title: GSM5365467: mES_m6A-REF_WT_IVT_rep2; Mus musculus; RIP-Seq
library_construction_protocol: Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, 15596026) and the mRNA was purified using Dynabeads mRNA Purification Kit (Thermo Fisher Scientific,  61006). mRNA was treated by TURBO DNase (Thermo Fisher, AM2238) at 37 ℃ for 30 min and purified by ethanol precipitation or RNA Clean & Concentrator-5 kit (Zymo Research, R1015) to remove genomic DNA. IVT RNA generation:  The 100 ng (in 3ul volume at most) extracted mRNA was first annealed with 1ul PolyT primer (12 uM) for 3 min at 75 ℃ and 2 min at 42 ℃. Then we prepared the first strand cDNA mix by adding 2ul 5× First-Strand buffer of SMARTScribe Reverse Transcriptase (Takara, 639536), 0.5 ul 100 mM DTT, 0.5 ul RNase inhibitor (Takara, 2313A), 1ul TSO T7 Primer (10uM), 1ul 50× Advantage UltraPure PCR Deoxynucleotide Mix (Takara, 639125) and 1ul SMARTScribe Reverse Transcriptase (Takara, 639536). After adding the first strand cDNA mix to the mRNA/PolyT Primer mixture, the reaction mixture was incubated at 42 ℃ for 1.5 h and stopped by heating at 68 ℃ for 10min for first strand cDNA synthesis. The primer extension was conducted by adding 10 ul 10× Advantage 2 PCR buffer and 1ul Advantage 2 PCR Polymerase mix (Takara, 639207), 2 ul 50× Advantage UltraPure PCR Deoxynucleotide Mix, 2 ul T7 Extension Primer (10 uM), 2ul RNaseH (NEB, M0297) and RNase-free water to 100 ul. The reaction was conducted at 37 ℃ for 15 min, 95 ℃ for 2 min, 60 ℃ for 1 min and 68 ℃ for 10 min. The double-strand cDNA was purified using NucleoSpin PCR cleanup (MACHEREY-NAGEL, 740609.50). The in vitro transcription step was performed using HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, E2040) and NTPs from Takara (cat. 6140). The sample was subjected to TURBO DNase (Thermo Fisher, AM2238) treatment at 37 ℃ for 30 min and purified by RNA Clean & Concentrator-5 kit (Zymo Research, R1015) to remove dsDNA. The concentration of IVT RNA was determined by Qubit RNA HS Assay kit (Thermo Fisher, Q32852). MeRIP-seq: Cellular mRNA and IVT RNA were fragmented by using Thermo Fisher's Fragmentation Kit (Thermo Fisher Scientific, AM8740) at 94 °C for 30s and then purified by ethanol precipitation. The commercial antibodies of NEB (EpiMark® N6-Methyladenosine Enrichment Kit, E1610S), SYSY (202003) and Abcam (ab190886) were used in m6A immunoprecipitation. The 25 ul protein G beads (Thermo Fisher Scientific, 1004D) were washed three times with Reaction buffer (150mM NaCl, 10mM pH 7.4 Tris-HCl, 0.1% Igepal CA-630 in nuclease free H2O). Then the protein G beads were resuspended in 250 ul Reaction buffer and 2 ul anti-m6A antibody was added. After incubation with orbital rotation at 4 ℃ for 1 h, the antibody-protein G beads mixture was washed twice with Reaction buffer. A total of 1ul mRNA for each sample was added to the mixture and then incubated with rotation at 4 ℃ for 3 h. After washed with Reaction buffer twice, the mRNA-antibody-protein G mixture was washed low salt buffer (50 mM NaCl, 10 mM pH 7.4 Tris-HCl, 0.1% Igepal CA-630 in nuclease-free H2O) and high salt buffer (500 mM NaCl, 10 mM pH 7.4 Tris-HCl, 0.1% Igepal CA-630 in nuclease-free H2O) twice, respectively. The IP products were eluted with 130 ul Elution buffer (150 mM NaCl, 10 mM pH 7.4 Tris-HCl, 0.1% Igepal CA-630, 6.7 mM m6A in nuclease-free H2O) at 37 °C, 1000 rpm for 1.5 hours. After purified by ethanol precipitation, the IP products and input sample kept before IP were used to constructed the NGS library using NEBNext® Ultra II Directional RNA Library Prep (NEB, E7760S). m6A-REF-seq: We followed the experimental procedural of m6A-RFE-seq in Zhang et al., (2019) and used IVT RNA as negative control. After heated at 85°C for 5 min and chilled on ice for 2 min, the equivalent endogenous mRNA and IVT RNA were incubated with MazF (Takara, 2415A) in the MazF reaction buffer at 37 °C for 30 min. After purified by RNA Clean & Concentrator-5 kit (Zymo Research, R1015), the fragment mRNA was end-repaired by T4 polynucleotide kinase (T4PNK; Vazyme Biotech). Then the fragment mRNA was purified by RNA Clean & Concentrator-5 kit, and the NGS libraries were constructed by using the VAHTS Small RNA Library Prep Kit (Vazyme Biotech, NR801) with 10 ng of fragmented RNA. m5C BS-seq: Cellular mRNA and IVT RNA samples were conducted bisulfite treatment following the instruction in Huang et al., (2019) using EZ RNA methylation kit (Zymo Research, R5001). High-stringency conditions: Total among of 1 ug cellular mRNA and IVT RNA were treated with three cycles of 70 °C for 10 min and 64 °C for 45 min in sulfonation step and then 37 °C for 30 min in desulfonation step. The NGS libraries were constructed by using NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7760S). RNA-seq, MeRIP-seq and m5C BS-seq libraries were constructed by using NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7760S). The m6A-REF-seq libraries were constructed by using the VAHTS Small RNA Library Prep Kit (Vazyme Biotech, NR801) with 10 ng of fragmented mRNA.