Journal: PLoS Biology
Article Title: Refined RIP-seq protocol for epitranscriptome analysis with low input materials
Figure Lengend Snippet: Comparison of 3 different m 6 A antibodies for MeRIP. (A) S/N ratio of GLuc/CLuc and SETD7 / GAPDH with different antibodies. The amount of 32 μg total RNA from human lung cancer cell line A549 with spiked-in control RNA GLuc and CLuc was used for m 6 A MeRIP using Method II. (B) m 6 A peak signals of SETD7 transcripts in 3 MeRIP-seq libraries. (C) Overlap of m 6 A peaks from the SySy, NEB, and Millipore libraries. (D) Number of m 6 A peaks called by subsampling to different read depths with different antibodies. (E) The percentages of m 6 A peaks in 5 nonoverlapping transcript segments: TSS; 5’UTR; CDS; stop codon; and 3’UTR. (F) Metagene profiles depicting sequence coverage in windows surrounding the TSS and stop codon demonstrated that m 6 A peaks were enriched in the vicinity of the stop codon. (G) Top enriched motifs identified in the SySy, NEB, and Millipore libraries. Data related to this figure can be found in S1 Data . CDS, coding sequence; CLuc, unmodified control RNA; GLuc, m 6 A-modified control RNA; m 6 A, N6-Methyladenosine; m 6 A MeRIP, m 6 A RNA immunoprecipitation followed by high-throughput sequencing; NEB, New England Biolabs; S/N, signal-to-noise; SySy, Synaptic Systems; TSS, transcription start site; UTR, untranslated region.
Article Snippet: Antibodies and plasmids We used the following antibodies to m6 A: rabbit polyclonal anti-m6 A (202 003, SySy, Germany; ABE572, Millipore, Germany) and rabbit monoclonal anti-m6 A supplied in EpiMark N6-Methyladenosine Enrichment Kit (E1610S, NEB, Ipswich, MA). pCMV-Flag-MS2-METTL3 full-length and pCMV-Flag-MS2-METTL3 1–200AA plasmids were generously provided by Professor Richard I. Gregory (Harvard University).
Techniques: Sequencing, Modification, Immunoprecipitation, Next-Generation Sequencing