Mouse embryonic RNA-seq

10.1038/nature18633

The study was aimed at interrogating the early stages of blood cell development within the embryo. Blood develops from mesoderm, which is specified at gastrulation. We therefore sorted cells from mouse embryos between E6.5 and E7.75 to capture gastrulation and the emergence of mesoderm, followed by specificiation of the first mature blood cells.

Single cell RNA-seq of adult human neural retina

10.15252/embj.2018100811

To comprehensively profile cell types in the human retina, we performed single cell RNA-sequencing on 20,009 cells obtained post-mortem from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct clusters representing all known retinal cells: rod photoreceptors, cone photoreceptors, Muller glia cells, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, retinal astrocytes and microglia.

Single-cell RNA sequencing of human steady-state immune cells from the lamina propria of colon and matching mesenteric lymph nodes

10.1038/s41590-020-0602-z

The human colon contains an extensively diverse microbial ecosystem and one of the most numerous communities of immune cells. Studies have highlighted dynamic crosstalk between immune cells and commensals. While studies have demonstrated increasing diversity of microbiota from stomach to stool, whether and how immune cell heterogeneity and microbiota diversity change across the colon is undefined. Furthermore, whether these changes are co-depended in the healthy colon is unknown. Here, tissue samples are collected from caecum, transverse colon, sigmoid colon and mLN of cadaveric donors by the Cambridge Biorepository of Translational Medicine (CBTM). We use single cell RNA sequencing (10X genomics) to assess the dynamics of immune cell populations across the colon and in matching lymph nodes. Associated microbiome 16S sequencing data is available.

Human fetal liver, skin and kidney single cell transcriptome data

10.1038/s41586-019-1652-y

This is a droplet-based single cell transcriptome data set from 13 human fetal livers (6-18 PCW) and 8 skin and kidney samples (6-12 PCW). It includes 199,642 cells with a mean detected gene number of 3000.

Single cell RNA on hemocytes from wandering larvae of Drosophila melanogaster not infested and infested by Leptopilina boulardi

10.15252/embj.2020104486

We aimed at characterizing the diverse hemocyte populations present in the hemolymph of the Drosophila larvae. The hemocytes were collected from wandering larvae infested by wasp (WI) or not infested (NI). The hemocytes were then sequenced using 10x genomics technology.

Spatio-temporal immune zonation of the human kidney

10.1126/science.aat5031

Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly, or via crosstalk with immune cells. We resolved the spatio-temporal immune topology of the human kidney with a scRNAseq dataset from 40,268 mature and 27,203 fetal kidney cells. Within the epithelial compartment there were zonated patterns of immune gene expression, with pelvic epithelial anti-microbial peptide expression in mature but not fetal kidneys. Tissue-resident immune cells were evident in both fetal and mature kidney, with post-natal acquisition of infection-defense capabilities. Epithelial-immune signalling orchestrated localization of macrophages and neutrophils to regions of the kidney susceptible to infection.

Transcriptional landscape of porcine circulating immune cells

10.3389/fgene.2021.689406

Bulk RNA-seq from immune sorted cells and single cell RNA sequencing data from porcine PBMCs were generated to determine the gene expression pattern of porcine immune cells. This study is part of the FAANG project, promoting rapid prepublication of data to support the research community. These data are released under Fort Lauderdale principles, as confirmed in the Toronto Statement (Toronto International Data Release Workshop. Birney et al. 2009. Pre-publication data sharing. Nature 461:168-170). Any use of this dataset must abide by the FAANG data sharing principles. Data producers reserve the right to make the first publication of a global analysis of this data. If you are unsure if you are allowed to publish on this dataset, please contact the FAANG Data Coordination Centre and FAANG consortium (email faang-dcc@ebi.ac.uk and cc faang@iastate.edu) to enquire. The full guidelines can be found at http://www.faang.org/data-share-principle."

