This dataset contains Spatial Transcriptomics (ST) data matching with Matrix Assisted Laser Desorption/Ionization - Mass Spetrometry Imaging (MALDI-MSI). This data is complementary to data contained in the same project. FIles with the same identifiers in the two datasets originated from the very same tissue section and can be combined in a multimodal ST-MSI object. For more information about the dataset please see our manuscript posted on BioRxiv (doi: https://doi.org/10.1101/2023.01.26.525195). This dataset includes ST data from 19 tissue sections, including human post-mortem and mouse samples. The spatial transcriptomics data was generated using the Visium protocol (10x Genomics). The murine tissue sections come from three different mice unilaterally injected with 6-OHDA. 6-OHDA is a neurotoxin that when injected in the brain can selectively destroy dopaminergic neurons. We used this mouse model to show the applicability of the technology that we developed, named Spatial Multimodal Analysis (SMA). Using our technology on these mouse brain tissue sections we were able to detect both dopamine with MALDI-MSI and the corresponding gene expression with ST. This dataset includes also one human post-mortem striatum sample that was placed on one Visium slide across the four capture areas. This sample was analyzed with a different ST protocol named RRST (Mirzazadeh, R., Andrusivova, Z., Larsson, L. et al. Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples. Nat Commun 14, 509 (2023). https://doi.org/10.1038/s41467-023-36071-5), where probes capturing the whole transcriptome are first hybridized in the tissue section and then spatially detected. Each tissue section contained in the dataset has been given a unique identifier that is composed of the Visium array ID and capture area ID of the Visium slide that the tissue section was placed on. This unique identifier is included in the file names of all the files relative to the same tissue section, including the MALDI-MSI files published in the other dataset included in this project. In this dataset you will find the following files for each tissue section: - raw files: these are the read one fastq files (containing the pattern *R1*fastq.gz in the file name), read two fastq files (containing the pattern *R1*fastq.gz in the file name) and the raw microscope images (containing the pattern Spot.jpg in the file name). These are the only files needed to run the Space Ranger pipeline, which is freely available for any user (please see the 10x Genomics website for information on how to install and run Space Ranger); - processed data files: we provide processed data files of two types: a) Space Ranger outputs that were used to produce the figures in our publication; b) manual annotation tables in csv format produced using Loupe Browser 6 (csv tables with file names ending _RegionLoupe.csv, _filter.csv, _dopamine.csv, _lesion.csv, _region.csv patterns); c) json files that we used as input for Space Ranger in the cases where the automatic tissue detection included in the pipeline failed to recognize the tissue or the fiducials. Using these processed files the user can reproduce the figures of our publication without having to restart from the raw data files. The MALDI-MSI analyses preceding ST was performed with different matrices in different tissue section. We used 1) 9-aminoacridine (9-AA) for detection of metabolites in negative ionization mode, 2) 2,5-dihydroxybenzoic acid (DHB) for detection of metabolites in positive ionization mode, 3) 4-(anthracen-9-yl)-2-fluoro-1-ethylpyridin-1-ium iodide (FMP-10), which charge-tags molecules with phenolic hydroxyls and/or primary amines, including neurotransmitters. The information about which matrix was sprayed on the tissue sections and other information about the samples is included in the metadata table. We also used three types of control samples: - standard Visium: samples processed with standard Visium (i.e. no matrix spraying, no MALDI-MSI, protocol as recommended by 10x Gemomics with no exeptions) - internal controls (iCTRL): samples not sprayed with any matrix, neither processed with MALDI-MSI, but located on the same Visium slide were other samples were processed with MALDI-MSI - FMP-10-iCTRL: sample sprayed with FMP-10, and then processed as an iCTRL. This and other information is provided in the metadata table.

Monitoring neutrophil gene expression is a powerful tool for understanding disease mechanisms, developing new diagnostics, therapies and optimizing clinical trials. Neutrophils are sensitive to the processing, storage and transportation steps that are involved in clinical sample analysis. This study is the first to evaluate the capabilities of technologies from 10X Genomics, PARSE Biosciences, and HIVE (Honeycomb Biotechnologies) to generate high-quality RNA data from human blood-derived neutrophils. Our comparative analysis shows that all methods produced high quality data, importantly capturing the transcriptomes of neutrophils. 10X FLEX cell populations in particular showed a close concordance with the flow cytometry data. Here, we establish a reliable single-cell RNA sequencing workflow for neutrophils in clinical trials: we offer guidelines on sample collection to preserve RNA quality and demonstrate how each method performs in capturing sensitive cell populations in clinical practice.

This dataset includes only the 10X Flex time course data and analysis.

