DOI: 10.5061/dryad.w0vt4b922
title: Data from: Three-dimensional single-cell transcriptome imaging of thick tissues

description: Multiplexed error-robust fluorescence in-situ hybridization
 (MERFISH) allows genome-scale imaging of RNAs in individual cells in
 intact tissues. To date, MERFISH has been applied to image thin tissue
 samples of ∼10-µm thickness. Here, we present a method to enable
 three-dimensional (3D) single-cell transcriptome imaging of thick tissue
 specimens by integrating MERFISH with confocal microscopy for optical
 sectioning and deep learning to increase imaging speed and quality. We
 demonstrated 3D MERFISH on mouse brain tissue sections of up to 200 µm
 thickness with high detection efficiency and accuracy. We anticipate that
 3D thick-tissue MERFISH imaging will broaden the scope of questions that
 can be addressed by spatial genomics.
description: # Data from: Three-dimensional single-cell transcriptome imaging of thick
 tissues This dataset includes processed data and code used to generate the
 results of the following study conducted by the Xiaowei Zhuang Lab at
 Harvard University and the Howard Hughes Medical Institute: Fang Rongxin*,
 Halpern Aaron R.*, Rahman Mohammed Mostafizur, Huang Zhengkai, Lei Zhiyun,
 Hell Sebastian J., Dulac Catherine, Zhuang Xiaowei (2023).
 Three-dimensional single-cell transcriptome imaging of thick tissues.
 eLife, 12.
 [https://doi.org/10.7554/eLife.90029.1](https://doi.org/10.7554/eLife.90029.1) (* equal contribution) This dataset includes three-dimensional, spatially resolved transcriptome imaging data from two experiments: * A 100 μm-thick slice of the mouse cortical region, collected from one adult P56 male animal. * A 200 μm-thick slice of the mouse hypothalamic region, collected from one adult P56 male animal. The 100 μm cortex slice was imaged using a 242-gene panel, while the 200 μm hypothalamus slice was imaged with a 156-gene panel. Both gene panels were analyzed using MERFISH, a technique that encodes individual genes with error-robust barcodes. The 242-gene panel used a 22-bit binary code with a Hamming distance of 4 and a Hamming weight of 4 as described in our previous study (10.1038/s41586-021-03705-x). Similarly, the 156-gene panel used a 20-bit binary code with comparable properties as described in our previous study (10.1126/science.aau5324). These binary barcodes were imprinted onto the RNAs using combinatorial labeling with encoding probes and measured bit-by-bit using sequential hybridization of readout probes with two-color imaging used in each round. ### Versioning **Dec 2024:** README updated, no significant changes. ## Data Organization The folder structure is shown below: ``` . ├── data │ ├── data_Figure1 │ │ ├── 100ms_DL_HFP.tif │ │ ├── 100ms_HPF.tif │ │ ├── barcodes_1000ms.csv │ │ ├── barcodes_100ms.csv │ │ └── barcodes_100ms_DL.csv │ ├── data_Figure1_S1_disk_vs_epi │ │ ├── dis_405.tif │ │ ├── dis_488.tif │ │ ├── dis_560.tif │ │ ├── dis_650.tif │ │ ├── epi_405.tif │ │ ├── epi_488.tif │ │ ├── epi_560.tif │ │ └── epi_650.tif │ ├── data_Figure2_S1_encoding_probe_concentration │ │ ├── encoding_probe_0.1nM.csv │ │ ├── encoding_probe_0.1nM.tif │ │ ├── encoding_probe_0.5nM.csv │ │ ├── encoding_probe_0.5nM.tif │ │ ├── encoding_probe_1.0nM.csv │ │ ├── encoding_probe_1.0nM.tif │ │ ├── encoding_probe_1.5nM.csv │ │ ├── encoding_probe_1.5nM.tif │ │ ├── encoding_probe_0.1nM_example_image.tif │ │ ├── encoding_probe_0.5nM_example_image.tif │ │ ├── encoding_probe_1.0nM_example_image.tif │ │ └── encoding_probe_1.5nM_example_image.tif │ ├── data_Figure2_S1_readout_probe_concentration │ │ ├── readout_probe_1.0nM.tif │ │ ├── readout_probe_5.0nM.tif │ │ ├── readout_probe_7.5nM.tif │ │ ├── readout_probe_10.0nM.tif │ │ ├── readout_probe_1.0nM_thunderSTORM.csv │ │ ├── readout_probe_5.0nM_thunderSTORM.csv │ │ ├── readout_probe_7.5nM_thunderSTORM.csv │ │ └── readout_probe_10.0nM_thunderSTORM.csv │ ├── mouse.cortex.242genes.100um │ │ ├── barcode_freq_fov_z_100um_100um_thin_tissue.csv │ │ ├── transcripts.csv │ │ ├── transcripts_per_feature.csv │ │ ├── codebook.csv │ │ ├── feature_metadata.csv │ │ ├── M1_bulk.csv │ │ ├── README.txt │ │ └── seurat_obj.rds │ └── mouse.hypothalamus.156genes.200um │ ├── transcripts.csv │ ├── transcripts_per_feature.csv │ ├── codebook.csv │ ├── feature_metadata.csv │ └── seurat_obj.rds ├── notebooks │ ├── Figure1.ipynb │ ├── Figure2.ipynb │ ├── Figure2-S1.ipynb │ ├── Figure3-MOP.ipynb │ ├── Figure3-MPOA.ipynb │ ├── Figure3-f_h.ipynb │ ├── Figure3-S1.ipynb │ ├── Figure3-S2a.ipynb │ ├── Figure3-S2b.ipynb │ ├── Figure3-S3.ipynb │ └── Figure3-S4.ipynb ├── README.md └── README.html ``` * data/data_Figure1/: Contains the data used to generate the results in Figure 1. * 100ms_HPF.tif: Image shown in Figure 1a. * 100ms_DL_HPF.tif: Image shown in Figure 1b. * barcodes_1000ms.csv: Identified transcripts for 242 genes from a MERFISH experiment in a 10μm thin brain tissue section, taken with an exposure time of 1 second. * barcodes_100ms.csv: Identified transcripts for 242 genes from a MERFISH experiment in a 10μm thin brain tissue section, taken with an exposure time of 0.1 seconds. * barcodes_100ms_DL.csv: Identified transcripts for 242 genes from a MERFISH experiment in a 10μm thin brain tissue section, taken with an exposure time of 0.1 seconds with deep learning enhancement. * data/data_Figure1_S1_disk_vs_epi/: Contains the data shown in Figure 1 supplementary 1. * dis_405.tif: Spinning disk confocal image for channel 405nm. * dis_488.tif: Spinning disk confocal image for channel 488nm. * dis_560.tif: Spinning disk confocal image for channel 560nm. * dis_650.tif: Spinning disk confocal image for channel 650nm. * epi_405.tif: Epifluorescence image for channel 405nm. * epi_488.tif: Epifluorescence image for channel 488nm. * epi_560.tif: Epifluorescence image for channel 560nm. * epi_650.tif: Epifluorescence image for channel 650nm. * data/data_Figure2_S1_encoding_probe_concentration/: Contains the images and data used for generating results in Figure 2 Supplementary Figure 1a-b. * encoding_probe_0.1nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex stainined with MERFISH library with concentration of 0.1nM per probe. * encoding_probe_0.1nM.csv: RNA molecules detected encoding_probe_0.1nM.tif using software thunderSTORM with default paramter setting. * encoding_probe_0.5nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex stainined with MERFISH library with concentration of 0.5nM per probe. * encoding_probe_0.5nM.csv: RNA molecules detected encoding_probe_0.5nM.tif using software thunderSTORM with default paramter setting. * encoding_probe_1.0nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex stainined with MERFISH library with concentration of 1.0nM per probe. * encoding_probe_1.0nM.csv: RNA molecules detected encoding_probe_1.0nM.tif using software thunderSTORM with default paramter setting. * encoding_probe_1.5nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex stainined with MERFISH library with concentration of 1.5nM per probe. * encoding_probe_1.5nM.csv: RNA molecules detected encoding_probe_1.5nM.tif using software thunderSTORM with default paramter setting. * encoding_probe_0.1nM_example_image.tif: Example image shown Figure 2 - figure supplementary 1a. * encoding_probe_0.5nM_example_image.tif: Example image shown Figure 2 - figure supplementary 1a. * encoding_probe_1.0nM_example_image.tif: Example image shown Figure 2 - figure supplementary 1a. * encoding_probe_1.5nM_example_image.tif: Example image shown Figure 2 - figure supplementary 1a. * data/data_Figure2_S1_readout_probe_concentration/: Contains the images and data used for generating results in Figure 2 Supplementary Figure 1c-d. * readout_probe_1.0nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex imaged with readout probe concentration 1.0nM. * readout_probe_5.0nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex imaged with readout probe concentration 5.0nM. * readout_probe_7.5nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex imaged with readout probe concentration 7.5nM. * readout_probe_10.0nM.tif: High-pass filtered image of one bit of 242-gene MERFISH in mouse cortex imaged with readout probe concentration 10.0nM. * readout_probe_1.0nM_thunderSTORM.csv: RNA molecules detected readout_probe_1.0nM.tif using software thunderSTORM with default paramter