Name: GSM7299911
Instrument: NextSeq 500
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Total RNA was extracted using TRIzol (Thermo Fisher) according to the supplier's protocol. TURBO DNA-free Kit (Thermo Fisher) was used to remove any contaminating DNA from RNA samples. Isolated RNA was further purified by ethanol precipitation and 30-40 µg of total RNA were subjected to poly(A)-selection with Dynabeads Oligo (dT)25 (Thermo Fisher) according to the manufacturer's instructions. poly(A)+ RNA from CHIKV-infected HEK293T cells (12 hr p.i. MOI of 4) was fragmented for 10 min at 70°C with RNA fragmentation reagent (Thermo Fisher). After fragmentation, the RNA was cleaned up through an ethanol precipitation step. At this stage, a sample of fragmented poly(A)+ RNA was saved as input RNA for later use in cDNA library construction while 5 μg of fragmented poly(A)+ RNA were used per m6A-IP. For each m6A-immunoprecipitation (m6A-IP), 50 μl of slurry of Magna ChIP Protein G magnetic beads (Sigma-Aldrich) were washed twice with IP/wash buffer 20 mM Tris HCl pH 7.4, 150 mM NaCl, and 0.1% NP-40 (v/v). Beads were re-suspended in 100 μl of IP/wash buffer and coated with 5 μg of rabbit anti-m6A antibody (Active motif, 61495) for 45 min at room temperature with rotation. Beads were then washed 3x with IP/wash buffer and m6A-IPs were prepared by mixing the antibody-coated beads with 910 μl of IP/wash buffer, 35 μl of 0.5 M EDTA pH 8.0, 4 μl of murine RNase inhibitor (New England Biolabs), and 40 μg of fragmented total RNA. 1% input samples (10 μl from the total 1 mL m6A-IP mixture) were removed before immunoprecipitation and stored at −80°C. m6A-IPs were incubated overnight at 4°C with rotation. Beads were then washed 3x with IP/wash buffer. IP samples were further incubated with 126 μl of IP/wash buffer, 15 μl of 10% SDS (v/v) and 9 μl of PCR-grade proteinase K (20 mg/mL) (Thermo Fisher) for 30 min at 55°C. After incubation, 150 μl of the supernatant containing the RNA was transferred to a new microcentrifuge tube and 100 μl of IP/wash buffer was added to each sample. RNA was purified with Trizol LS (Thermo Fisher) and ethanol-precipitated together with 1.5 µl of RNA-grade glycogen (Thermo Fisher). Input samples were processed together with m6A-IPs from the proteinase K treatment onwards. 1 to 5 ng of RNA from input and corresponding m6A-IPs were used for NGS library production following the protocol NEBNext Ultra II directional RNA library prep kit for Illumina (New England Biolabs), treating samples as rRNA-depleted and fragmented RNAs.