The Fly Cell Atlas: single-cell transcriptomes of the entire adult Drosophila - 10x dataset

10.1126/science.abk2432

Single-cell technologies have ushered in a new era for Drosophila research, allowing researchers to obtain transcriptomes for all stable cell types and dynamic cell states. Here we present the first release of the adult Fly Cell Atlas (FCA), which includes 580k cells from 15 individually dissected sexed tissues, as well as from the entire head and body. We annotated more than 250 distinct cell types across all tissues. Few cell types were uniquely detected in the entire head and body samples, suggesting high cell-type saturation. We provide an in-depth analysis of gene signatures and transcription factor combinations, as well as sexual dimorphism, across the whole animal. Finally, we studied cell type lineages that are shared between tissues, such as blood cells and muscle cells, allowing the retrieval of rare cell types and tissue- specific subtypes. This atlas provides a valuable resource for the entire Drosophila community as a reference to study genetic perturbations and disease models at single-cell resolution.

snRNA-seq of chicken (Gallus gallus) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 8,413 nuclei in chicken adult testis. This dataset includes two samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of mouse (Mus musculus) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 5,264 nuclei in mouse adult testis. This dataset includes two samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of platypus (Ornithorhynchus anatinus) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 6,751 nuclei in platypus adult testis. This dataset includes two samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of opossum (Monodelphis domestica) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 9,926 nuclei in opossum adult testis. This dataset includes three samples from three different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of gorilla (Gorilla gorilla) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 7,359 nuclei in gorilla adult testis. This dataset includes three samples from three different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of macaque (macaca mulatta) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 7,631 nuclei in macaque adult testis. This dataset includes two samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of chimpanzee (Pan troglodytes) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 12,352 nuclei in chimpanzee adult testis. This dataset includes three samples from three different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of human (Homo sapiens) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 16,015 nuclei in human adult testis. This dataset includes five samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

snRNA-seq of marmoset (Callithrix jacchus) adult testis

10.1038/s41586-022-05547-7

Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 7,318 nuclei in marmoset adult testis. This dataset includes two samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.

A spatial multi-omics atlas of the human lung reveals a novel immune cell survival niche

10.1038/s41588-022-01243-4

Multiple distinct cell types of the human lung and airways have been defined by single cell RNA sequencing (scRNAseq). Here we present a multi-omics spatial lung atlas to define novel cell types which we map back into the macro- and micro-anatomical tissue context to define functional tissue microenvironments. Firstly, we have generated single cell and nuclei RNA sequencing, VDJ-sequencing and Visium Spatial Transcriptomics data sets from 5 different locations of the human lung and airways. Secondly, we define additional cell types/states, as well as spatially map novel and known human airway cell types, such as adult lung chondrocytes, submucosal gland (SMG) duct cells, distinct pericyte and smooth muscle subtypes, immune-recruiting fibroblasts, peribronchial and perichondrial fibroblasts, peripheral nerve associated fibroblasts and Schwann cells. Finally, we define a survival niche for IgA-secreting plasma cells at the SMG, comprising the newly defined epithelial SMG-Duct cells, and B and T lineage immune cells. Using our transcriptomic data for cell-cell interaction analysis, we propose a signalling circuit that establishes and supports this niche. Overall, we provide a transcriptional and spatial lung atlas with multiple novel cell types that allows for the study of specific tissue microenvironments such as the newly defined gland-associated lymphoid niche (GALN).

Timecourse single-cell RNA-seq of whole rabbit embryos sampled across gestational days 7, 8 and 9

10.1038/s41556-023-01174-0

We characterised the transcriptomic profiles of 146,133 individual cells (post-QC) from whole rabbit embryos spanning gestational days 7, 8 and 9. These experiments were performed to elucidate the molecular programmes underlying gastrulation and early organogenesis in a non-rodent mammal. Combined with existing datasets of early mouse development, our rabbit developmental atlas facilitates a broad cross-species approach to deciphering early human development. Cell libraries were prepared using the 10X Genomics Chromium platform.

Single-cell RNA-Seq reveals dynamic, random monoallelic gene expression in mammalian cells

10.1126/science.1245316

In the diploid genome, genes come in two copies, which can have different DNA sequence and where one is maternal and one is paternal. In a particular cell, a gene could potentially be expressed from both copies (biallelic expression) or only one (monoallelic). We performed RNA-Sequencing on individual cells, from zygote to the cells of the late blastocyst, and also individual cells from the adult liver. Using first generation crosses between two distantly related mouse strains, CAST/Ei and C57BL/6, we determined the expression separately from the maternal and paternal alleles. We found that half of the genes were expressed by only one allele, randomly so that some cells would express the paternal allele, some the maternal and a few cell both alleles. We also observed the spread of the progressive inactivation of the paternal X chromosome. Overall design: First generation mouse strain crosses were used to study monoallelic expression on the single cell level

