This data collection contains spatially resolved single-cell transcriptomics datasets acquired using MERFISH on the mouse nucleus accumbens (NAc) collected by the Xiaowei Zhuang Lab and Yi Zhang Lab at Harvard University and Howard Hughes Medical Institute. * The data collection is composed of 8 experiments, which include 27 coronal slices of the NAc region (10 µm thick slices, spaced at a 100 µm interval anterior to posterior) collected from 2 biological replicates. For each mouse, 4 experiments were performed, and each experiment was named with a {sample_id}, comprised of a mouse replicate number and a sample number, e.g. mouse1_sample1, mouse2_sample3. For each {sample_id}, multiple coronal slices (1-4 slices) were included on the same coverslip and were imaged together. Each slice is identified by a {slice_id}, e.g. slice7, where the number corresponds to its ordinal position in anterior-to-posterior sectioning. Each slice was imaged as a group of tiled fields-of-view (FOVs). Each FOV covered 223 µm x 223 µm in x and y, and the centers of each FOV belonging to an individual slice were separated by 200 µm. * In this dataset, a total of 253 genes were imaged. Among the 253 genes, 251 genes were imaged using MERFISH, which encodes individual genes with error-robust barcodes (20-bit binary codes in this case), imprints the barcodes onto the RNAs using combinatorial labeling with encoding probes, and measures the barcodes bit-by-bit using sequential hybridization of readout probes. The 20 bits are imaged in 10 hybridization rounds with two-color imaging each round. The remaining 2 genes were imaged by 1 round of two-color FISH and an additional such round was acquired using no fluorescent probes to estimate local background. In addition to gene imaging, additional imaging rounds were included to image markers (polyT and DAPI) for cell segmentation. In one experiment, mouse2_sample2, nucRed was substituted for DAPI. All bits used for MERFISH imaging of the 251 genes and all segmentation markers were imaged in 7 z-planes spaced apart by 1.5 µm, starting at 0 µm and ending at 9.0 µm above the surface of the coverglass. All bits used for the remaining 2 genes imaged by two-color FISH were imaged in a single z-plane 4.5 µm above the surface of the coverglass. * Each of the subdirectory folders contains either the raw (e.g. mouse1_sample1_raw) or processed (e.g. mouse1_sample1_processed) images of one experiment. Each experiment contains many fields of view (FOVs), with the images from each FOV present as a single file, with the images from all the bits, segmentation markers, and z planes combined into a single multi-frame tiff file that was then compressed with zstandard. The raw image files are named as aligned_images plus the FOV id (e.g. aligned_images0.tif.zstd); the processed image files are named as processed_images plus the FOV id (e.g. processed_images100.tif.zstd). • {sample_id}_raw: Folders containing raw images. Each image has been corrected for x-y drift that occurred during the rounds of imaging using fiducial beads. Aligned images are named aligned_images{FOV}.tif.zstd, where {FOV} is the number of the FOV used for that set of images. The tiff file contains the images of all z-planes from all bits, background, and segmentation imaging rounds. See data_organization_raw.csv file for detailed channel information. • {sample_id}_processed: Folders containing processed images. Each of the aligned images are processed with a high-pass filter followed by deconvolution. Processed images are named processed_images{FOV}.tif.zstd. For each FOV, the corresponding processed image tiff file contains the images of all z-plans from the bits used in the combinatorial imaging as well as the unprocessed images from the segmentation imaging rounds. See data_organization_processed.csv file for detailed channel information, and preprocessing.json file for parameters used in image processing. * The processed_data folder contains the following files: • segmented_cells_{sample_id}.csv: Segmented cell boundary coordinates (x,y,z) in the unit of microns for each cell and its corresponding slice id. The first column (cellID) is a unique cell identifier, the second column (z_height) is height of the 7 z planes relative to the coverslip. The third and forth columns (x-boundary, y_boundary) are the x and y boundary coordinates, respectively, arranged as a 3-tiered list (list of lists of lists), wherein the top tier corresponds to the z-heights (as ordered in the second column), the second tier corresponds to the number of discrete areas of the segmentation for each z plane (note that in most cases, this is a single area, but in some instances the cell boundary was split into more than one discrete area), and the third tier (lowest) contains a list of the boundary coordinates. Finally, the fifth column (slice_id) gives the slice number to which each cell belongs. Note that the same coordinate system is used for the cell boundaries and decoded spots (below). • spots_{sample_id}.csv: Decoded spot location (x,y,z) in the unit of microns and their target gene identity for each experiment. The same coordinate system was used in segmented_cells_{sample_id}.csv and spots_{sample_id}.csv for each experiment, and hence the spots can be parsed into the segmented cells (and also the slices) by comparison of their coordinates with the cell boundary locations. • counts.h5ad: Cell by gene matrix of the whole dataset. We also provide these files that are associated to this dataset: • genes_combinatorial.txt: List of target genes that are imaged in combinatorial imaging rounds. • genes_sequential.txt: List of target genes that are imaged sequentially after the combinatorial imaging rounds. • probes.fasta: Provides sequences all encoding probes used for hybridization. • codebook.csv: Provides the barcodes that encode individual genes measured in the combinatorial imaging rounds. • data_organization_raw.csv: Provides information on how individual channels and z-planes are ordered in the multi-frame tiff file for each field of view of raw images for all sample_ids except mouse2_sample2. • dataorganization_raw_mouse2_sample2.csv: A separate data_organization_raw file specific to mouse2_sample2 (wherein nucRed was used in place of DAPI) • data_organization_processed.csv: Provides information on how individual channels and z-planes are ordered in the multi-frame tiff file for each field of view of processed images for all sample_ids except mouse2_sample2. • dataorganization_processed_mouse2_sample2.csv: A separate data_organization_processed file specific to mouse2_sample2 (wherein nucRed was used in place of DAPI) • microscope.json: Provides parameters of the microscope that was used with pixel size and orientation of images. • preprocessing.json: Provides values and filters used in generating the processed images. • sliceFOVs: Provides the set of FOVs that correspond to each slice according to its slice id. • "fov_coordinates" folder: contains a csv file for each slice, named by {sample_id}_{slice_id} as described above. Each file contains three columns, the first column is the number of the FOV, the second and third columns are the x and y coordinates of the FOV (in µm), respectively. These coordinates can be used to arrange the FOVs of an experiment as they were arranged on the coverslip.