SRX20230537 GSM7299915_r1 SRP436082 PRJNA967597 SRS17559538 GSM7299915 GSM7299915 RIP-Seq TRANSCRIPTOMIC other total RNA was isolated, DNase-treated, ethanol-precipitated and fragmented with RNA fragmentation reagent for 10 min at 70°C. Then, the RNA was ethanol-precipitated followed by a ribodepletion step using the human FFPE/degraded RNA riboPOOL kit (siTOOLs Biotech) according to the supplier's protocol. 5 μg of fragmented and ribodepleted RNA was used for 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. NextSeq 500 SRX20230538 GSM7299916_r1 SRP436082 PRJNA967597 SRS17559541 GSM7299916 GSM7299916 RIP-Seq TRANSCRIPTOMIC other total RNA was isolated, DNase-treated, ethanol-precipitated and fragmented with RNA fragmentation reagent for 10 min at 70°C. Then, the RNA was ethanol-precipitated followed by a ribodepletion step using the human FFPE/degraded RNA riboPOOL kit (siTOOLs Biotech) according to the supplier's protocol. 5 μg of fragmented and ribodepleted RNA was used for 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. NextSeq 500 SRX20230539 GSM7299917_r1 SRP436082 PRJNA967597 SRS17559539 GSM7299917 GSM7299917 RIP-Seq TRANSCRIPTOMIC other total RNA was isolated, DNase-treated, ethanol-precipitated and fragmented with RNA fragmentation reagent for 10 min at 70°C. Then, the RNA was ethanol-precipitated followed by a ribodepletion step using the human FFPE/degraded RNA riboPOOL kit (siTOOLs Biotech) according to the supplier's protocol. 5 μg of fragmented and ribodepleted RNA was used for 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. NextSeq 500 SRX20230540 GSM7299918_r1 SRP436082 PRJNA967597 SRS17559540 GSM7299918 GSM7299918 RIP-Seq TRANSCRIPTOMIC other total RNA was isolated, DNase-treated, ethanol-precipitated and fragmented with RNA fragmentation reagent for 10 min at 70°C. Then, the RNA was ethanol-precipitated followed by a ribodepletion step using the human FFPE/degraded RNA riboPOOL kit (siTOOLs Biotech) according to the supplier's protocol. 5 μg of fragmented and ribodepleted RNA was used for 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. NextSeq 500