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Fig.
1. Conceptual diagram of barcoding. (A) Schematic representation
of the use of color groups to encode unique gene identities. Combinations
shown are only two colors chosen from a total of four used throughout
our experiments. A minimum of two colors is used to reduce the chance
of single-color false positive signals. Each detected site is therefore
representative of two or more independent hybridization events that
are spatially colocalized, thus increasing fidelity. (B) Schema of the
actual probe hybridizations at the transcription site leading to detection
by barcode. The gene locus is represented (green) with several polymerases
(blue) transcribing nascent messages (pink). A shotgun approach is used
such that each specific probe may be of any of the colors in the barcode
(red, yellow, magenta). The transcription site is shown below with hybridization
data against the nuclear background (blue). Scale bar, 3 µm.
(C) An example of the signal readout interpreted by the transcription
detection algorithm, showing the pixel intensities for the area of the
transcription sites in all five color bandwidths. In this example, there
is signal for each of the probe components used (red, yellow, and magenta)
and only background levels for the color not in the barcode (purple).
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