kit (Invitrogen, Cat NO. Q32852). Next, 500 ng of mRNA was taken as the Input control for sequencing. A total of 950 ng of fragmented mRNA was incubated with 1 μL of m6A antibody (NEB, Cat. E1610S) at 4°C for 1 h. After incubation, the mixture was washed twice in reaction buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40), twice in low salt buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40), and twice in high salt buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% NP-40). Bound mRNA was eluted using 100 μL of elution buffer (QIAGEN, Cat. 1015762). Library preparation was conducted using the SMARTer Stranded Total RNA-Seq Kit version 2-Pico Input Mammalian (Takara) per the manufacturer's instructions. Briefly, the immunoprecipitated mRNA fragments and an equal amount of input control were subjected to cDNA synthesis using the kit components SMART Pico Oligos Mix v2, 5X First-Strand Buffer, SMART TSO Mix v2, and SMARTScribe Reverse Transcriptase. The cDNA was then ligated with Illumina adapters and indexes, followed by PCR amplification. The libraries were then purified using AMPure beads to remove excess primers and nucleotides. Ribosomal cDNA removal was performed using the ZapR v2 and R-Probes v2 reagents provided in the kit. The library fragments underwent a second round of PCR enrichment to amplify the libraries to sufficient quantity for sequencing. Before sequencing on an Illumina HiSeq4000, the libraries were analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Library preparation was performed using the SMARTer Stranded Total RNA-Seq Kit version 2-Pico Input Mammalian (Takara), according to the manufacturer's protocol. m6a-seq