Skeletal muscle repair is driven by the coordinated self-renewal and fusion of myogenic stem and progenitor cells. Single-cell gene expression analyses of myogenesis have been hampered by the poor sampling of rare and transient cell states that are critical for muscle repair, and do not inform the spatial context that is important for myogenic differentiation. Here, we demonstrate how large-scale integration of single-cell and spatial transcriptomic data can overcome these limitations. We created a single-cell transcriptomic dataset of mouse skeletal muscle by integration, consensus annotation, and analysis of 23 newly collected scRNAseq datasets and 88 publicly available single-cell (scRNAseq) and single-nucleus (snRNAseq) RNA-sequencing datasets. The resulting dataset includes more than 365,000 cells and spans a wide range of ages, injury, and repair conditions. Together, these data enabled identification of the predominant cell types in skeletal muscle, and resolved cell subtypes, in...

This dataset details the scRNASeq and TCR-Seq analysis of sorted PD-1+ CD8+ T cells from patients with melanoma treated with checkpoint therapy (anti-PD-1 monotherapy and anti-PD-1 & anti-CTLA-4 combination therapy) at baseline and after the first cycle of therapy. A major publication using this dataset is accessible here: (reference)

*experimental design

Single-cell RNA sequencing was performed using 10x Genomics with feature barcoding technology to multiplex cell samples from different patients undergoing mono or dual therapy so that they can be loaded on one well to reduce costs and minimize technical variability. Hashtag oligomers (oligos) were obtained as purified and already oligo-conjugated in TotalSeq-C format from BioLegend. Cells were thawed, counted and 20 million cells per patient and time point were used for staining. Cells were stained with barcoded antibodies together with a staining solution containing antibodies against CD3, CD4, CD8, PD-1/IgG4 and fixable viability dye (eBioscience) prior to FACS sorting. Barcoded antibody concentrations used were 0.5 µg per million cells, as recommended by the manufacturer (BioLegend) for flow cytometry applications. After staining, cells were washed twice in PBS containing 2% BSA and 0.01% Tween 20, followed by centrifugation (300 xg 5 min at 4 °C) and supernatant exchange. After the final wash, cells were resuspended in PBS and filtered through 40 µm cell strainers and proceeded for sorting. Sorted cells were counted and approximately 75,000 cells were processed through 10x Genomics single-cell V(D)J workflow according to the manufacturer’s instructions. Gene expression, hashing and TCR libraries were pooled to desired quantities to obtain the sequencing depths of 15,000 reads per cell for gene expression libraries and 5,000 reads per cell for hashing and TCR libraries. Libraries were sequenced on a NovaSeq 6000 flow cell in a 2X100 paired-end format.

*extract protocol

PBMCs were thawed, counted and 20 million cells per patient and time point were used for staining. Cells were stained with barcoded antibodies together with a staining solution containing antibodies against CD3, CD4, CD8, PD-1/IgG4 and fixable viability dye (eBioscience) prior to FACS sorting. Barcoded antibody concentrations used were 0.5 µg per million cells, as recommended by the manufacturer (BioLegend) for flow cytometry applications. After staining, cells were washed twice in PBS containing 2% BSA and 0.01% Tween 20, followed by centrifugation (300 xg 5 min at 4 °C) and supernatant exchange. After the final wash, cells were resuspended in PBS and filtered through 40 µm cell strainers and proceeded for sorting. Sorted cells were counted and approximately 75,000 cells were processed through 10x Genomics single-cell V(D)J workflow according to the manufacturer’s instructions.

*library construction protocol

Sorted cells were counted and approximately 75,000 cells were processed through 10x Genomics single-cell V(D)J workflow according to the manufacturer’s instructions. Gene expression, hashing and TCR libraries were pooled to desired quantities to obtain the sequencing depths of 15,000 reads per cell for gene expression libraries and 5,000 reads per cell for hashing and TCR libraries. Libraries were sequenced on a NovaSeq 6000 flow cell in a 2X100 paired-end format.

*library strategy

scRNA-seq and scTCR-seq

*data processing step

Pre-processing of sequencing results to generate count matrices (gene expression and HTO barcode counts) was performed using the 10x genomics Cell Ranger pipeline.

Further processing was done with Seurat (cell and gene filtering, hashtag identification, clustering, differential gene expression analysis based on gene expression).

*genome build/assembly

Alignment was performed using prebuilt Cell Ranger human reference GRCh38.

*processed data files format and content

RNA counts and HTO counts are in sparse matrix format and TCR clonotypes are in csv format.

Datasets were merged and analyzed by Seurat and the analyzed objects are in rds format.

