GSM8247420: Normal_brain13_RNAseq; Homo sapiens; RNA-Seq
Total cellular RNAs were extracted by TRIzol (Thermo Fisher) or Zymo RNA miniprep kit (Zymo Research).  RNA-seq and MeRIP-seq were performed according to our previous report with some modifications (Xiong et al. 2021. PMID: 34108665). Total RNA (5 μg) was fragmented at 70 °C for 6 min in the fragmentation buffer (10 mM ZnCl2, 10 mM TrisHCl pH7.4), following precipitation the RNA were resuspended with ice-cold IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% NP-40, 20 U/mL Superase-In, 1× Protease Inhibitor) to a ﬁnal volume of 1050 μL. Of this, 50 μL of diluted RNA was utilized for RNA-seq (5% input for m6A IP) and the remaining 1000 μL was reserved for MeRIP-seq (Methylated RNA immunoprecipitation sequencing). To prepare anti-m6A antibody conjugated beads, 20 μL of Dynabeads Protein G beads (Thermo Fisher) was washed 3 times with 1ml IP buffer and then incubated with 1 μg of m6A antibody (Synaptic Systems, Cat 202-003) at room temperature for 30min. The conjugated anti-m6A-Dynabeads were washed in 1ml IP buffer 3 times. Subsequently, the RNA sample was mixed with the beads and rotated at 4 °C for 3 hours. The beads were then washed in the IP buffer 5 times. The immunoprecipitated RNAs were extracted from the beads by TRIzol-LS (Thermo Fisher). The extracted RNA was subjected to the next generation sequencing library preparation for RNA-seq and MeRIP-seq, as well as qPCR, to quantify the m6A levels. RNA samples were collected by one of two methods: lysis in TRIzol reagent (Thermo Fisher) for RNA-seq and MeRIP-seq, followed by standard phenol-chloroform extraction methods; alternatively, lysis was performed using Zymo Research Quick-RNA MiniPrep kit (Genesee Scientific) for regular qPCR following the manufacturer protocol. Libraries for RNA-Seq and MeRIP-Seq were made with NEBNext Ultra Directional Library Prep Kit for Illumina (NEB, E7760) following the manufacturer's instructions. Ribosome RNA was depleted with NEBNext rRNA Depletion Kit (NEB, E6301S). The generated libraries were quantified using the Qubit-3 system and/or qPCR. In some cases (such as Chip-seq), the libraries were examined by TBE PAGE gel and Bioanalyzer 2100. Sequencing of the libraries was performed using a NextSeq 550 platform (Illumina) with paired-end sequencing.