manual_ISS_FixedFlex
Manually in-situ-sequenced test tissue, nuclei/cell transcriptome probed with 10x Genomics Fixed/Flex kit (extra modality method development)
Instrument-free in situ sequencing, pioneered in the lab of Mats Nilsson (SciLifeLab, Stockholm), uses direct mRNA detection, with barcoded custom padlock probes hybridizing to target mRNA molecules, ligation of the padlocks, and rolling circle amplification (similar to the SCRINSHOT method of Sountoulidis et al., 2020; doi: 10.1371/journal.pbio.3000675). This is followed by cycles of labelling the barcoded part of the amplified padlocks with adapters and fluorophores, imaging at high resolution, stripping adapters and fluorophores, and re-probing, through which barcodes on the padlocks can be de-convoluted bio-informatically. As part of method development to add an extra modality (transcriptome) to the in situ sequencing, animal tissue is used (e.g. mouse brain sections). After in situ sequencing, tissue is taken off the slide, nuclei or cells isolated, and these used for transcriptome analysis with the 10x Genomics Fixed/Flex kit. This has added probe sets for the detection of the rolling circle amplification products to enable the transcriptome results to be mapped back to the in situ sequencing results (matching optical label signals per nucleus to probe readout per nucleus).
Wellcome Sanger Institute