10X Genomics Visium spatial transcriptome sample and library generation

Whole hearts were dissected, placed in sterile HBSS, and perfused through the apex to remove blood. Fresh samples were embedded in Optimal Cutting Compound (OCT) and frozen with liquid-nitrogen-cooled isopentane before sectioning into 10 µm slices using Thermo Scientific™ CryoStar NX50 cryostat. Sections were mounted on −20 °C-cooled Visium slides. Five sections were mounted for day 4, four sections for day 7, two sections for day 10, and one section for day 14 post fertilization. cDNA libraries were generated using 10X Genomics Visium Spatial Gene Expression 3ʹ Library Construction V1 Kit. A 10X Genomics Visium Spatial Gene Expression slide has four capture areas. Each capture area has 5000 circular spots that are 55 µm in diameter with a center-to-center distance of 110 µm. Each spot contains DNA oligos consisting of a PCR handle, spot-specific spatial barcodes, unique molecular identify (UMI), and poly-dT-VN tail for mRNA capture. Hematoxylin and eosin stained heart sections were imaged using Zeiss PALM MicroBeam laser capture microscope and images were processed using Fiji from ImageJ. Illumina NextSeq 500/550 was used to sequence the spatially tagged cDNA libraries with 150 cycle high-output kits (Read 1 = 120, Read 2 = 5, Index 1 = 14, and Index 2 = 8). Sequencing files were processed using 10X Genomics Space Ranger pipeline. TAR annotations were generated by running groHMM on the combined Space-Ranger-derived alignment files across the four time points. TAR and GRCg6a Ensembl v96 annotations were combined to create one set of genome annotation. Spatially tagged TAR and gene expression matrices were generated with the combined annotation set following the Space Ranger pipeline.