Fig. 1: Overview of co-profiling of DNA methylation and transcriptome in tissues.

a, Spatial-DMT workflow. A tissue section is fixed, permeabilized, treated with HCl treatment, and subjected to Tn5 transposition and reverse transcription (RT). Spatial barcodes are sequentially ligated, after which cDNA and gDNA are separated using streptavidin beads. cDNA is processed using template switching, after which a cDNA library is prepared; gDNA is processed using enzymatic methyl-seq (EM-seq) conversion and splint ligation, after which a library is constructed. b, Chemistry workflow of DNA methylation and RNA library preparation. BCA, barcode A; BCB, barcode B; TSO, template switch oligo. c, Comparison of the number of CpGs per tissue pixel per cell across spatial-DMT and other single-cell DNA-methylation datasets^5,10,12,14 (number of cells: mouse muscle stem cells¹⁰, n = 260; mouse brain⁵, n = 103,560; human brain LA¹², n = 1,049; human brain SL¹², n = 1,920; mouse brain¹², n = 491; number of pixels: E11 embryo (10 µm), n = 2,493; E11 embryo 1 (50 μm), n = 1,954; E11 embryo 2 (50 µm), n = 1,947; E13 embryo (50 µm), n = 1,699; P21 brain (20 µm), n = 2,235). The solid line indicates the maximum number of CpGs in the mouse genome, large dashed line indicates 10% of the total number of CpGs and small dashed line indicates 1% of the total number of CpGs. d, Box plots showing CG/CH retention rates in E11 and E13 embryos and P21 brain tissues. Number of pixels are as stated in part c for these datasets. e, Number of genes (left) and UMIs (right) per pixel. For data from previous studies^1,2, the numbers of pixels are as follows (left to right): ref. ¹, n = 901, 1,789, 1,837, 1,840; ref. ², n = 2,373, 2,187. For the other datasets, number of pixels are as stated in part c. The box plots show the median (centre line), first and third quartiles (box limits) and 1.5× interquartile range (whiskers). f, Spatial clusters (top) and UMAP analysis (bottom) of DNA methylation (DNAm, left), RNA transcription (RNA, middle) and integrated data (spatial DNAm + RNA (WNN), right) in E11 mouse embryo (pixel size, 50 μm; ROI (red dashed box), 5 × 5 mm²; technical replicates, n = 2). Integrated analysis reveals more-refined spatial clusters and distinct anatomical regions, including brain, spinal cord, heart and craniofacial regions. Scale bars, 500 µm. D, DNA; R, RNA; W, WNN. g, Spatial mapping and joint clustering of DNA methylation and RNA data in E11 facial and forebrain regions (pixel size, 10 μm; ROI (red dashed box), 1 × 1 mm²). Scale bar, 500 µm. h, Spatial mapping of GABAergic cortical interneurons and telencephalon cells on the basis of deconvolution of transcriptomic pixels using a scRNA-seq reference²⁵. Cell-type distributions align with the anatomic references from the Allen developing mouse atlases⁵⁵. Pallm, mantle zone of the pallium; Pallv, ventricular zone of the pallium; POA, preoptic alar plate; POHv, ventricular zone of preopto-hypothalamic band; POm, mantle zone of preoptic area; POv, ventricular zone of preoptic area; PThEm, mantle zone of prethalamic eminence; PThEv, ventricular zone of prethalamic eminence; p2A, alar plate of prosomere 2; p3A, alar plate of prosomere 3; p3R, roof plate of prosomere 3; SPallm, mantle zone of the subpallium; SPallv, ventricular zone of the subpallium; TelA, alar plate of evaginated telencephalic vesicle; TelR, roof plate of evaginated telencephalic vesicle; TH, thalamus; THyA, alar part of terminal hypothalamus; THyB, basal part of terminal hypothalamus; TPaAv, ventricular zone of TPaA (terminal paraventricular area of THyA); TSPaAv, ventricular zone of TSPaA (terminal subparaventricular area of THyA).