The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells

10.1016/j.cell.2015.05.015

Overall design: In total, we analyzed the transcriptomes of 233 individual male and female human PGCs from 15 embryos at between 4 and 19 weeks of gestation as well as 86 neighboring somatic cells from 13 of these embryos using a single-cell RNA-Seq method that we developed. Furthermore, we analyzed the DNA methylomes of both male and female human gonadal PGCs as well as the neighboring somatic cells of eleven of these embryos at between 7 and 19 weeks of gestation using whole-genome bisulfite sequencing (WGBS). In total, we generated 1.3 billion 100 bp paired-end reads for transcriptome analyses and 4.1 billion 100 bp or 150 bp paired-end reads for DNA methylome analyses.

Genomic profiling of human spermatogonial stem cells [scRNA-Seq]

10.1016/j.stem.2017.09.003

To better understand human spermatogonial stem cells (SSCs), we profiled their transciptome and epigenome, which revealed the mechanism how human SSCs regulates their self-renewal versus differentiation dermination, as well as how latent pluripotency is established in human SSCs. Remarkly, we discovered signaling pathways (e.g. LIF, BMP, WNT) that differentially regulated self-renewal vesus differentiation in SSCs. We also discovered that SSCs repress core pluripotent factors (Sox2, Pou5f1 and Nanog) yet activate ancillary factors (e.g. Klf4, Mbd3, Tcf3, Sall4) transcriptionally and epigenetically. Overall design: Using SSEA4 as self-renewal marker and Kit as differentiating marker, we isolated self-renewal and differentiation SSCs by magnetic antibody cell sorting (MACS). SSEA4+ or Kit+ cells were loaded into 5-10 µm integrated fluidic circuits (IFCs) using Fluidigm C1 instrument. Single cells in IFCs were lysed and total RNA was harvested for polyadenylation selection, reverse transcription and PCR amplification. Library constructions were performed according to Fluidigm Library preparation with Nextera XT protocol and sequenced on a 50-cycle single end run.

A single-cell transcriptome atlas of the ageing Drosophila brain

10.1016/j.cell.2018.05.057

Overall design: scRNA-seq (10x Genomics CHROMIUM Single Cell 3' Solution V2 Chemistry and Drop-seq) of adult brains from Drosophila melanogaster at various ages; SMART-seq2 on FAC-sorted R23E10-Gal4 positive neurons of adult brains from Drosophila melanogaster; Adapted SMART-seq2 on FAC-sorted R23E10-Gal4 positive neurons of adult brains from Drosophila melanogaster; CEL-seq2 on FAC-sorted R23E10-Gal4 positive neurons of adult brains from Drosophila melanogaster; Bulk RNA-seq of adult brains from Drosophila melanogaster; ATAC-seq of young (0 days) and old (50 days) adult brains from Drosophila melanogaster

Tabula Muris: Transcriptomic characterization of 20 organs and tissues from Mus musculus at single cell resolution

10.1038/s41586-018-0590-4

We have created a resource of single cell transcriptome data from the model organism Mus musculus. Contributor: The Tabula Muris Consortium The full list of contributors to this dataset can be found in the corresponding publication. Overall design: Single cell RNA sequencing of single cells across 20 tissues of 3 month aged mice

Single cell RNA-seq analysis on human endometrium across the natural menstrual cycle

10.1038/s41591-020-1040-z

To unbiasedly and systematically characterize endometrial transformation across the human menstrual cycle in preparation for embryo implantation, we analyzed the transcriptomic transformation of human endometrium at single cell resolution, dissecting multidimensional cellular heterogeneity of the tissue across the entire natural menstrual cycle. Overall design: We collected single cell transcriptome profile of endometrium biopsies sampled from 19 healthy and fertile females, at 4-27 days of their menstrual cycle. [10x dataset] - collected to validate main findings drawn from the original C1 dataset of this study. In this dataset, we collected additional data from 10 endometrial biopsies using the 10x Chromium system. Importantly, we collected both C1 and 10x data for two biopsies, one from mid-secretory phase and the other early-secretory. They served as anchors for a direct comparison between the two datasets, and were referred as ?anchor biopsies? in the manuscript of this study. Biopsies in the 10x dataset were obtained following the same protocol used for the C1 dataset, as described in the manuscript.