This project contains the datasets used to evaluate and reproduce the results of ENACT (End-to-End Analysis and Cell Type
Annotation for Visium HD Slides). The dataset consists of:

• a sample Visium HD sample of human colorectal cancer, courtesy of 10X Genomics (Visium HD Spatial Gene Expression Library, Human Colorectal Cancer (FFPE) - 10x Genomics). All credit goes to 10X Genomics.
• configuration files to be used to reproduce the results provided in the ENACT publication,
• evaluation plots used in the ENACT publication, and
• results obtained after running ENACT on the human colorectal cancer sample using the four bin-to-cell assignment methods (naive, weighted_by_area, weighted_by_transcript, and weighted_by_cluster) and the three cell annotation methods (Sargent, CellAssign, CellTypist)

Additionally, results from running ENACT on the following three public VisiumHD samples are provided to showcase ENACT’s tissue-agnostic nature:

Arsenic exposure via drinking water is a serious environmental health concern. Epidemiological studies suggest a strong association between prenatal arsenic exposure and subsequent childhood respiratory infections, as well as morbidity from respiratory diseases in adulthood, long after systemic clearance of arsenic. We investigated the impact of exclusive prenatal arsenic exposure on the inflammatory immune response and respiratory health after an adult influenza A (IAV) lung infection. C57BL/6J mice were exposed to 100 ppb sodium arsenite in utero, and subsequently infected with IAV (H1N1) after maturation to adulthood. Assessment of lung tissue and bronchoalveolar lavage fluid (BALF) at various time points post IAV infection reveals greater lung damage and inflammation in arsenic exposed mice versus control mice. Single-cell RNA sequencing analysis of immune cells harvested from IAV infected lungs suggests that the enhanced inflammatory response is mediated by dysregulation of innate immune function of monocyte derived macrophages, neutrophils, NK cells, and alveolar macrophages. Our results suggest that prenatal arsenic exposure results in lasting effects on the adult host innate immune response to IAV infection, long after exposure to arsenic, leading to greater immunopathology. This study provides the first direct evidence that exclusive prenatal exposure to arsenic in drinking water causes predisposition to a hyperinflammatory response to IAV infection in adult mice, which is associated with significant lung damage.

Methods Whole lung homogenate preparation for single cell RNA sequencing (scRNA-seq).

Lungs were perfused with PBS via the right ventricle, harvested, and mechanically disassociated prior to straining through 70- and 30-µm filters to obtain a single-cell suspension. Dead cells were removed (annexin V EasySep kit, StemCell Technologies, Vancouver, Canada), and samples were enriched for cells of hematopoetic origin by magnetic separation using anti-CD45-conjugated microbeads (Miltenyi, Auburn, CA). Single-cell suspensions of 6 samples were loaded on a Chromium Single Cell system (10X Genomics) to generate barcoded single-cell gel beads in emulsion, and scRNA-seq libraries were prepared using Single Cell 3’ Version 2 chemistry. Libraries were multiplexed and sequenced on 4 lanes of a Nextseq 500 sequencer (Illumina) with 3 sequencing runs. Demultiplexing and barcode processing of raw sequencing data was conducted using Cell Ranger v. 3.0.1 (10X Genomics; Dartmouth Genomics Shared Resource Core). Reads were aligned to mouse (GRCm38) and influenza A virus (A/PR8/34, genome build GCF_000865725.1) genomes to generate unique molecular index (UMI) count matrices. Gene expression data have been deposited in the NCBI GEO database and are available at accession # GSE142047.

Preprocessing of single cell RNA sequencing (scRNA-seq) data

Count matrices produced using Cell Ranger were analyzed in the R statistical working environment (version 3.6.1). Preliminary visualization and quality analysis were conducted using scran (v 1.14.3, Lun et al., 2016) and Scater (v. 1.14.1, McCarthy et al., 2017) to identify thresholds for cell quality and feature filtering. Sample matrices were imported into Seurat (v. 3.1.1, Stuart., et al., 2019) and the percentage of mitochondrial, hemoglobin, and influenza A viral transcripts calculated per cell. Cells with < 1000 or > 20,000 unique molecular identifiers (UMIs: low quality and doublets), fewer than 300 features (low quality), greater than 10% of reads mapped to mitochondrial genes (dying) or greater than 1% of reads mapped to hemoglobin genes (red blood cells) were filtered from further analysis. Total cells per sample after filtering ranged from 1895-2482, no significant difference in the number of cells was observed in arsenic vs. control. Data were then normalized using SCTransform (Hafemeister et al., 2019) and variable features identified for each sample. Integration anchors between samples were identified using canonical correlation analysis (CCA) and mutual nearest neighbors (MNNs), as implemented in Seurat V3 (Stuart., et al., 2019) and used to integrate samples into a shared space for further comparison. This process enables identification of shared populations of cells between samples, even in the presence of technical or biological differences, while also allowing for non-overlapping populations that are unique to individual samples.

