Input Image	Input
	Channel group: 1 Sequences: ALL Flatfield Correction: Basic Brightfield Correction Stack Processing: Maximum Projection
Find Texture Regions	Input	Method	Output
	Channel: Alexa 488 ROI: None	Method: Split into Classes Number of Classes: 2 Texture Scale: 1 px Region Scale: 1 px Training Region Radius: 2 px Include Intensity Information	Output Population A: DNA Output Population B: Background (Figure S2)
Calculate Morphology Properties	Input	Method	Output
	Population: DNA Region: Region	Method: Standard Area	Property Prefix: Region
Calculate Morphology Properties (2)	Input	Method	Output
	Population: Background Region: Region	Method: Standard Area	Property Prefix: Region
Calculate Intensity Properties	Input	Method	Output
	Channel: Alexa 488 Population: DNA Region: Region	Method: Standard Mean Standard Deviation Sum	Property Prefix: Intensity Region Alexa 488
Calculate Intensity Properties (2)	Input	Method	Output
	Channel: Alexa 647 Population: DNA Region: Region	Method: Standard Mean Standard Deviation Sum	Property Prefix: Intensity Region Alexa 647
Calculate Intensity Properties (3)	Input	Method	Output
	Channel: Alexa 647 Population: Background Region: Region	Method: Standard Mean Sum	Property Prefix: Intensity Region Alexa 647
Filter Image	Input	Method	Output
	Channel: Alexa 647	Method: Sliding Parabola Curvature: 20	Output Image: Sliding Parabola 647
Find Image Region	Input	Method	Output
	Channel: Sliding Parabola 647 ROI: DNA ROI Regin: Region	Method: Absolute Threshold Lowest Intensity: ≥250 Highest Intensity: ≤inf Split into Objects Area: >5 px2	Output population: Red/DNA Region Absolute threshold Output Region: Image Region
Find Image Region (2)	Input	Method	Output
	Channel: Sliding Parabola 647 ROI: Background ROI Regin: Region	Method: Absolute Threshold Lowest Intensity: ≥250 Highest Intensity: ≤inf Split into Objects Area: >5 px2	Output population: Red/Background Region Absolute threshold Output Region: Image Region
Find Spots	Input	Method	Output
	Channel: Alexa 647 ROI: DNA ROI Region: Region	Method: A Relative Spot Intensity: >0.07 Splitting Sensitivity: 1 Calculate Spot Properties	Output Population: Red spots in DNA region
Find Spots (2)	Input	Method	Output
	Channel: Alexa 647 ROI: Background ROI Region: Region	Method: A Relative Spot Intensity: >0.07 Splitting Sensitivity: 1 Calculate Spot Properties	Output Population: Red spots in Background
Define results	Results
	Method: List of Outputs Population: Red/DNA region Ab thresh Number of Objects
	Population: Red/Background Region Ab thresh Number of Objects
	Population: DNA Number of Objects Region Area [μm2]: Sum Intensity Region Alexa 488 Mean: Sum Intensity Region Alexa 488 Sum: Sum Intensity Region Alexa 647 Mean: Sum Intensity Region Alexa 647 Sum: Sum Number of Spots: Sum Number of Spots per Area of Region: Sum
	Population: Red spots in DNA region Number of Objects Apply to All: None
	Population: Background Region Area [μm2]: Sum Intensity Region Alexa 647 Mean: Sum Intensity Region Alexa 647 Sum: Sum Number of Spots: Sum Number of Spots per Area of Region: Sum
	Population: Red spots in background Number of Objects
	Method: Formula Output Formula: a/b Population Type: Objects Variable a: DNA—Intensity Region Alexa 488 Sum Sum Variable b: DNA—Region Area [μm2] Sum Output Name: DNA 488 sum/area
	Method: Formula Output Formula: a/b Population Type: Objects Variable a: Background—Intensity Region Alexa 647 Sum Sum Variable b: Background—Region Area [μm2] Sum Output Name: Background: Red sum/Background area
	Method: Formula Output Formula: a/b Population Type: Objects Variable a: DNA—Intensity Region Alexa 647 Sum Sum Variable b: DNA—Region Area [μm2] Sum Output Name: DNA: Red sum/DNA area
	Method: Formula Output Formula: a/b Population Type: Objects Variable a: DNA—Number of Spots Sum Variable b: DNA—Region Area [μm2] Sum Output Name: DNA: Red Spots/DNA area
	Method: Formula Output Formula: a/b Population Type: Objects Variable a: Red spots in background—Number of Objects Variable b: Background—Region Area [μm2] Sum Output Name: Background: Red Spots/Background area
	Method: Formula Output Formula: a/b Population Type: Objects Variable a: Red/DNA region Absolute threshold—Number of Objects Variable b: DNA—Region Area [μm2] Sum Output Name: Bright Red Spots/DNA area
	Method: Formula Output Formula: a/b Population Type: Objects Variable a: Red/Background Region Absolute threshold—Number of Objects Variable b: Background—Region Area [μm2] Sum Output Name: Bright Red Spots/Background area
	Object Results Population: Red/DNA region Ab thresh: None Population: Red/Background Region Ab thresh: None Population: DNA: ALL Population: Red spots in DNA region: None Population: Background: None Population: Red spots in background: None