NIS-Seq Analysis Suite v1.0
JSB lab 2020-2024

1. Enter experiment or well name:


2. Enter number of cycles:


3. Load In-situ images (TIFF, 4 channels, 2048x2048, 16bit, sorted by cycle > tile > channel):

Download example data HeLa (1 tile)

4. Load nuclear masks (Generate with CellPose, TIFF, 1 channel, 2048x2048, 16 bit, sorted by tile):


5. Load or calculate NIS-Seq cycle alignment:
Load: (tab delimited, x (px) - y (px), no header)
Or calculate: Calculate alignment (7 seconds per cycle)

6. Load or detect spots:
Load: (tab delimited, tile - x (px) - y (px), no header)
Or calculate:
Brightness threshold: au
Detect spots

7. Load compensation matrix:
(no header)
Download compensation_matrix_NextSeq2000_jsb-lab_2022.txt

8. Perform sequence calling:
Start sequence calling

9. Load reference library and filter matching spots:
(tab delimited, gene - sequence, no header)
Download Brunello human sgRNA library and scrambled control
Filter

10. Determine maximum NIS-Seq intensity per nucleus across cycles and channels:
Start measurement

11. Collapse sequences to nuclei
Minimum NIS-Seq intensity per nucleus (from 10.): au
Minimum relative intensity of top sequence: %
Assign library-matching sequences to nuclei

Inspect raw images:



Cycle
Tile
Channel
Brightness
High-pass frequency filter

test