Qualitative enrichment of modified vs. nonmodified RNAs using quantitative immunoprecipitation and a thin-layer chromatography-based assay. (A) Schematic overview of the experimental setup. Modified (m5C as example) and unmodified or differently modified (depicted as X) chemically synthesized oligonucleotides of 12 nt in length were radioactively 5′ labeled. Oligonucleotides were subjected to immunoprecipitation using the indicated antibodies. After washing, the radioactive signals (cpm-values) of the immunoprecipitated RNAs as well as the input samples were measured using a scintillation detector (Cerenkov measurement), and enrichment factors were calculated. (B–F) Depiction of the enrichment of the antibodies α-m6A 9B7, polyclonal α-m6A (Synaptic Systems), α-m5C 32E2, α-m5C (Diagenode), α-m5C (Cell Signaling), α-Ψ 27C8, and α-m26A 60G3 by using the RNA-IP-based approach, shown in A. (B) Results for the m6A antibodies 9B7 and polyclonal Synaptic Systems, (C) the enrichment with antibody α-m5C 32E2, (D) enrichment of α-m5C from Diagenode or Cell Signaling, (E) of antibody clone Ψ 27C8, and (F) depicts the enrichment of the antibody α-m26A 60G3. Experiments were conducted in triplicate. (G) Workflow for enrichment factor using in vitro transcription (ivt), antibody enrichment, and thin layer chromatography (TLC). Ivt with modified (m5CTP as example) and radiolabeled NTPs and immunoprecipitation, the RNA was hydrolyzed with the nuclease P1 and analyzed by TLC. (H) Evaluation of m6A IP-TLC experiments as described above using 1% (upper panel) or 50% (lower panel) m6ATP for the ivt and analysis of the monoclonal m6A antibody clone 9B7 and the polyclonal m6A-antibody from Synaptic Systems. The light gray bars show the normalized signal intensities, corresponding to the precipitated unmodified RNA; the dark gray bars show the same for the precipitated modified RNAs. Experiments were conducted in triplicate. (I) Evaluation of m5C IP-TLC experiments using 1% (upper panel) and 50% (lower panel) m5CTP for the ivt and analysis of m5C antibody clone 32E2 and commercially available m5C-specific antibodies from Diagenode and Cell Signaling. Experiments were conducted in triplicate. (J) Determination of the enrichment factors of antibody α-Ψ 27C8 based on the RNA-IP and TLC experiments. For these experiments, 1% (upper panel) and 50% (lower panel) ΨTP was used for the ivt. TLCs and further evaluations of other antibodies that we tested can be found in Supplemental Figure 2.
