DOI: 10.5061/dryad.p8cz8wb1m
title: Example Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) data from the mouse colon

description: Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) is a
 massively multiplexed single-RNA-molecule imaging technique capable of
 spatially resolved single-cell transcriptomic profiling of thousands of
 genes in millions of cells within intact tissue slices. Initially
 introduced for brain tissues, MERFISH has since been extended to other
 tissues, where rapid RNA degradation during the preparation process can
 pose challenges. The companion paper to these example data outlines the
 application of MERFISH in one such challenging tissue, the mammalian
 gastrointestinal tract. The example data contained here represent raw
 MERFISH images and associated metadata necessary to illustrate the
 analysis pipeline that identifies RNAs from such data. 
description: # Imaging the intestinal transcriptome with Multiplexed Error-Robust
 Fluorescence In Situ Hybridization (MERFISH) Paolo Cadinu Jeffrey R.
 Moffitt Boston Children's Hospital, 2025 ## Description This
 repository contains a subset of a raw MERFISH measurement of the mouse
 gastrointestinal tract. The images, metadata, and example scripts are
 designed to illustrate the use of an existing analysis pipeline for
 MERFISH raw data decoding. These data are provided as a companion dataset
 to the detailed instructions and protocols provided in Cadinu, Yang, Xu,
 et al., Current Protocols 2025. ## Main folder This folder contains a set
 of raw image and metadata files derived from a MERFISH measurement. \--
 .tif files represent image stacks taken from individual fields of view at
 different hybridization rounds and saved on one of two different cameras
 \-- .xml files contain metadata associated with the hardware settings used
 to collect the image stack ### Settings folder This folder contains files
 that describe the MERFISH measurement. ### data organization.csv This file
 details the organization of the raw data. It contains the following
 fields: * bitName: Identifier for the readout oligo probe * imageType: The
 base name of the image file * imageRegExp: Regular expression defining the
 file name, constructed using the imageType, field of view number, and
 imaging camera ID * bitNumber: Numeric identifier for the readout bit *
 imagingRound: Hybridization round, ranging from -1 to 13 * color: Color
 channel associated with the image * imagingCameraID: Identifier for the
 imaging camera * frame: List of acquired frames * zPos: List of z-stack
 plane positions relative to the coverslip surface, expressed in microns *
 fiducialImageType: Analogous to imageType, but specific to fiducial beads
 * fiducialRegExp: Regular expression for fiducial bead file names *
 fiducialImagingRound: Hybridization round for fiducial beads *
 fiducialFrame: List of acquired frames for fiducial beads * fiducialColor:
 Channel associated with fiducial beads * fiducialCameraID: Camera ID used
 for fiducial beads ### Fibl2v1E1\_codebook.csv This file includes a list
 of genes and blanks present in the Fibl2v1E1 library, along with their
 corresponding binary barcodes. It contains the following fields: * name:
 The gene name * id: The Ensembl isoform ID * barcode: The binary barcode
 associated with each gene ### Transforms.matb This file contains a set of
 microscope-specific geometrical transformations used to correct the
 chromatic aberration in the X-Y planes between the channels used to
 acquire the MERFISH signals. It is stored in a format accessible by the
 analysis code. ### FPKM.matb This file provides the abundance of all genes
 present in the library, as measured by bulk RNA-seq. It is stored in a
 format accessible by the analysis code. It includes the following fields:
 * FPKM: abundance expressed in FPKM * geneName: gene name ## Example
 Script folder Code in this folder is designed to be used with public
 analysis code stored here:
 [https://github.com/ZhuangLab/MERFISH_analysis](https://github.com/ZhuangLab/MERFISH_analysis) ### startup.m When placed in the directory from which MATLAB is launched, MATLAB will run this script. It will configure the necessary paths to all needed analysis. NOTE: This script must be updated to reflect paths specific to each computer on which it is run. ### MERFISH\_Decoding.m This MATLAB file provides the complete routine for decoding a MERFISH dataset. ### plot\_barcodes\_space.py This Python script is used to visualize all the decoded barcodes in their spatial positions.