The Human Testis Cell Atlas via Single-cell RNA-seq (Healthy men scRNA-seq data set)

10.1038/s41422-018-0099-2

Overall design: We isolated single testicular cell from three healthy men of peak reproductive age. Two technical replicates were performed for each individual.

Dissecting the human liver cellular landscape by single cell RNA-seq reveals novel intrahepatic monocyte/ macrophage populations

10.1038/s41467-018-06318-7

Overall design: Primary liver samples from 5 patients were used for single cell RNA sequencing by 10X Genomics

Single cell RNAseq of mouse olfactory bulb reveals cellular heterogeneity and activity dependent molecular census of adult-born neurons

10.1038/s41467-021-25444-3

Cellular heterogeneity within the mammalian brain poses a challenge towards understanding its complex functions. Within the olfactory bulb (OB), odor information is processed by subtypes of inhibitory interneurons whose heterogeneity and functionality is influenced by ongoing adult neurogenesis. To investigate this cellular heterogeneity, and to better understand the developmental programs of adult-born neurons, we utilized single cell RNA sequencing and computational modeling to reveal diverse and transcriptionally distinct neuronal and non-neuronal cell types. We also analyzed molecular changes during adult-born interneuron maturation, and uncovered developmental programs within their gene expression profiles. Finally, we discovered that distinct neuronal subtypes are differentially affected by sensory experience. Together, these data provide a transcriptome-based foundation for investigating subtype-specific neuronal function in the OB, charting the molecular profiles that arise during the maturation and integration of adult-born neurons, and documenting activity-dependent changes in the cellular composition of the olfactory system. Overall design: To develop a comprehensive profile of cellular heterogeneity within the OB, here we employed high-throughput single-cell RNA sequencing (scRNA-seq) (Zheng et al. 2017) of activity manipulated and wildtype mouse olfactory bulbs. This technique allows an in-depth categorization of single cell molecular signatures, and provides an analysis of potential developmental and activity-dependent changes that occur in adult-born neuron populations. Together, our data inform the heterogeneity of interneurons within the OB, provide a molecular blueprint of stereotyped developmental programs for OB interneurons, and reveal the transcriptional changes that govern activity-dependent circuit integration of adult-born neurons. Furthermore, our results suggest that distinct molecular mechanisms act on different subsets of adult-born neurons, driving diversity and survival of adult-born interneuron subsets in an activity-dependent manner.

Modular transcriptional programs separately define axon and dendrite connectivity

10.7554/eLife.50822

Patterns of synaptic connectivity are remarkably precise and complex. Single-cell RNA sequencing has revealed a vast transcriptional diversity of neurons. Nevertheless, a clear logic underlying the transcriptional control of neuronal connectivity has yet to emerge. Here, we focused on Drosophila T4/T5 neurons, a class of closely related neuronal subtypes with different wiring patterns. Eight subtypes of T4/T5 neurons are defined by combinations of two patterns of dendritic inputs and four patterns of axonal outputs. Single-cell profiling during development revealed distinct transcriptional programs defining each dendrite and axon wiring pattern. These programs were defined by the expression of a few transcription factors and different combinations of cell surface proteins. Gain and loss of function studies provide evidence for independent control of different wiring features. We propose that modular transcriptional programs for distinct wiring features are assembled in different combinations to generate diverse patterns of neuronal connectivity. Overall design: Single-cell profiling of FACS purified populations of Drosophila melanogaster T4/T5 neurons during pupal development (24h and 48h APF).

Single-cell sequencing of enteroendocrine cells (EEs) in Drosophila midgut.

10.1016/j.celrep.2019.11.048

Enteroendocrine cells (EEs) in the intestinal epithelium have important endocrine functions, yet this cell lineage exhibits great local and regional variations that have hampered detailed characterization of EE subtypes. Through single cell RNA-sequencing analysis, combined with a collection of neuropeptide/receptor knock-in strains, here we provide a comprehensive analysis of cellular diversity, spatial distribution and transcription factor (TF) code of EEs in adult Drosophila midgut. We identify 10 major EE subtypes that totally produced 14 different classes of hormone peptides. Each EE on average co-produces 2-5 different classes of hormone peptides. Functional screen with subtype-enriched TFs suggests a combinatorial TF code that controls EE cell diversity, with class-specific TFs Mirr and Ptx1that respectively define two major classes of EEs, and regional TFs such as Esg, Drm, Exex and Fer1that further define regional EE identity. Our single-cell data should greatly facilitate Drosophila modeling of EE differentiation and function. Overall design: FACS sort EE cells using CG32547-KI-GAL4> uas-GFP line and perform 10X genomics single cell sequencing. Raw data was further processed by Seurat algorithm and EE subtypes were visualized by generating a t-distributed stochastic neighborhood embedding (t-SNE) plot.

