This dataset contains all the Seurat objects that were used for generating all the figures in Pal et al. 2021 (https://doi.org/10.15252/embj.2020107333). All the Seurat objects were created under R v3.6.1 using the Seurat package v3.1.1. The detailed information of each object is listed in a table in Chen et al. 2021.

These datasets are generated by ReapTEC (read-level pre-filtering and transcribed enhancer call) using 5' single-cell RNA-seq data on human heterogenous CD4+ T cells. By taking advantage of a unique “cap signature” derived from the 5′-end of a transcript, ReapTEC simultaneously profiles gene expression and enhancer activity at nucleotide resolution using 5′-end single-cell RNA-sequencing (5′ scRNA-seq). The detail of ReapTEC pipeline is described in https://github.com/MurakawaLab/ReapTEC.

This is the Seurat object in .rds format with the raw matrix information (after filtering) , cell type annotation information and the UMAP coordinates. Users can use R readRDS function to load this .rds file. If you are using this dataset, please cite our paper: Qian, Peipei, Jiahui Kang, Dong Liu, and Gangcai Xie. "Single cell transcriptome sequencing of Zebrafish testis revealed novel spermatogenesis marker genes and stronger Leydig-germ cell paracrine interactions." Frontiers in genetics 13 (2022): 851719.

The dataset contains an integrated, annotated Seurat v4 object. One can load the dataset into the R environment using the code below:

seurat_obj <- readRDS('PATH/TO/DOWNLOAD/seurat.rds')

The object has three assays: (I) RNA, (II) SCT and (III) integrated.

GSE264573 single-cell seurat RDS

This is the accompanying data set for the paper entitled ‘T cell receptor-centric approach to streamline multimodal single-cell data analysis.’, which is currently available as a preprint (https://www.biorxiv.org/content/10.1101/2023.09.27.559702v2). Details on the origin of the datasets, and processing steps can be found there.

The purpose of this atlas both the full dataset and down sampling version is to aid in improving the interpretability of other T cell based datasets. This can be done by adding in the down sampled object that contains up to 500 cells per annotation model or all 12 dataset to your new sample. This dataset aims to improve the capacity to identify TCR-specific signature by ensuring a well covered background, which will improve the robustness of the FindMarker Function in Seurat package.

Seurat matrix referring to scRNA-seq of Mm1 mouse tumors in CyC manuscript

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, including endothelial subtypes distinguished by vessel-type of origin, fibro/adipogenic progenitors defined by functional roles, and many distinct immune populations. The representation of different experimental conditions and the depth of transcriptome coverage enabled robust profiling of sparsely expressed genes. We built a densely sampled transcriptomic model of myogenesis, from stem cell quiescence to myofiber maturation and identified rare, transitional states of progenitor commitment and fusion that are poorly represented in individual datasets. We performed spatial RNA sequencing of mouse muscle at three time points after injury and used the integrated dataset as a reference to achieve a high-resolution, local deconvolution of cell subtypes. We also used the integrated dataset to explore ligand-receptor co-expression patterns and identify dynamic cell-cell interactions in muscle injury response. We provide a public web tool to enable interactive exploration and visualization of the data. Our work supports the utility of large-scale integration of single-cell transcriptomic data as a tool for biological discovery.

Methods Mice. The Cornell University Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and experiments were performed in compliance with its institutional guidelines. Adult C57BL/6J mice (mus musculus) were obtained from Jackson Laboratories (#000664; Bar Harbor, ME) and were used at 4-7 months of age. Aged C57BL/6J mice were obtained from the National Institute of Aging (NIA) Rodent Aging Colony and were used at 20 months of age. For new scRNAseq experiments, female mice were used in each experiment.