Clustering and reference-based cell identity labeling of single immune cells from IAV-infected lung with scRNA-seq

Principal components were identified from the integrated dataset and were used for Uniform Manifold Approximation and Projection (UMAP) visualization of the data in two-dimensional space. A shared-nearest-neighbor (SNN) graph was constructed using default parameters, and clusters identified using the SLM algorithm in Seurat at a range of resolutions (0.2-2). The first 30 principal components were used to identify 22 cell clusters ranging in size from 25 to 2310 cells. Gene markers for clusters were identified with the findMarkers function in scran. To label individual cells with cell type identities, we used the singleR package (v. 3.1.1) to compare gene expression profiles of individual cells with expression data from curated, FACS-sorted leukocyte samples in the Immgen compendium (Aran D. et al., 2019; Heng et al., 2008). We manually updated the Immgen reference annotation with 263 sample group labels for fine-grain analysis and 25 CD45+ cell type identities based on markers used to sort Immgen samples (Guilliams et al., 2014). The reference annotation is provided in Table S2, cells that were not labeled confidently after label pruning were assigned “Unknown”.

Differential gene expression by immune cells

Differential gene expression within individual cell types was performed by pooling raw count data from cells of each cell type on a per-sample basis to create a pseudo-bulk count table for each cell type. Differential expression analysis was only performed on cell types that were sufficiently represented (>10 cells) in each sample. In droplet-based scRNA-seq, ambient RNA from lysed cells is incorporated into droplets, and can result in spurious identification of these genes in cell types where they aren’t actually expressed. We therefore used a method developed by Young and Behjati (Young et al., 2018) to estimate the contribution of ambient RNA for each gene, and identified genes in each cell type that were estimated to be > 25% ambient-derived. These genes were excluded from analysis in a cell-type specific manner. Genes expressed in less than 5 percent of cells were also excluded from analysis. Differential expression analysis was then performed in Limma (limma-voom with quality weights) following a standard protocol for bulk RNA-seq (Law et al., 2014). Significant genes were identified using MA/QC criteria of P < .05, log2FC >1.

Analysis of arsenic effect on immune cell gene expression by scRNA-seq.

Sample-wide effects of arsenic on gene expression were identified by pooling raw count data from all cells per sample to create a count table for pseudo-bulk gene expression analysis. Genes with less than 20 counts in any sample, or less than 60 total counts were excluded from analysis. Differential expression analysis was performed using limma-voom as described above.

Additional file 3. Cell type specific isoforms in psl format.

This dataset contains files reconstructing single-cell data presented in 'Reference transcriptomics of porcine peripheral immune cells created through bulk and single-cell RNA sequencing' by Herrera-Uribe & Wiarda et al. 2021. Samples of peripheral blood mononuclear cells (PBMCs) were collected from seven pigs and processed for single-cell RNA sequencing (scRNA-seq) in order to provide a reference annotation of porcine immune cell transcriptomics at enhanced, single-cell resolution. Analysis of single-cell data allowed identification of 36 cell clusters that were further classified into 13 cell types, including monocytes, dendritic cells, B cells, antibody-secreting cells, numerous populations of T cells, NK cells, and erythrocytes. Files may be used to reconstruct the data as presented in the manuscript, allowing for individual query by other users. Scripts for original data analysis are available at https://github.com/USDA-FSEPRU/PorcinePBMCs_bulkRNAseq_scRNAseq. Raw data are available at https://www.ebi.ac.uk/ena/browser/view/PRJEB43826. Funding for this dataset was also provided by NRSP8: National Animal Genome Research Program (https://www.nimss.org/projects/view/mrp/outline/18464). Resources in this dataset:Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - All Cells 10X Format. File Name: PBMC7_AllCells.zipResource Description: Zipped folder containing PBMC counts matrix, gene names, and cell IDs. Files are as follows:

matrix of gene counts* (matrix.mtx.gx) gene names (features.tsv.gz) cell IDs (barcodes.tsv.gz)