Droplet-based single-cell RNA-sequencing of mouse and naked mole-rat spleen and circulating immune cells, in natural conditions, following lipopolysaccharide (LPS) challenge, and saline control

10.1371/journal.pbio.3000528

Single-cell RNA-sequencing was performed in the mouse and naked mole-rat immune systems in order to map and compare their immune cell repertoirs and their cell-specific gene-expression responses to LPS challenge Overall design: Single-cell RNA-sequencing was obtained from: 1. 4 adult mouse and 4 adult naked mole-rat spleens in duplicate; 2. 4 adult mouse and 3 adult naked mole-rat circulating immune cells; 3. 2 adult mouse and 2 adult naked mole-rat spleens following LPS challenege in duplicate; 4. 2 adult mouse and 2 adult naked mole-rat spleens following saline administration in duplicate; 5. 2 adult mouse and 2 adult naked mole-rat circulating immune cells following LPS challenege in duplicate; 4. 2 adult mouse and 2 adult naked mole-rat circulating immune cells following saline administration in duplicate;

Single-cell Transcriptomic Atlas of the Human Retina Identifies Cell Types Associated with Age-Related Macular Degeneration [Microfluidics]

10.1038/s41467-019-12780-8

Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Overall design: Three replicates of macula and peripheral retinas.

Single-cell transcriptomics uncovers human corneal limbal stem cells and its differentiation trajectory

10.1016/j.jtos.2020.12.004

Limbal stem cells (LSCs), known as corneal epithelial stem cells, are located at the basal epithelial layer of the corneal limbus and serve an important function in maintaining the homeostasis of the corneal epithelium. Several putative molecular markers of LSCs have been previously identified. However, the specificity of these markers remains largely controversial. To address this gap in the current understanding of LSCs, we performed a transcriptome profiling of heterogeneous corneal limbal basal cells using single-cell transcriptomics technology to identify LSCs and their exclusive markers. We isolated limbal basal cells from two young donors, constructed scRNA-seq libraries, generated RNA-sequencing data using the 10x Genomics platform, and then finally obtained the transcriptome of about 18,000 individual single-cells using Cell ranger (https://10xgenomics.com). Next, we performed quality control, filtering and data integration using Seurat package (https://satijalab.org/seurat), about 16,400 cells were retained for further downstream analysis such as dimensional reduction, unsupervised clustering, and Differentially expressed gene selection, trajectory analysis and visualization. We identified 11 unique clusters of cells assigned to a putative cell type based on published known biomarkers for differentiation, proliferation and putative epithelial stem cells. As a results, we found 2 Terminally Differentiated Cell (TDC), 3 Post-Mitotic Cell (PMC), 1 Transient Amplifying Cell (TAC), 2 Limbal Progenitor Cell (LPC), Putative Limbal Stem Cell (LSC) and 2 Melanocyte (MC) sub-cell type clusters. Furthermore, we confirmed the trajectory order of LSC differentiation for 9 clusters using Pseudotemporal and Functional PCA analysis. Lastly, we validated each assigned cell subtypes and determined their location in human corneal limbus tissue with 9 markers using RNAscope We were able to reveal the heterogeneity of corneal limbal basal epithelium by defining novel dynamic trajectories for cell types in a pseudotemporal manner. This approach allowed us to identify the distinct clusters of LSCs and progenitors with exclusively expressed markers, and might apply for translational research on regenerating a normal corneal epithelium and restoring vision. Overall design: We isolated limbal basal cells from two young donors, constructed scRNA-seq libraries, generated RNA-sequencing data using the 10x Genomics platform, and then finally obtained the transcriptome of about 18,000 individual single-cells using Cell ranger (https://10xgenomics.com). Next, we performed quality control, filtering and data integration and further downstream analysis such as dimensional reduction, unsupervised clustering, and Differentially expressed gene selection, trajectory analysis and visualization.