Mouse injuries and single-cell isolation. To induce muscle injury, both tibialis anterior (TA) muscles of old (20 months) C57BL/6J mice were injected with 10 µl of notexin (10 µg/ml; Latoxan; France). At 0, 1, 2, 3.5, 5, or 7 days post-injury (dpi), mice were sacrificed and TA muscles were collected and processed independently to generate single-cell suspensions. Muscles were digested with 8 mg/ml Collagenase D (Roche; Switzerland) and 10 U/ml Dispase II (Roche; Switzerland), followed by manual dissociation to generate cell suspensions. Cell suspensions were sequentially filtered through 100 and 40 μm filters (Corning Cellgro #431752 and #431750) to remove debris. Erythrocytes were removed through incubation in erythrocyte lysis buffer (IBI Scientific #89135-030).

Single-cell RNA-sequencing library preparation. After digestion, single-cell suspensions were washed and resuspended in 0.04% BSA in PBS at a concentration of 106 cells/ml. Cells were counted manually with a hemocytometer to determine their concentration. Single-cell RNA-sequencing libraries were prepared using the Chromium Single Cell 3’ reagent kit v3 (10x Genomics, PN-1000075; Pleasanton, CA) following the manufacturer’s protocol. Cells were diluted into the Chromium Single Cell A Chip to yield a recovery of 6,000 single-cell transcriptomes. After preparation, libraries were sequenced using on a NextSeq 500 (Illumina; San Diego, CA) using 75 cycle high output kits (Index 1 = 8, Read 1 = 26, and Read 2 = 58). Details on estimated sequencing saturation and the number of reads per sample are shown in Sup. Data 1.

Spatial RNA sequencing library preparation. Tibialis anterior muscles of adult (5 mo) C57BL6/J mice were injected with 10µl notexin (10 µg/ml) at 2, 5, and 7 days prior to collection. Upon collection, tibialis anterior muscles were isolated, embedded in OCT, and frozen fresh in liquid nitrogen. Spatially tagged cDNA libraries were built using the Visium Spatial Gene Expression 3’ Library Construction v1 Kit (10x Genomics, PN-1000187; Pleasanton, CA) (Fig. S7). Optimal tissue permeabilization time for 10 µm thick sections was found to be 15 minutes using the 10x Genomics Visium Tissue Optimization Kit (PN-1000193). H&E stained tissue sections were imaged using Zeiss PALM MicroBeam laser capture microdissection system and the images were stitched and processed using Fiji ImageJ software. cDNA libraries were sequenced on an Illumina NextSeq 500 using 150 cycle high output kits (Read 1=28bp, Read 2=120bp, Index 1=10bp, and Index 2=10bp). Frames around the capture area on the Visium slide were aligned manually and spots covering the tissue were selected using Loop Browser v4.0.0 software (10x Genomics). Sequencing data was then aligned to the mouse reference genome (mm10) using the spaceranger v1.0.0 pipeline to generate a feature-by-spot-barcode expression matrix (10x Genomics).

Download and alignment of single-cell RNA sequencing data. For all samples available via SRA, parallel-fastq-dump (github.com/rvalieris/parallel-fastq-dump) was used to download raw .fastq files. Samples which were only available as .bam files were converted to .fastq format using bamtofastq from 10x Genomics (github.com/10XGenomics/bamtofastq). Raw reads were aligned to the mm10 reference using cellranger (v3.1.0).

