Extended Data Fig. 4: RNA fractionation and mass spectrometry.

a, Representative gel (6% polyacrylamide, 7 M urea) showing the three RNA fractions analyzed in Fig. 4: total RNA (no fractionation), small RNAs < 200 nts (column-based size selection), and tRNAs (gel purification). The band corresponding to tRNAs (~70 nts) is indicated by the arrow. b, Mass spectrometry chromatograms of nucleosides fragmented into nucleobases. Data were acquired by isolating a precursor ion (nucleoside), fragmenting the precursor ion, and then isolating and detecting a known fragment ion (nucleobase). The top two chromatograms show C (244.093 → 112.050 m/z) and a spiked-in heavy C standard. The bottom two chromatograms show hm5C (256.103 → 142.061 m/z) and a spiked-in heavy hm5C standard (277.103 → 145.061 m/z). The representative chromatograms shown were obtained from the same run on tRNAs from Tet1/2/3 tKO cells transiently transfected with TET2 WT (Fig. 4D). Only one known nucleoside, 5-aminomethyluridine (nm5U), is isobaric with hm5C, and can be easily distinguished from hm5C by retention time. c, Western blot for TET2 comparing WT (+/+) and presumptive KO (–/–) clones as determined by PCR screening and Sanger sequencing. Tubulin is shown as loading control. d, Mass spectrometry quantification of hm5C in size-selected small RNAs < 200 nts from WT (left) Tet2 single KO (middle) and Tet1/2/3 triple KO (right) mESCs. Bars represent mean + s.e.m. ***, P < 0.001. P-values are from one-way ANOVA followed by Holm-Sidak test. Uncropped gel and blot images for a and c are shown in Supplementary Fig. 1.