*The ‘raw’ count matrix is actually gene counts obtained following ambient RNA removal. During ambient RNA removal, we specified to calculate non-integer count estimations, so most gene counts are actually non-integer values in this matrix but should still be treated as raw/unnormalized data that requires further normalization/transformation. Data can be read into R using the function Read10X().Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - All Cells Metadata. File Name: PBMC7_AllCells_meta.csvResource Description: .csv file containing metadata for cells included in the final dataset. Metadata columns include:

nCount_RNA = the number of transcripts detected in a cell nFeature_RNA = the number of genes detected in a cell Loupe = cell barcodes; correspond to the cell IDs found in the .h5Seurat and 10X formatted objects for all cells prcntMito = percent mitochondrial reads in a cell Scrublet = doublet probability score assigned to a cell seurat_clusters = cluster ID assigned to a cell PaperIDs = sample ID for a cell celltypes = cell type ID assigned to a cellResource Title: Herrera-Uribe & Wiarda et al. PBMCs - All Cells PCA Coordinates. File Name: PBMC7_AllCells_PCAcoord.csvResource Description: .csv file containing first 100 PCA coordinates for cells. Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - All Cells t-SNE Coordinates. File Name: PBMC7_AllCells_tSNEcoord.csvResource Description: .csv file containing t-SNE coordinates for all cells.Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - All Cells UMAP Coordinates. File Name: PBMC7_AllCells_UMAPcoord.csvResource Description: .csv file containing UMAP coordinates for all cells.Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - CD4 T Cells t-SNE Coordinates. File Name: PBMC7_CD4only_tSNEcoord.csvResource Description: .csv file containing t-SNE coordinates for only CD4 T cells (clusters 0, 3, 4, 28). A dataset of only CD4 T cells can be re-created from the PBMC7_AllCells.h5Seurat, and t-SNE coordinates used in publication can be re-assigned using this .csv file.Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - CD4 T Cells UMAP Coordinates. File Name: PBMC7_CD4only_UMAPcoord.csvResource Description: .csv file containing UMAP coordinates for only CD4 T cells (clusters 0, 3, 4, 28). A dataset of only CD4 T cells can be re-created from the PBMC7_AllCells.h5Seurat, and UMAP coordinates used in publication can be re-assigned using this .csv file.Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - Gamma Delta T Cells UMAP Coordinates. File Name: PBMC7_GDonly_UMAPcoord.csvResource Description: .csv file containing UMAP coordinates for only gamma delta T cells (clusters 6, 21, 24, 31). A dataset of only gamma delta T cells can be re-created from the PBMC7_AllCells.h5Seurat, and UMAP coordinates used in publication can be re-assigned using this .csv file.Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - Gamma Delta T Cells t-SNE Coordinates. File Name: PBMC7_GDonly_tSNEcoord.csvResource Description: .csv file containing t-SNE coordinates for only gamma delta T cells (clusters 6, 21, 24, 31). A dataset of only gamma delta T cells can be re-created from the PBMC7_AllCells.h5Seurat, and t-SNE coordinates used in publication can be re-assigned using this .csv file.Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - Gene Annotation Information. File Name: UnfilteredGeneInfo.txtResource Description: .txt file containing gene nomenclature information used to assign gene names in the dataset. 'Name' column corresponds to the name assigned to a feature in the dataset.Resource Title: Herrera-Uribe & Wiarda et al. PBMCs - All Cells H5Seurat. File Name: PBMC7.tarResource Description: .h5Seurat object of all cells in PBMC dataset. File needs to be untarred, then read into R using function LoadH5Seurat().

Datasets and Code accompanying the new release of RCA, RCA2. The R-package for RCA2 is available at GitHub: https://github.com/prabhakarlab/RCAv2/

The datasets included here are:

Datasets required for a characterization of batch effects:

merged_rna_seurat.rds

de_list.rds

mergedRCAObj.rds

merged_rna_integrated.rds

10X_PBMCs.RDS: Processed 10X PBMC data RCA2 object (10X PBMC example data sets )

NBM_RDS_Files.zip: Several RDS files containing RCA2 object of Normal Bone Marrow (NBM) data, umap coordinates, doublet finder results and metadata information (Normal Bone Marrow use case)

Dataset used for the Covid19 example:

blish_covid.seu.rds

rownames_of_glocal_projection_immune_cells.txt

Blish_RCA_no_QC_filtering_project_to_multiple_panels.rds

Data sets used to outline the ability of supervised clustering to detect disease states:

809653.seurat.rds

blish_covid.seu.rds

Performance benchmarking results:

Memory_consumption.txt

rca_time_list.rds

ScanPY input files:

input_data.zip

The R script provides R code to regenerate the main paper Figures 2 to 7 modulo some visual modifications performed in Inkscape.