Preprocessing and batch correction of single-cell RNA sequencing datasets. First, ambient RNA signal was removed using the default SoupX (v1.4.5) workflow (autoEstCounts and adjustCounts; github.com/constantAmateur/SoupX). Samples were then preprocessed using the standard Seurat (v3.2.1) workflow (NormalizeData, ScaleData, FindVariableFeatures, RunPCA, FindNeighbors, FindClusters, and RunUMAP; github.com/satijalab/seurat). Cells with fewer than 750 features, fewer than 1000 transcripts, or more than 30% of unique transcripts derived from mitochondrial genes were removed. After preprocessing, DoubletFinder (v2.0) was used to identify putative doublets in each dataset, individually. BCmvn optimization was used for PK parameterization. Estimated doublet rates were computed by fitting the total number of cells after quality filtering to a linear regression of the expected doublet rates published in the 10x Chromium handbook. Estimated homotypic doublet rates were also accounted for using the modelHomotypic function. The default PN value (0.25) was used. Putative doublets were then removed from each individual dataset. After preprocessing and quality filtering, we merged the datasets and performed batch-correction with three tools, independently- Harmony (github.com/immunogenomics/harmony) (v1.0), Scanorama (github.com/brianhie/scanorama) (v1.3), and BBKNN (github.com/Teichlab/bbknn) (v1.3.12). We then used Seurat to process the integrated data. After initial integration, we removed the noisy cluster and re-integrated the data using each of the three batch-correction tools.

Cell type annotation. Cell types were determined for each integration method independently. For Harmony and Scanorama, dimensions accounting for 95% of the total variance were used to generate SNN graphs (Seurat::FindNeighbors). Louvain clustering was then performed on the output graphs (including the corrected graph output by BBKNN) using Seurat::FindClusters. A clustering resolution of 1.2 was used for Harmony (25 initial clusters), BBKNN (28 initial clusters), and Scanorama (38 initial clusters). Cell types were determined based on expression of canonical genes (Fig. S3). Clusters which had similar canonical marker gene expression patterns were merged.

Pseudotime workflow. Cells were subset based on the consensus cell types between all three integration methods. Harmony embedding values from the dimensions accounting for 95% of the total variance were used for further dimensional reduction with PHATE, using phateR (v1.0.4) (github.com/KrishnaswamyLab/phateR).

Deconvolution of spatial RNA sequencing spots. Spot deconvolution was performed using the deconvolution module in BayesPrism (previously known as “Tumor microEnvironment Deconvolution”, TED, v1.0; github.com/Danko-Lab/TED). First, myogenic cells were re-labeled, according to binning along the first PHATE dimension, as “Quiescent MuSCs” (bins 4-5), “Activated MuSCs” (bins 6-7), “Committed Myoblasts” (bins 8-10), and “Fusing Myoctes” (bins 11-18). Culture-associated muscle stem cells were ignored and myonuclei labels were retained as “Myonuclei (Type IIb)” and “Myonuclei (Type IIx)”. Next, highly and differentially expressed genes across the 25 groups of cells were identified with differential gene expression analysis using Seurat (FindAllMarkers, using Wilcoxon Rank Sum Test; results in Sup. Data 2). The resulting genes were filtered based on average log2-fold change (avg_logFC > 1) and the percentage of cells within the cluster which express each gene (pct.expressed > 0.5), yielding 1,069 genes. Mitochondrial and ribosomal protein genes were also removed from this list, in line with recommendations in the BayesPrism vignette. For each of the cell types, mean raw counts were calculated across the 1,069 genes to generate a gene expression profile for BayesPrism. Raw counts for each spot were then passed to the run.Ted function, using

Brain Transcriptome Single-cell Atlas (BTS) Anndata, Seurat object, and Celltypist model for further use of the atlas. The Celltypist model can be utilized to accurately annotate cell types in new datasets based on the atlas. Plots illustrating the expression profile for 3,380 neurological disorder risk genes across the atlas are also uploaded. Further availability for the data can be requested by the corresponding author.

This dataset is published in Kim, S., Lee, J., Koh, I.G. et al. An integrative single-cell atlas for exploring the cellular and temporal specificity of genes related to neurological disorders during human brain development. Exp Mol Med 56, 2271–2282 (2024). https://doi.org/10.1038/s12276-024-01328-6

This project is a collection of files to allow users to reproduce the model development and benchmarking in "Dawnn: single-cell differential abundance with neural networks" (Hall and Castellano, under review). Dawnn is a tool for detecting differential abundance in single-cell RNAseq datasets. It is available as an R package here. Please contact us if you are unable to reproduce any of the analysis in our paper. The files in this collection correspond to the benchmarking dataset based on simulated linear trajectories.

