Extended Data Fig. 2: Data analysis and comparison of MAGIC-seq with other methods.

a. Sequencing and data analysis of MAGIC-seq. After staining, permeabilization and reverse transcription (RT), sequencing libraries were prepared and sequenced by paired-end reads. The images of spots and H&E staining were aligned using AFIR algorithm. The sequencing reads were transformed into a spot-gene count matrix which was then remapped to the spots on the tissue sections. AFIR, affine transformation and image registration. b. Sample preparation device and fluorescence images for a triple-grid chip. The sample preparation device (top) consists of an aluminum alloy clamp, a three-chamber silicone adaptor and a barcoded slide with tissue stained. After assembly with screws in the corner, enzymatic reactions and washing steps could be performed through the well on the top. Representative fluorescence images of barcoded arrays in triple-grid chip were shown (bottom left). The fluorescence of each spot in each region was measured and plotted (bottom right). Boxes, interquartile range. Centerlines, median. Whiskers, values within 1.5× interquartile range of the top and bottom quartiles. c. Violin plot showing the number of UMIs per spot captured by MAGIC-seq in comparison with DBiT-seq, Decoder-seq and our sequenced 10x Visium datasets. d. Violin plot showing the number of genes and UMIs per spot captured by MAGIC-seq in comparison with Decoder-seq for mouse olfactory bulb (MOB) at different resolutions. For c and d, boxes, interquartile range. Centerlines, median. Whiskers, values within 1.5× interquartile range of the top and bottom quartiles. e. Saturation curves of median UMI counts per spot using MAGIC-seq and other spatial transcriptomic methods under different sequencing depths. f Saturation curves of median gene (left) and UMI (right) counts per spot for mouse olfactory bulb using MAGIC-seq and Decoder-seq under different sequencing depths.