10x Visium spatial transcriptomics library preparation

We used the 10x Genomics Spatial RNAseq Visium platform for the spatial transcriptomics experiments. A 10x Genomics Visium Gene Expression slide has 4 capture areas each with an array of 5000 circular spots containing printed DNA oligos for mRNA capture. These oligos on each spot have a PCR handle, unique spatial barcode, Unique Molecular Identifier (UMI), and a poly-dT-VN tail for capturing the 3’ end of mRNA molecules. Each spot with a unique spatial barcode is 55 µm in diameter and the center to center distance between the spots is 110 µm. One 55 µm spot captures mRNA from 10 to 20 cells depending on cell size and packing density which is variable across the tissue. Tissue sections from fresh frozen chicken embryonic hearts were mounted for 4 stages (five sections from day 4-HH24, four sections for day 7-HH31, two sections for day 10-HH36, and one section for day 14-HH40) with one stage per capture area on a 10x Visium gene expression slide. The sample for day 4 had three biological replicates with two of them having technical replicates and the samples for day 7 and day 10 had four and two biological replicates, respectively. These sections are then fixed in pre-chilled methanol for 30 min and then H&E stained and imaged. H&E staining is later used by the 10x Genomics Cell Ranger software to detect the spots which are covered by the tissue. Optimal permeabilization time for 10 µm thick chicken heart sections was found to be 12 min using 10x Genomics Visium Tissue Optimization Kit. Spatially tagged cDNA libraries were built using the 10x Genomics Visium Spatial Gene Expression 3’ Library Construction V1 Kit. H&E stained heart tissue sections were imaged using Zeiss PALM MicroBeam laser capture microdissection system and the images were stitched and processed using Fiji ImageJ software. cDNA libraries were sequenced on an Illumina NextSeq 500/550 using 150 cycle high output kits (Read 1 = 28, Read 2 = 120, Index 1 = 10, and Index 2 = 10). Fluidigm frames around the capture area on the Visium slide were aligned manually and spots covering the tissue were selected using Loop Browser 4.0.0 software (10x Genomics). Overall, tissue sections from day 4 to day 14 covered a total of 747, 1966, 1916, and 1967 spots on the capture area, respectively. Sequencing data were then aligned to the chicken reference genome using the Space Ranger 1.0.0 pipeline to derive a feature spot-barcode expression matrix (10x Genomics).