FILES: Data processing code

adapted_traj_sim_milo_paper.R Lightly adapted code from Dann et al. to simulate single-cell RNAseq datasets that form linear trajectories . generate_test_data_linear_traj_sim_milo_paper.R R code to assign simulated labels to datatsets generated from adapted_traj_sim_milo_paper.R. Seurat objects saved as cells_sim_linear_traj_gex_seed_*.rds. Simulated labels saved as benchmark_dataset_sim_linear_traj.csv.

Resulting datasets

cells_sim_linear_traj_gex_seed_*.rds Seurat objects generated by generate_test_data_linear_traj_sim_milo_paper.R. benchmark_dataset_sim_linear_traj.csv Cell labels generated by generate_test_data_linear_traj_sim_milo_paper.R.

Context

Dataset from NeurIPS2021 challenge similar to Kaggle 2022 competition: https://www.kaggle.com/competitions/open-problems-multimodal "Open Problems - Multimodal Single-Cell Integration Predict how DNA, RNA & protein measurements co-vary in single cells"

It is https://en.wikipedia.org/wiki/ATAC-seq#Single-cell_ATAC-seq single cell ATAC-seq data. And single cell RNA-seq data: https://en.wikipedia.org/wiki/Single-cell_transcriptomics#Single-cell_RNA-seq

Single cell RNA sequencing, i.e. rows - correspond to cells, columns to genes (or vice versa). value of the matrix shows how strong is "expression" of the corresponding gene in the corresponding cell. https://en.wikipedia.org/wiki/Single-cell_transcriptomics

See tutorials: https://scanpy.readthedocs.io/en/stable/tutorials.html ("Scanpy" - main Python package to work with scRNA-seq data). Or https://satijalab.org/seurat/ "Seurat" - "R" package

(For companion dataset on CITE-seq = scRNA-seq + Proteomics, see: https://www.kaggle.com/datasets/alexandervc/citeseqscrnaseqproteins-challenge-neurips2021)

Particular data

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE194122

Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Summary Single-cell multiomics data collected from bone marrow mononuclear cells of 12 healthy human donors. Half the samples were measured using the 10X Multiome Gene Expression and Chromatin Accessability kit and half were measured using the 10X 3' Single-Cell Gene Expression kit with Feature Barcoding in combination with the BioLegend TotalSeq B Universal Human Panel v1.0. The dataset was generated to support Multimodal Single-Cell Data Integration Challenge at NeurIPS 2021. Samples were prepared using a standard protocol at four sites. The resulting data was then annotated to identify cell types and remove doublets. The dataset was designed with a nested batch layout such that some donor samples were measured at multiple sites with some donors measured at a single site. In the competition, participants were tasked with challenges including modality prediction, matching profiles from different modalities, and learning a joint embedding from multiple modalities.

Overall design Single-cell multiomics data collected from bone marrow mononuclear cells of 12 healthy human donors.

Contributor(s) Burkhardt DB, Lücken MD, Lance C, Cannoodt R, Pisco AO, Krishnaswamy S, Theis FJ, Bloom JM Citation https://datasets-benchmarks-proceedings.neurips.cc/paper/2021/hash/158f3069a435b314a80bdcb024f8e422-Abstract-round2.html

Related datasets:

Other single cell RNA seq datasets can be found on kaggle: Look here: https://www.kaggle.com/alexandervc/datasets Or search kaggle for "scRNA-seq"

Inspiration

Single cell RNA sequencing is important technology in modern biology, see e.g. "Eleven grand challenges in single-cell data science" https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-1926-6

Also see review : Nature. P. Kharchenko: "The triumphs and limitations of computational methods for scRNA-seq" https://www.nature.com/articles/s41592-021-01171-x