The molecular landscape of neural differentiation inthe developing Drosophila brain revealed by targeted scRNA-seq and multi-informatic analysis

10.1016/j.celrep.2021.109039

In this work, Michki et. al. isolate neurons and neural progenitors from the developing Drosophila brain. Through a combination of in silico and in situ techniques, they map out the expression dynamics of a variety of molecular factors that may play a role in specifying terminal neural fates at the RNA level, and provide tools to enable similar analyses to be performed in other systems. Overall design: R9D11-Gal4 genetic driver was used with a UAS:hH2B-2xmNG reporter to label type-II neural lineages in third instar larval brains. 20 brains were dissected, dissociated, and sorted on a Sony MA900 FACS machine. 10,000 cells were sequenced in a single lane of a 10Xchromium chip using their v3 chemistry. Cells were sequenced on an Illumina NovaSeq/HiSeq4000 with 400M reads allocated to this library. 6092 cells were identified with a mean read count of 97,604 reads/cell when aligned to the BDGP6(2014-07) Drosophila genome assembly from ENSEMBL using STAR. Downstream filtering and analysis were performed using scanpy.

Single cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenic lineages. [single nucleus sequencing]

10.7554/eLife.67436

Purpose: While great progress has been made in understanding the differences in regional stem cell potential using viral and genetic lineage tracing strategies, the core molecular heterogeneity that underlies these regional differences is largely unknown. Methods: Here we present a single nucleus sequencing dataset of four microdissected regions of adult CD1 wild type mouse ventricular-subventricular zones (V-SVZ). Four samples (anterior-ventral, AV; anterior dorsal, AD; posterior ventral, PV; posterior dorsal, PD) were generated at the same time from the same mice, and each sample was run on its own lane of a 10x Chromium Single Cell Controller chip for single nucleus barcoding. Results: Here we present single nucleus and single whole cell sequencing datasets of microdissected adult mouse V-SVZ, and evidence for the existence of two broad subtypes of adult neural stem cells. By using spatially resolved microdissections in the single nucleus sequencing dataset as a reference, and mapping marker gene expression in the V-SVZ, we find that these two populations reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. Furthermore, we identified two subpopulations of newly born neurons that have gene expression consistent with dorsal or ventral origins. Finally, we identify genes expressed by both stem cells and the neurons they generate that specifically mark either the dorsal or ventral adult neurogenic lineage. These datasets, methods and findings will enable future study of region-specific regulation of adult neurogenesis. Overall design: Single nucleus profiles of regionally micro-dissected ventricular-subventricular zones of 17 postnatal day 35 male (8 mice) and female (9 mice) CD1 wild type mice.

Single cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenic lineages. [single whole-cell]

10.7554/eLife.67436

Purpose: While great progress has been made in understanding the differences in regional stem cell potential using viral and genetic lineage tracing strategies, the core molecular heterogeneity that underlies these regional differences is largely unknown. Methods: Here we present a single whole-cell sequencing dataset of microdissected adult hGFAP:GFP mouse V-SVZ. Four samples were (two samples from male mice, two samples from female mice) multiplexed & combined using MULTI-Seq barcodes (McGinnes et al. 2019), labeled with TotalSeq antibodies against VCAM1 and CD24 proteins (Biolegend), and were split into two technical replicate lanes on a 10x Chromium Single Cell Controller chip for single cell barcoding. Genomic libraries were prepared using standard Illumina protocols, and barcode libraries (MULTI-Seq, TotalSeq) were prepared using MULTI-Seq protocols. Results: Here we present single whole-cell and single nucleus sequencing datasets of microdissected adult mouse V-SVZ, and evidence for the existence of two broad subtypes of adult neural stem cells. By using spatially resolved microdissections in the single nucleus sequencing dataset as a reference, and mapping marker gene expression in the V-SVZ, we find that these two populations reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. Furthermore, we identified two subpopulations of newly born neurons that have gene expression consistent with dorsal or ventral origins. Finally, we identify genes expressed by both stem cells and the neurons they generate that specifically mark either the dorsal or ventral adult neurogenic lineage. These datasets, methods and findings will enable future study of region-specific regulation of adult neurogenesis. Overall design: Single cell profiles of micro-dissected ventricular-subventricular zones of male (postnatal day 35) and female (postnatal day 29) hGFP:GFP mice.

