Refined RIP-seq protocol for epitranscriptome analysis with low input materials
Fig 5
(A) Transcriptome-wide distribution of m6A sites. (B) Top motif and their distance to summit of m6A peaks; ***p < 1 × 10−4. (C) Volcano plot for peaks with differential m6A intensity between tumor1 and tumor2. RTumor1 and RTumor2 stand for the ratio of IP over Input for sample tumor1 and tumor2, respectively. Peaks with p < 1 × 10−10 were reassigned to be p = 1 × 10−10. (D) Correlation between the ratios (tumor1/tumor2) at RNA and protein levels for genes with differential m6A peaks. Red dots represent genes with discordance ratio at mRNA and protein levels, and dot size represents m6A odds ratio between tumor1 and tumor2. (E) Top: the IP (orange for tumor1 and cyan for tumor2) and Input (light green) signal at m6A peak (marked by red box) near SLC2A1 stop codon in the 2 tumors. Bottom: ratio between tumor1 and tumor2 at mRNA and protein levels for SLC2A1. (F) Western blotting analysis of tumor1 and tumor2 samples. (G) Real-time PCR analysis of METTL3 and SLC2A1 mRNA expression levels in A549 upon silencing of METTL3 using 2 different siRNAs. *p < 0.05; ***p < 1 × 10−4. (H) Western blotting analysis of METTL3 and SLC2A1 protein levels in the A549 upon METTL3 knockdown. (I) Real-time PCR analysis of METTL3 mRNA in rescue assays. METTL3 primer recognized both full-length and 1–200AA METTL3 mutant. METTL3 knockdown cells were transfected with either full-length METTL3 (siM3_1+full) or 1–200AA METTL3 mutant overexpressing plasmid (siM3_1+200). siM3_1 is the abbreviation for siMETTL3_1; ***p < 1 × 10−4. (J) Western blotting analysis of SLC2A1 in rescue assays. Data related to this figure can be found in S1 Data. ADC, adenocarcinoma; CDS, coding sequence; IP, immunoprecipitation; lincRNA, long intergenic noncoding RNA; m6A, N6-Methyladenosine; METTL3, Methyltransferase Like 3; siRNA, small interfering RNA; SLC2A1, Solute Carrier family 2, Facilitated Glucose Transporter member 1; TSS, transcription start site; UTR, untranslated region.