Search scholar.google "challenges in single cell rna sequencing" https://scholar.google.fr/scholar?q=challenges+in+single+cell+rna+sequencing&hl=en&as_sdt=0&as_vis=1&oi=scholart gives many interesting and highly cited articles

(Cited 968) Computational and analytical challenges in single-cell transcriptomics Oliver Stegle, Sarah A. Teichmann, John C. Marioni Nat. Rev. Genet., 16 (3) (2015), pp. 133-145 https://www.nature.com/articles/nrg3833

Remark 0: See https://arxiv.org/abs/2208.05229 "Computational challenges of cell cycle analysis using single cell transcriptomics" Alexander Chervov, Andrei Zinovyev For the cell cycle analysis

Remark 1:

Full dataset in https://www.kaggle.com/datasets/alexandervc/scrnaseq-crisprperturbseq-normanweissman But it is huge, and loading crashes memory, so here are cropped pieces to start with.

Remark 2:

dataset used in: "GEARS: Predicting transcriptional outcomes of novel multi-gene perturbations" Yusuf Roohani, Kexin Huang, Jure Leskovec https://www.biorxiv.org/content/10.1101/2022.07.12.499735v1 https://twitter.com/yusufroohani/status/1547965695744360448 Accepted in ICLR

Data and Context

Data - results of single cell RNA sequencing, i.e. rows - correspond to cells, columns to genes (or vice versa). value of the matrix shows how strong is "expression" of the corresponding gene in the corresponding cell. https://en.wikipedia.org/wiki/Single-cell_transcriptomics

See tutorials: https://scanpy.readthedocs.io/en/stable/tutorials.html ("Scanpy" - main Python package to work with scRNA-seq data). Or https://satijalab.org/seurat/ "Seurat" - "R" package

Particular data - Perturb-seq: Single-cell, pooled CRISPR screening experiment comparing the transcriptional effects of overexpressing genes alone or in combination

Paper: Norman TM, Horlbeck MA, Replogle JM, Ge AY et al. Exploring genetic interaction manifolds constructed from rich single-cell phenotypes. Science 2019 Aug 23;365(6455):786-793. PMID: 31395745 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746554/

Data: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133344

Related datasets:

Other single cell RNA seq datasets can be found on kaggle: Look here: https://www.kaggle.com/alexandervc/datasets Or search kaggle for "scRNA-seq"

Inspiration

Single cell RNA sequencing is important technology in modern biology, see e.g. "Eleven grand challenges in single-cell data science" https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-1926-6

Also see review : Nature. P. Kharchenko: "The triumphs and limitations of computational methods for scRNA-seq" https://www.nature.com/articles/s41592-021-01171-x

Search scholar.google "challenges in single cell rna sequencing" https://scholar.google.fr/scholar?q=challenges+in+single+cell+rna+sequencing&hl=en&as_sdt=0&as_vis=1&oi=scholart gives many interesting and highly cited articles

(Cited 968) Computational and analytical challenges in single-cell transcriptomics Oliver Stegle, Sarah A. Teichmann, John C. Marioni Nat. Rev. Genet., 16 (3) (2015), pp. 133-145 https://www.nature.com/articles/nrg3833

Challenges in unsupervised clustering of single-cell RNA-seq data https://www.nature.com/articles/s41576-018-0088-9 Review Article 07 January 2019 Vladimir Yu Kiselev, Tallulah S. Andrews & Martin Hemberg Nature Reviews Genetics volume 20, pages273–282 (2019)

Challenges and emerging directions in single-cell analysis https://link.springer.com/article/10.1186/s13059-017-1218-y Published: 08 May 2017 Guo-Cheng Yuan, Long Cai, Michael Elowitz, Tariq Enver, Guoping Fan, Guoji Guo, Rafael Irizarry, Peter Kharchenko, Junhyong Kim, Stuart Orkin, John Quackenbush, Assieh Saadatpour, Timm Schroeder, Ramesh Shivdasani & Itay Tirosh Genome Biology volume 18, Article number: 84 (2017)

