Extended Data Fig. 1: Gating on a secondary reporter eliminates noise from the primary reporter fluorescence distribution. | Nature Protocols

Extended Data Fig. 1: Gating on a secondary reporter eliminates noise from the primary reporter fluorescence distribution.

From: SorTn-seq: a high-throughput functional genomics approach to discovering regulators of bacterial gene expression

Extended Data Fig. 1

The primary reporter (eYFP) fluorescence distribution is shown for without (a) or with (b) gating on a secondary reporter (mCherry). In a and b, events are first gated on SSC and FSC parameters area (A) and height (H) to isolate individual bacteria (‘singlets’ and ‘cells’). a, A characteristic secondary peak is observed centered around zero. Many of the events comprising this secondary peak exhibit negative fluorescence levels and therefore cannot be sorted. This non-fluorescent population is probably comprised of dead/dormant cells, cellular debris, bubbles or electronic noise generated by the instrument. b, The addition of a gate around mCherry-positive (mCherry+) events results in the removal of the secondary peak from the eYFP fluorescence distribution. This improved distribution allows for more accurate placement of gates during cell sorting.

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