INSCRIBE STAR Protocols: Code and demonstration data for multiplexed smFISH and ratiometric barcode readout

Abstract

This repository contains the analysis code and demonstration data accompanying the INSCRIBE STAR Protocols manuscript. The workflow processes multiplexed smFISH imaging followed by ratiometric barcode readout in HEK293 cells, integrating gene-expression measurements and barcode editing levels at single-cell resolution. The deposit includes numbered Jupyter notebooks and Fiji/ImageJ macros, projected and registered images, segmentation masks, probability maps, ilastik project files, and quantified single-cell measurements. The computational workflow covers focus selection and projection, registration across imaging rounds, cell segmentation and mask matching, transcript quantification, ratiometric barcode quantification, and integration of measurements across rounds. The demonstration starts from the supplied projected images. The focus-projection notebook requires original CZI z-stacks, which are not included because they exceed the repository’s standard upload limit. All original raw imaging data are available from the corresponding author upon request. Refer to the included README for software requirements, folder organization, and execution instructions.