Fig 2: A platform for single-cell m6A level measurement(A) Workflow of m6A detection from single-cell transcriptomes. (1) Fluorescence dye-stained cells (K562, SYTO9 [green]; YAC1, SYTO87 [red]) were allowed to settle into the wells of a nanowell plate by gravity. The plate was scanned by epifluorescence microscopy to count and phenotype cells prior to lysis. (2) After cell lysis, the nanowell plate was immediately sealed with an oligo-dT-coated coverslip to capture polyA+ RNAs confined in each well. A flow cell was assembled with the detached coverslip. Captured polyA+ RNA molecules were labeled with Cy3-dATP by using Klenow exo-. m6A-modified RNA molecules were detected as described in Figure 1A. The flow cell was scanned by using TIRF microscopy to measure the amounts of captured polyA+ RNA and m6A-modified RNA molecules. (3) The nanowell and flow cell scanning images were used to generate a COM and single-molecule density matrix of captured RNA (RDM), respectively. Occupied wells were registered by finding the outlier of correlation coefficient between the matrices.(B) Scatterplot of single-cell fluorescence intensity from nanowell scanning images with K562 (green) and YAC1 (red) cells.(C) Box plots depicting the number of captured polyA+ RNA molecules per imaging area (15,400 µm2). Number of imaging areas is indicated above box plots.(D) Matrices of single-molecule density of m6A-modified polyA+ RNA (top) and the ratio of m6A-modified polyA+ RNA to total polyA+ RNA molecules (bottom).(E) Box plots showing m6A levels for K562 and YAC cells.(F) Representative fluorescence images of nanowells occupied with MUTZ3 cells stained with SYTO9 (blue), anti-CD14 (red), and anti-CD34 (green).(G and H) Scatterplot of single-cell fluorescence intensity (G) from nanowell scanning images of stained MUTZ3 cells. Each cell was colored by corresponding m6A level (H).(I and J) Box plots showing the number of captured polyA+ RNA molecules (I) and m6A levels (J) in CD14-CD34+ and CD14+CD34- cell populations.See also Figure S2.

Fig 3: METTL3 depletion leads to an increase in the m6A DIP peaks in hPSCs.METTL3 depletion leads to an increase in the m6A DIP peaks in hPSCs. (a) SID-UPLC-MS/MS quantification of m6A in the total nucleic acids (left panel), and in ultrafiltrate fractions released upon RNase H treatment of siCTL and siMETTL3 hPSCs (right panel). The data are means of 2 independent experiments. (b) The total numbers of consensus m6A peaks in WT and siMETTL3 hiPSCs. (c) Heatmaps showing the distribution of density of indicated reads across peak-containing genomic regions (3 kb around peak center) for S9.6 DRIP in WT and m6A DIP in WT and siMETTL3 hPSCs. The colour of each line represents the density of reads for a given peak. The width of the heatmaps is normalized by peak length. (d) Pie chart showing the percentages of transcripts differentially expressed between siCTL and siMETTL3 hPSCs (p<0.01) and without significant changes in expression amongst genes containing siMETTL3-specific m6A peaks. P values were calculated using the Standard ballgown parametric F-test. (e) Pie chart demonstrating the percentages of genes differentially expressed between siCTL and siMETTL3 hPSCs containing siMETTL3-specific m6A peaks (diff peaks) and without such peaks (No diff peaks). (f) Average profile of m6A peak densities for all genes sorted based on levels of their expression in siMETTL3 hPSCs. The colour gradient represents log10 of mean RPKM per bin. (g) Average profile of m6A peak densities for all genes sorted based on the fold change of their expression between siCTL and siMETTL3 hPSCs. The colour gradient represents log10 of mean fold change (FC) per bin.

Fig 4: YTHDF2 depletion leads to accumulation of R-loops, increased accretion of m6A on RNA:DNA hybrids and cell growth retardation. (a) Immunostaining of WT and YTHDF2 KO HAP1 for YTHDF2 and phospho-Histone H3. The experiments were repeated independently 3 times with similar results. (b) Immunostaining of WT and YTHDF2 KO HAP1 for R-loops alongside the quantification of S9.6 nuclear signal. (c) DRIP qPCR of the indicated sequences performed on WT and YTHDF2 KO HAP1. RANBP17 and HECW1 downstream regions lacking DRIP peaks were used as controls. (d) SID-UPLC-MS/MS quantification of m6A and ribo-m5C in S9.6-IPs performed on WT and YTHDF2 KO HAP1 and normalized for dA or rA. RNase H-pre-treated samples were used as controls. Data are means ± SD, n=5 (left) and n=7 (right panel) measurements of 4 independent samples. (e) The growth curves of WT and YTHDF2 KO HAP1. (f) Immunostaining of WT/Ythdf2 KO mNSCs for R-loops alongside the quantification of S9.6 nuclear signal. (g) DRIP and m6A DIP qPCR of mouse LINE-1 ORF1 performed on WT and Ythdf2 KO mNSCs. Individual channels (a) or S9.6 channel (b, f) with merged views are shown. Scale bars are 10 µm. Data are means ± SD, n=3 independent experiments in (c, e, g). The elements of the box plots (b, f) are: centre line, median; box limits, upper and lower quartiles; whiskers, minimum and maximum of all the data; n, sample size, ***p < 0.0001. Significance was determined by unpaired two-tailed Student’s (b, f) or unpaired two-tailed Welch's (e) t-test.

Fig 5: METTL14 R255K decreases mRNA m6A modification.a Western blot showing the protein level of METTL14 in WT and METTL14 R255K knock-in mESCs. Representative figures of three blots are shown. The band intensity of METTL14 relative to GAPDH is shown in the right-hand panel, and data are mean ± s.d. from three independent replicates. b Dot blot showing the m6A mRNA modification levels from WT and METTL14 R255K mESCs. Methylene blue (MB) was used as a loading control. The experiment was performed once. c LC–MS/MS quantification of m6A abundance in mRNA from WT, R255K, or R255K with METTL14 overexpressed cells. Data are mean ± s.d. of three independent experiments. Two-sided Student’s t test. d Distribution of m6A peaks from WT and METTL14 R255K cells in transcriptomic regions. e Profile of m6A distribution on Nedd4 and Zfp36l1 in WT and R255K cell lines. Shown is normalized density of IP (green or red) versus input (gray). Values on the left show the range of IP and input density. f MeRIP-qPCR verifying decreased m6A peaks on Nedd4, Zfp36l1, Gas2l3, and Trim71. Enrichment of MeRIP versus input RNA was normalized against Gapdh. Data are mean ± s.d. from three independent experiments. Two-sided Student’s t test. g Abundance of m6A signals around the m6A peak center. Solid line, downregulated m6A peaks in R255K. Dotted line, constitutive m6A peaks. h Box plot showing the expression levels of genes with decreased m6A abundance in R255K cells, other m6A modified genes, and non-m6A modified genes. The center line indicates median value, and the box and whiskers represent the interquartile range (IQR) and 1.5 IQR, respectively. Two-sided Mann–Whitney U test. Source data for (a), (c), (f), and (h) are provided as a Source Data file.