Provided R scripts are:

ComputePairWiseDE_v2.R (Required code for pairwise DE computation)

RCA_Figure_Reproduction.R

Provided python Code for Scanpy analysis:

RA_Scanpy.ipynb

CITESeq_Scanpy.ipynb

The single-cell sequencing technology platform market is experiencing robust growth, projected to reach a market size of $115 million in 2025 and exhibiting a Compound Annual Growth Rate (CAGR) of 10.1% from 2025 to 2033. This expansion is driven by several key factors. The increasing demand for personalized medicine and the rising prevalence of complex diseases like cancer are major catalysts. Single-cell sequencing allows researchers to analyze individual cells within a complex biological sample, providing unparalleled insights into cellular heterogeneity and disease mechanisms. This granular level of analysis is crucial for developing targeted therapies and improving diagnostic accuracy. Furthermore, technological advancements, such as the development of more efficient and cost-effective sequencing platforms, are accelerating market adoption. The emergence of novel applications in areas like immunology, neuroscience, and developmental biology further fuels market growth. Competition among leading companies such as 10x Genomics, BD, BGI, Singleron, and others is fostering innovation and driving down costs, making the technology more accessible to a wider range of researchers and clinicians. The market's segmentation is likely diverse, encompassing various sequencing platforms (e.g., microfluidic, plate-based), applications (e.g., oncology, immunology), and end-users (e.g., academic research institutions, pharmaceutical companies, hospitals). While precise segment breakdown data is unavailable, it's reasonable to expect that oncology and immunology applications currently represent the largest market segments, given their high research activity and the potential for therapeutic advancements. The geographic distribution is likely concentrated in North America and Europe initially, given the higher levels of research funding and technological infrastructure in these regions. However, the market is expected to expand rapidly in Asia and other developing economies as research capabilities and funding increase. Market restraints include the high cost of sequencing, the complexity of data analysis, and the need for skilled personnel. However, ongoing technological advancements and decreasing costs are gradually mitigating these challenges, paving the way for wider adoption.

The single-cell genomics market is experiencing robust growth, driven by advancements in sequencing technologies, decreasing costs, and increasing applications across diverse research areas. The market's expansion is fueled by the rising demand for personalized medicine, improved diagnostic capabilities, and a deeper understanding of complex biological processes at a cellular level. Key applications include cancer research (identifying cancer stem cells and understanding tumor heterogeneity), immunology (analyzing immune cell populations and responses), and drug discovery (identifying novel drug targets and predicting drug efficacy). The market is segmented by technology (e.g., microfluidics, next-generation sequencing), application, and end-user (e.g., pharmaceutical companies, research institutions). While technological complexities and high initial investment costs present some barriers, ongoing innovations in sample preparation, data analysis, and bioinformatics are mitigating these challenges. The competitive landscape is marked by the presence of both established players like Thermo Fisher Scientific and Qiagen, and emerging companies developing innovative single-cell solutions. This dynamic market is poised for continued expansion, driven by collaborative efforts between academia and industry to advance the field and broaden its applications. The forecast period of 2025-2033 promises substantial growth for single-cell genomics, with a projected CAGR (let's assume a conservative 15% based on industry trends). This expansion reflects the increasing adoption of single-cell technologies across various sectors. The market’s considerable size in 2025 (let’s assume $2 Billion based on the scale of related markets) indicates a strong foundation for future growth. Further market penetration is likely to be witnessed in developing economies as research capabilities and funding increase. Key growth drivers include the development of more accessible and user-friendly platforms, improved data analysis software, and increased investment in research and development. Understanding the complex cellular heterogeneity in diverse diseases will continue to drive demand, making single-cell genomics an essential tool in advancing biomedical research and clinical applications.

This page includes the data and code necessary to reproduce the results of the following paper: Yang Liao, Dinesh Raghu, Bhupinder Pal, Lisa Mielke and Wei Shi. cellCounts: fast and accurate quantification of 10x Chromium single-cell RNA sequencing data. Under review. A Linux computer running an operating system of CentOS 7 (or later) or Ubuntu 20.04 (or later) is recommended for running this analysis. The computer should have >2 TB of disk space and >64 GB of RAM. The following software packages need to be installed before running the analysis. Software executables generated after installation should be included in the $PATH environment variable.

R (v4.0.0 or newer) https://www.r-project.org/ Rsubread (v2.12.2 or newer) http://bioconductor.org/packages/3.16/bioc/html/Rsubread.html CellRanger (v6.0.1) https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome STARsolo (v2.7.10a) https://github.com/alexdobin/STAR sra-tools (v2.10.0 or newer) https://github.com/ncbi/sra-tools Seurat (v3.0.0 or newer) https://satijalab.org/seurat/ edgeR (v3.30.0 or newer) https://bioconductor.org/packages/edgeR/ limma (v3.44.0 or newer) https://bioconductor.org/packages/limma/ mltools (v0.3.5 or newer) https://cran.r-project.org/web/packages/mltools/index.html

Reference packages generated by 10x Genomics are also required for this analysis and they can be downloaded from the following link (2020-A version for individual human and mouse reference packages should be selected): https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest After all these are done, you can simply run the shell script ‘test-all-new.bash’ to perform all the analyses carried out in the paper. This script will automatically download the mixture scRNA-seq data from the SRA database, and it will output a text file called ‘test-all.log’ that contains all the screen outputs and speed/accuracy results of CellRanger, STARsolo and cellCounts.

