Sequential barcoding. (a) Schematic of sequential barcoding. In each round of hybridization, 24 probes are hybridized on each transcript, imaged and then stripped by DNAse I treatment. The same probe sequences are used in different rounds of hybridization, but probes are coupled to different fluorophores. (b) Composite four-color FISH Data from 3 rounds of hybridizations on multiple yeast cells. Twelve genes are encoded by 2 rounds of hybridization, with the third hybridization using the same probes as hybridization 1. The boxed regions are magnified in the bottom right corner of each image. The matching spots are shown and barcodes are extracted. Spots without colocalization are due to nonspecific binding of probes in the cell as well as mis-hybridization. The number of each barcode can be quantified to provide the abundances of the corresponding transcripts in single cells.