Single-Cell RNA Sequencing in Cancer: Lessons Learned and Emerging Challenges https://www.sciencedirect.com/science/article/pii/S1097276519303569 Molecular Cell Volume 75, Issue 1, 11 July 2019, Pages 7-12 Journal home page for Molecular Cell

Processed Seurat objects in .rds format with single-cell dataset obtained from Bassez et al. (2021) Nature Medicine (https://www.nature.com/articles/s41591-021-01323-8).BassezA_2021_33958794_downsampled: Seurat object including all samples (42) with random downsampling to max 1000 cells per sample, without any further filtering.BassezA_2021_33958794_3patients: Seurat object with 3 patient samples from the original data set: "BIOKEY_11", "BIOKEY_30", and "BIOKEY_4" from "Pre" condition, without downsampling or further per sample filtering.

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.

In the context of the Human Cell Atlas, we have created a single-cell taxonomy of cell types and states in human tonsils. This repository contains the Seurat objects derived from this effort. In particular, we have datasets for each modality (scRNA-seq, scATAC-seq, CITE-seq, spatial transcriptomics), as well as cell type-specific datasets. Most importantly, this is the input that we used to create the HCATonsilData package, which allows programmatic access to all this datasets within R.

This data is used for the Seurat version of the batch correction and integration tutorial on the Galaxy Training Network. The input data was provided by Seurat in the 'Integrative Analysis in Seurat v5' tutorial. The input dataset provided here has been filtered to include only cells for which nFeature_RNA > 1000. The other datasets were produced on Galaxy. The original dataset was published as: Ding, J., Adiconis, X., Simmons, S.K. et al. Systematic comparison of single-cell and single-nucleus RNA-sequencing methods. Nat Biotechnol 38, 737–746 (2020). https://doi.org/10.1038/s41587-020-0465-8.

Summary: Dendritic cells (DCs) orchestrate innate and adaptive immunity, by translating the sensing of distinct danger signals into the induction of different effector lymphocyte responses, to induce different defense mechanisms suited to face distinct types of threats. Hence, DCs are very plastic, which results from two key characteristics. First, DCs encompass distinct cell types specialized in different functions. Second, each DC type can undergo different activation states, fine-tuning its functions depending on its tissue microenvironment and the pathophysiological context, by adapting the output signals it delivers to the input signals it receives. Hence, to better understand DC biology and harness it in the clinic, we must determine which combinations of DC types and activation states mediate which functions, and how.
To decipher the nature, functions and regulation of DC types and their physiological activation states, one of the methods that can be harnessed most successfully is ex vivo single cell RNA sequencing (scRNAseq). However, for new users of this approach, determining which analytics strategy and computational tools to choose can be quite challenging, considering the rapid evolution and broad burgeoning of the field. In addition, awareness must be raised on the need for specific, robust and tractable strategies to annotate cells for cell type identity and activation states. It is also important to emphasize the necessity of examining whether similar cell activation trajectories are inferred by using different, complementary methods. In this chapter, we take these issues into account for providing a pipeline for scRNAseq analysis and illustrating it with a tutorial reanalyzing a public dataset of mononuclear phagocytes isolated from the lungs of naïve or tumor-bearing mice. We describe this pipeline step-by-step, including data quality controls, dimensionality reduction, cell clustering, cell cluster annotation, inference of the cell activation trajectories and investigation of the underpinning molecular regulation. It is accompanied with a more complete tutorial on Github. We anticipate that this method will be helpful for both wet lab and bioinformatics researchers interested in harnessing scRNAseq data for deciphering the biology of DCs or other cell types, and that it will contribute to establishing high standards in the field.