The emerging single-cell technology market is experiencing rapid growth, driven by advancements in genomics, proteomics, and bioinformatics. This technology allows researchers to analyze individual cells, providing unprecedented insights into cellular heterogeneity and function across various biological systems. The market's expansion is fueled by increasing demand for personalized medicine, drug discovery, and disease diagnostics. Applications span oncology, immunology, neuroscience, and infectious diseases, with single-cell RNA sequencing (scRNA-seq) currently dominating the market share. The high cost of instrumentation and data analysis remains a barrier to wider adoption, but ongoing technological innovations are driving down costs and improving accessibility. Furthermore, the development of new analytical tools and bioinformatics pipelines is enhancing data interpretation and accelerating research progress. This burgeoning field is attracting significant investment and collaborative efforts from both established players and innovative startups, fostering a competitive yet collaborative landscape. The projected market growth signifies a transformative impact on healthcare and life sciences, promising significant advancements in disease understanding and treatment. The forecast period from 2025 to 2033 anticipates substantial market expansion, propelled by increasing adoption across research institutions, pharmaceutical companies, and biotechnology firms. Key growth drivers include the development of more affordable and user-friendly single-cell technologies, the integration of multi-omics approaches (combining genomics, proteomics, and metabolomics), and expanding collaborations between academia and industry. Competitive pressures are driving innovation in areas like sample preparation, data analysis software, and the development of novel single-cell applications, such as spatial transcriptomics. Although challenges such as data complexity and the need for specialized expertise persist, the potential for single-cell technologies to revolutionize biological research and healthcare remains immense. This is reflected in the continuous influx of funding and the emergence of new market participants. By 2033, the market is poised to be significantly larger and more diverse, with a wider range of applications and technological advancements shaping the future of biological research and medicine.

The Spatial Omics market, valued at $335.60 million in 2025, is poised for significant growth, exhibiting a Compound Annual Growth Rate (CAGR) of 10.60% from 2025 to 2033. This robust expansion is driven by several key factors. Advancements in technologies like spatial transcriptomics, spatial genomics, and spatial proteomics are providing researchers with unprecedented insights into the spatial organization of biological systems. This detailed understanding is crucial for accelerating drug discovery and development, enabling more precise diagnostics, and advancing translational research. The increasing adoption of single-cell analysis techniques further fuels market growth, as researchers strive to understand cellular heterogeneity and interactions within tissues and organs. Furthermore, the growing demand for high-throughput screening and personalized medicine is creating a strong pull for these technologies across pharmaceutical and biotechnology companies as well as academic research institutions. The availability of comprehensive software solutions for data analysis and interpretation is also a significant contributing factor, simplifying the complex data generated by these technologies. The market segmentation reveals a diverse landscape. Instruments currently dominate the product segment, reflecting the high capital expenditure associated with acquiring advanced spatial omics platforms. However, consumables and software are expected to experience substantial growth, driven by increasing research activity and the continuous need for reagents and analytical tools. Formalin-Fixed Paraffin-Embedded (FFPE) samples currently hold a significant share, leveraging existing pathology workflows. However, fresh frozen samples are gaining traction due to their superior preservation quality for certain applications. In terms of applications, diagnostics is a rapidly growing segment, promising to revolutionize personalized medicine by allowing for spatially-resolved diagnoses of diseases. North America currently holds a major share of the market, benefiting from a strong presence of key players and robust funding for biomedical research. However, Asia Pacific is projected to witness significant growth driven by increasing investment in life sciences and healthcare infrastructure. The competitive landscape is marked by the presence of established players such as 10x Genomics and NanoString Technologies, alongside emerging companies developing innovative spatial omics technologies. This dynamic ecosystem is indicative of a field ripe for continued innovation and expansion. Recent developments include: March 2024: 10x Genomics initiated commercial distribution of its eagerly anticipated Visium HD Spatial Gene Expression instrument. Visium HD is engineered to empower researchers with the ability to thoroughly analyze FFPE tissue samples by quantifying spatial gene expression across the entire transcriptome at a single-cell resolution., January 2024: SimBioSys obtained 510(k) clearance from the US Food and Drug Administration (FDA). This clearance permits the marketing of TumorSight Viz, a software application designed to generate three-dimensional spatial visualizations of breast tumors.. Key drivers for this market are: High Burden of Cancer and Genetic Diseases, Advancement in Omics Technologies and Increasing Demand for Personalized Medicine; Rising Government Initiatives and Funding Activities. Potential restraints include: High Burden of Cancer and Genetic Diseases, Advancement in Omics Technologies and Increasing Demand for Personalized Medicine; Rising Government Initiatives and Funding Activities. Notable trends are: The Spatial Transcriptomics Segment is Expected to Hold a Significant Market Share Over the Forecast Period.

