a, Western blot showing knockdown efficiency of SETD2 using shSETD2#1, and the decrease of H3K36me3 in HepG2 and HeLa cells. GAPDH and H3 were used as loading controls. b, LC–MS/MS quantification of m6A abundance in poly(A) RNA from HeLa cells after shRNA knockdown of SETD2 (shSETD2#1) or of individual m6A MTC genes (METTL3, METTL14, WTAP) in comparison to control cells. Data are mean ± s.d. of two independent experiments. c, Knockdown efficiency of shRNAs was verified by qRT–PCR. Data are mean ± s.d. of three independent experiments. d, Dot blot (left) and quantification (right, data are mean ± s.d.) of m6A in total RNA from control cells or cells with knockdown of SETD2 or individual m6A MTC genes. Methylene blue (MB) was used as a loading control. e, shRNA knockdown of SETD2 (shSETD2#1 and shSETD2#2) was verified by western blot in HepG2 cells. f, Dot blot (right) and quantification (left, data are mean ± s.d.) of m6A in total RNA after shRNA knockdown of SETD2 in HepG2 cells. g, Western blot showing depletion of SETD2 and H3K36me3 in wild-type (WT) or SETD2-knockout (KO) HeLa cells. h, LC–MS/MS quantification of m6A in poly(A) RNA from wild-type or SETD2-knockout HeLa cells. Data are mean ± s.d. of two independent experiments. i, Dot blot (right) and quantification (left, data are mean ± s.d.) of m6A in total RNA from wild-type or SETD2-knockout HeLa cells. j, Western blot confirmed the overexpression of HA-tagged KDM4A and a decrease in H3K36me3 in HEK293T cells. k, Quantification (left, data are mean ± s.d.) and dot blot (right) showing hypomethylation of m6A in poly(A) RNA from HEK293T cells with KDM4A overexpression compared to control cells. l, Genotyping of wild-type (+/+) and SETD2 heterozygous knockout (+/−) mice. c-kit+ bone marrow (BM) cells were used. m, Western blot showing H3K36me3 level in c-kit+ bone marrow cells from wilde-type or SETD2 heterozygous knockout mice. n, o, LC–MS/MS quantification (n) and dot blot analysis (o, data are mean ± s.d. on left) of m6A in poly(A) RNA from c-kit+ bone marrow cells of wild-type and SETD2 heterozygous knockout mice. Data are mean ± s.d. of two technical replications and three mice were used for each group in n. p, Western blot showing expression of SUV39H1 and H3K9me3 in HeLa cells transfected with short interfering RNA (siRNA) against SUV39H1 (siSUV39H1) or negative control (siNC). q, r, Dot blot (right) and quantification (left, data are mean ± s.d.) of m6A in total RNA (q) or poly(A) RNA (r) from HeLa cells transfected with SUV39H1 siRNA or control. s, A498 cells were transfected with GFP-fused wild-type or mutated SETD2, and were selected for GFP-positive cells after 48 h by cell sorting. The increase in SETD2 mRNA expression in SETD2-overexpressed cells was confirmed by qRT–PCR. Data are mean ± s.d. of three independent experiments. t, Western blot showing H3K36me3 level in A498 cells after overexpression of wild-type (WT) or mutated (that is, R1625C and ΔSRI) SETD2 compared to empty pEGFP vector (vector). HEK293T served as a positive control. u, Dot blot detecting m6A in poly(A) RNA from A498 cells after overexpression of wild-type or mutated SETD2 compared to empty pEGFP vector. v, Western blot showing that silencing of individual m6A MTC genes (METTL3, METTL4 or WTAP) did not affect cellular H3K36me3 levels in HepG2 cells. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed student’s t-test. N.S., not significant. Images are representative of three (a, d, g and i) or two (e, f, j, k, m, o–r, t–v) independent experiments. Source data