Fluorescent in situ sequencing

[2] Sequencing data is then generated through an intensive interleaved microscopy and biochemistry protocol and subsequent image processing and bioinformatics.

FISSEQ is compatible with diverse sample types including cell culture, tissue sections, and whole mount embryos.

FISSEQ is an example of an extremely dense form of in-situ nucleic acid readout: every letter along the RNA chain is read.

However, the requirement to sequence RNA in intact tissue—rather than isolated and purified DNA, as in conventional bulk sequencing—posed additional challenges.

These limitations were overcome,[2] and FISSEQ allows the joint, high-throughput readout of sequence and spatial information.

FISSEQ in fibroblast cells. Each spot is an RNA molecule reverse transcribed and amplified. The spot color indicates the base . The image represents one cycle in a sequencing run. 30 cycles would yield 30 bases of the RNA sequence. Scale bar 20um.
FISSEQ Schematic: A tagged random hexamer primer is used to prime M-MuLV reverse transcriptase to generate aminoallyl dUTP- modified cDNA fragments in fixed cells or tissues from RNA. BS(PEG)9 permanently cross-links the modified cDNA and the cellular protein matrix. After cDNA circularization, Phi29 DNA polymerase generates cDNA amplicons . Amplicons in 3D in situ RNA sequenced within the cell.