The spatial transcriptomic technology market is experiencing robust growth, projected to reach $3912 million in 2025 and maintain a Compound Annual Growth Rate (CAGR) of 11.5% from 2025 to 2033. This expansion is driven by the increasing need for high-resolution spatial profiling of gene expression in biological tissues, enabling a deeper understanding of complex biological processes and disease mechanisms. Key drivers include advancements in imaging technologies, the development of more sensitive and efficient detection methods, and the growing adoption of spatial transcriptomics in diverse research areas such as oncology, neuroscience, and immunology. The rising prevalence of chronic diseases, coupled with increasing investments in biomedical research and development, further fuels market growth. Leading companies like 10x Genomics, NanoString, and Vizgen are at the forefront of innovation, continuously developing and improving spatial transcriptomic platforms and analysis tools. Competitive pressures are promoting technological advancements, making spatial transcriptomics more accessible and affordable for a broader range of researchers and clinical laboratories. Despite significant progress, market penetration is still relatively low compared to other genomics technologies. This presents substantial future growth opportunities. Continued advancements in data analysis tools, along with a decreasing cost-per-sample, will significantly impact broader adoption. The development of standardized protocols and data sharing initiatives are also crucial factors for promoting wider uptake within the scientific community. Furthermore, regulatory approvals and reimbursement policies will influence the market trajectory in the clinical diagnostics space. The market will likely witness increased collaboration between research institutions, biotechnology companies, and healthcare providers to maximize the clinical translation of spatial transcriptomics findings.

Supplementary data supporting the SpatialOne: End-to-End Analysis of Spatial Transcriptomics at Scale publication

To showcase the capabilities of SpatialOne, two human lung cancer formalin-fixed, paraffin-embedded (FFPE) samples are analyzed. These samples are prepared following the CG000495 protocol (Figure 1b), sequenced with the 10x Visium CytAssist, and processed using the 10x SpaceRanger version 2. We also present analysis of two adult mouse samples sequenced using 10x Visium samples (one fresh frozen brain tissue section processed using SpaceRanger v2 and one FFPE kidney sample processed using the SpaceRanger v1), and 75 internal samples.

For the human lung cancer samples, single-cell data from the the Lung Cancer Atlas (Salcher et al., 2022) is used as reference. This dataset is filtered to include only Chromium-generated data. For the mice samples, the GSE107585 single-cell dataset serves as reference. In the human lung cancer datasets, a pathologist annotated regions of interest corresponding to tumors, blood vessels, and alveolar regions.

Changelog:

Added a README file describing the zip content.

Raw gene expression counts are provided for:

all viable cells from 4T1 tumors (4T1_vaibale_raw_counts.txt),
CAFs from 4T1-Thy1.1 tumor (4T1_Thy1.1_CAF_raw.txt),
CAFs from mT3 tumor (MT3_CAF_raw.txt) and
normal mammary fibroblasts (Normal_mammary_fibroblasts_raw.txt).

As a part of the HuBMAP Consortium, the Skin Tissue Mapping Center (TMC), a joint effort by University of Pittsburgh (Pitt)-Department of Dermatology & the GE HealthCare team, aims to re-create functional units of skin and its cellular organization thereby generating a comprehensive spatial cellular atlas in 2D and 3D using a range of spatial and non-spatial tissue analysis tools. Our cohort includes skin samples from chronic sun-exposed and under-sun-exposed anatomical regions from healthy donors representing various age groups, skin color, and gender. These samples are analyzed using multiplexed imaging (Cell DIVE), scRNA sequencing and spatial transcriptomics. The resultant data are integrated and used for 2D cell classification & 3D volume reconstruction of skin structures in sequential sections. Additional characterization of immune cells, lymphovascular and neural components are performed in collaboration with other HuBMAP mapping centers.

Dataset for the manuscript Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing. Contains fastq files for 4 brain samples obtained from short-read NovaSeq 6000 v1.5 Illumina and long-read Oxford Nanopore PromethION sequencing. Single-cell suspensions were generated using 10x Genomics Chromium Next GEM Single Cell 3'Kit v3.1