Correction to ‘Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue’

PHYTOMap: Multiplexed single-cell 3D spatial gene expression analysis in plant tissue

“…Target genes were manually selected based on their cell type-specific expression. Probes were constructed by combining the probe design used in STARmap 18 and HYBISS 12 (Fig. S1a).…”

“…A SNAIL probe is a pair of a padlock probe (PLP) and a primer designed as follows: (1) For each gene, 40-50 nt sequences with a GC content of 40-60% were selected and confirmed that there is no homologous region in the other transcripts by blasting against TAIR10 Arabidopsis genome; (2) selected sequences were split into halves of 20-25 nt (the 5’ halves for PLPs and the 3’ halves for primers), with 2 nt gap in between, and ensured that the melting temperature ( T m ) of each half is around 60°C; (3) PLPs have complementary sequences for target specific bridge-probes; (4) for each gene, four SNAIL probes were designed; (5) PLPs and primers have complementary sequences to form a circular structure. Bridge probes and detection read-out probes were designed as described before 12 and detailed in Table 1. All probes were manufactured by Integrated DNA Technologies (IDT).…”

“…1b and 1c). A previous study that used the SBH chemistry to detect amplified DNA probes in situ showed that signal was maintained at least over ten sycles 12 .…”

“…The amplification of DNA barcodes provides high signal-to-noise ratio, enabling signal detection from cleared whole-mount tissue. The location of mRNA molecules is defined by the sequence-by-hybridization (SBH) chemistry 12 that targets the barcode sequences of DNA amplicons across sequential rounds of probing, imaging, and stripping (Fig. 1b).…”

Single cell‐ and spatial ‘Omics revolutionize physiology

“…However, like other spatial technologies, in situ sequencing-based methods are still rapidly evolving. Several studies presented variations of the workflow, such as direct hybridization of padlock probes to mRNAs, 111,112 stabilization of the DNA nanoballs by crosslinking, 113,114 additional tissue clearing and the use of a hybridizationbased barcode read-out 115,116 all with the aim to increase sensitivity and throughput.…”