setting. * readout_probe_5.0nM_thunderSTORM.csv: RNA molecules detected readout_probe_5.0nM.tif using software thunderSTORM with default paramter setting. * readout_probe_7.5nM_thunderSTORM.csv: RNA molecules detected readout_probe_7.5nM.tif using software thunderSTORM with default paramter setting. * readout_probe_10.0nM_thunderSTORM.csv: RNA molecules detected readout_probe_10.0nM.tif using software thunderSTORM with default paramter setting. * data/mouse.cortex.242genes.100um/transcripts.csv: Lists the identified RNA transcript locations. * barcode_id: Indicates the barcode ID for each identified transcript. * global_x, global_y, and global_z: Represent the coordinates of the transcript in micrometers. * x and y: Represent the pixel coordinates of the transcript in the field of view. * fov: Refers to the field of view ID the transcript belongs to. * data/mouse.cortex.242genes.100um/transcripts_per_feature.csv: Contains the cell-by-gene matrix, where each row represents a cell and each column represents a gene. * data/mouse.cortex.242genes.100um/M1_bulk.csv: Bulk RNA-seq data from mouse primary motor cortex. * data/mouse.cortex.242genes.100um/barcode_freq_fov_z_100um_100um_thin_tissue.csv: Number of transcripts per 100^2um^2 for 10um per zplane measured by MERFISH in the 10um thin tissue slices. * data/mouse.cortex.242genes.100um/feature_metadata.csv: Contains the segmented cell coordinates. * id: Represents the cell ID. * fov: Refers to the field of view number. * num_z: Indicates the number of z-planes that capture the cell. * center_x, center_y, and center_z: Represent the coordinates in micrometers. * x and y: Represent the pixel coordinates in each field of view. * data/mouse.cortex.242genes.100um/codebook.csv: Contains the gene names and their corresponding MERFISH binary barcodes. * data/mouse.cortex.242genes.100um/seurat_obj.rds: A Seurat object containing the processed single-cell analysis results. * data/mouse.hypothalamus.156genes.200um/transcripts.csv: Lists the identified RNA transcript locations. * barcode_id: Indicates the barcode ID for each identified transcript. * global_x, global_y, and global_z: Represent the coordinates of the transcript in micrometers. * x and y: Represent the pixel coordinates of the transcript in the field of view. * fov: Refers to the field of view ID the transcript belongs to. * data/mouse.hypothalamus.156genes.200um/feature_metadata.csv: Contains the segmented cell coordinates. * id: Represents the cell ID. * fov: Refers to the field of view number. * num_z: Indicates the number of z-planes that capture the cell. * center_x, center_y, and center_z: Represent the coordinates in micrometers. * x and y: Represent the pixel coordinates in each field of view. * data/mouse.hypothalamus.156genes.200um/codebook.csv: Contains the gene names and their corresponding MERFISH binary barcodes. * data/mouse.hypothalamus.156genes.200um/seurat_obj.rds: A Seurat object containing the processed single-cell analysis results. * notebooks/Figure1.ipynb - notebook for generating results in Figure 1. * notebooks/Figure2.ipynb - notebook for generating results in Figure 2. * notebooks/Figure2-S1.ipynb - notebook for generating results in Figure 2 – figure supplement 1. * notebooks/Figure3-f_h.ipynb - notebook for generating results in Figure 3f,3h. * notebooks/Figure3-MOP.ipynb - notebook for generating results in Figure 3a,3e. * notebooks/Figure3-MPOA.ipynb - notebook for generating results in Figure 3c,3g. * notebooks/Figure3-S1.ipynb - notebook for generating results in Figure 3 – figure supplement 1. * notebooks/Figure3-S2a.ipynb - notebook for generating results in Figure 3 – figure supplement 2a. * notebooks/Figure3-S2b.ipynb - notebook for generating results in Figure 3 – figure supplement 2b. * notebooks/Figure3-S3.ipynb - notebook for generating results in Figure 3 – figure supplement 3. * notebooks/Figure3-S4.ipynb - notebook for generating results in Figure 3 – figure supplement 4. Our raw imaging data is approximately 3TB, which exceeds the maximum upload limit for Dryad (300GB per submission). If you are interested in accessing the raw imaging data, please contact the authors directly for further assistance.