Data:

MDAlab_cDC1_maturation.tar : Docker image used for the analysis

Context

Dataset from NeurIPS2021 challenge similar to Kaggle 2022 competition: https://www.kaggle.com/competitions/open-problems-multimodal "Open Problems - Multimodal Single-Cell Integration Predict how DNA, RNA & protein measurements co-vary in single cells"

CITE-seq - joint single cell RNA sequencing + single cell measurements of CD** proteins. (https://en.wikipedia.org/wiki/CITE-Seq) (For companion dataset on scRNA-seq + scATAC-seq, see: https://www.kaggle.com/datasets/alexandervc/scrnaseq-scatacseq-challenge-at-neurips-2021 )

Single cell RNA sequencing, i.e. rows - correspond to cells, columns to genes (or vice versa). value of the matrix shows how strong is "expression" of the corresponding gene in the corresponding cell. https://en.wikipedia.org/wiki/Single-cell_transcriptomics

See tutorials: https://scanpy.readthedocs.io/en/stable/tutorials.html ("Scanpy" - main Python package to work with scRNA-seq data). Or https://satijalab.org/seurat/ "Seurat" - "R" package

Particular data

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE194122

Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Summary Single-cell multiomics data collected from bone marrow mononuclear cells of 12 healthy human donors. Half the samples were measured using the 10X Multiome Gene Expression and Chromatin Accessability kit and half were measured using the 10X 3' Single-Cell Gene Expression kit with Feature Barcoding in combination with the BioLegend TotalSeq B Universal Human Panel v1.0. The dataset was generated to support Multimodal Single-Cell Data Integration Challenge at NeurIPS 2021. Samples were prepared using a standard protocol at four sites. The resulting data was then annotated to identify cell types and remove doublets. The dataset was designed with a nested batch layout such that some donor samples were measured at multiple sites with some donors measured at a single site. In the competition, participants were tasked with challenges including modality prediction, matching profiles from different modalities, and learning a joint embedding from multiple modalities.

Overall design Single-cell multiomics data collected from bone marrow mononuclear cells of 12 healthy human donors.

Contributor(s) Burkhardt DB, Lücken MD, Lance C, Cannoodt R, Pisco AO, Krishnaswamy S, Theis FJ, Bloom JM Citation https://datasets-benchmarks-proceedings.neurips.cc/paper/2021/hash/158f3069a435b314a80bdcb024f8e422-Abstract-round2.html

Related datasets:

Other single cell RNA seq datasets can be found on kaggle: Look here: https://www.kaggle.com/alexandervc/datasets Or search kaggle for "scRNA-seq"

Inspiration

Single cell RNA sequencing is important technology in modern biology, see e.g. "Eleven grand challenges in single-cell data science" https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-1926-6

Also see review : Nature. P. Kharchenko: "The triumphs and limitations of computational methods for scRNA-seq" https://www.nature.com/articles/s41592-021-01171-x

Search scholar.google "challenges in single cell rna sequencing" https://scholar.google.fr/scholar?q=challenges+in+single+cell+rna+sequencing&hl=en&as_sdt=0&as_vis=1&oi=scholart gives many interesting and highly cited articles

(Cited 968) Computational and analytical challenges in single-cell transcriptomics Oliver Stegle, Sarah A. Teichmann, John C. Marioni Nat. Rev. Genet., 16 (3) (2015), pp. 133-145 https://www.nature.com/articles/nrg3833

We profile the transcriptomes of ~30,000 mouse single cells to deconvolve the hepatic mesenchyme in healthy and fibrotic liver at high resolution. We reveal spatial zonation of hepatic stellate cells across the liver lobule, designated portal vein-associated HSC and central vein-associated HSC, and uncover an equivalent functional zonation in a mouse model of centrilobular fibrosis. Our work illustrates the power of single-cell transcriptomics to resolve key collagen-producing cells driving liver fibrosis with high precision. We provide the contents of these data as Seurat R objects.

RDS files containing processed Seurat objects for multiome analysis of neuroblastoma cell lines. File names reflect the cell line.