A human breast transcriptome atlas

10.1016/j.devcel.2022.05.003

The breast is a dynamic organ whose response to physiological and pathophysiological conditions alters its disease susceptibility, yet the specific effects of these clinical variables on cell state remain poorly annotated. We present a unified, high-resolution breast atlas by integrating single-cell RNA-seq, mass cytometry, and cyclic immunofluorescence, encompassing a myriad of states. We define cell subtypes within the alveolar, hormone-sensing, and basal epithelial lineages, delineating associations of several subtypes with cancer risk factors, including age, parity, and BRCA2 germline mutation. Of particular interest is a subset of alveolar cells termed basal-luminal (BL) cells, which exhibit poor transcriptional lineage fidelity, accumulate with age, and carry a gene signature associated with basal-like breast cancer. We further utilize a medium-depletion approach to identify molecular factors regulating cell-subtype proportion in organoids. Together, these data are a rich resource to elucidate diverse mammary cell states. Overall design: A total of 16 breast samples were assayed (4 samples from reductive mammoplasties and 12 from prophylactic mastectomies).

scRNA sequencing analysis for human ureters and patient derived ureter oragnoid

10.1016/j.devcel.2022.07.004

Tissue engineering offers a promising treatment strategy for ureteral strictures. Successful ureter engineering is necessitated by detailed understanding of the tissue architecture, cellular heterogeneity, and signaling pathways underlying regeneration. We define and spatially map cell populations within the human ureter by using a combinatorial approach: single-cell RNA sequencing, 10X Visium spatial transciptomics, and immunofluorescence. The stromal and urothelial cell populations are analyzed in detail, and we infer potential cell-cell communication networks underpinning the bi-directional crosstalk between these compartments. Specifically, we analyze and experimentally validate the importance of Sonic Hedgehog (SHH) signaling pathway in adult stem cell maintenance. The SHH-expressing basal cells support organoid generation in vitro, and accurately predict the differentiation trajectory of basal stem cells, to terminally differentiated umbrella cells, in vivo. Our results highlight essential processes involved in adult ureter tissue homeostasis, and provide a toolkit for guiding ureter tissue engineering. Overall design: 10 normal human ureters each were subjected to RNA sequencing at single cell resolution on Novaseq using 10X Gemonics library preparation platform. Data was processed using Cellranger 4.0.0 and anayzed using Seurat v3.2.1 to characterize various cell types and understand finer details of the regenrative pathways in the ureter urothelium. This was further explored by analyzing single cell RNA sequencing data from human ureter derived organoid (generated in the same manner as the 10 ureter samples). Freshly processed and thawed tissue sample from the same human subject was used as control to further understand the urothelial regenerative pathways.

Transcriptomic Taxonomy and Neurogenic Trajectories of Adult Human, Macaque and Pig Hippocampal and Entorhinal Cells

10.1016/j.neuron.2021.10.036

The hippocampal-entorhinal system supports cognitive functions, has lifelong neurogenic capabilities in many species, and is selectively vulnerable to Alzheimer's disease. To investigate neurogenic potential and cellular diversity, we profiled single-nucleus transcriptomes in five hippocampal-entorhinal subregions in human, macaque, and pig. Integrated cross-species analysis revealed robust transcriptomic and histologic signatures of neurogenesis in adult mouse, pig and macaque, but not humans. Doublecortin (DCX), a widely accepted marker of newly generated granule cells, was detected in diverse human neurons, but it did not define immature neuron populations. To explore species differences in cellular diversity and implications for disease, we characterized subregion-specific transcriptomically-defined cell types and transitional changes from the three-layered archicortex to the six-layered neocortex. Notably, METTL7B defined subregion-specific excitatory neurons and astrocytes in primates, associated with endoplasmic reticulum and lipid droplet proteins, including Alzheimer's disease-related proteins. Together this resource reveals cell-type- and species-specific properties shaping hippocampal-entorhinal neurogenesis and function. Overall design: snRNA-seq of hippocampal-entorhinal system in 6 human donors, dentate gyrus in 3 rhesus macaque donors, and adult hippocampus in 9 pig donors with different postmortem intervals (30 minutes, 1 hour, 7 hour)

Cross-species single-nuclei transcriptome atlas of dorsal root ganglion sensory neurons [humanDRG]

10.1038/s41467-023-36014-0

Sensory neurons of the dorsal root ganglion (DRG) play a crucial role in maintaining tissue homeostasis by sensing and initiating responses to stimuli. While most preclinical studies of DRGs are conducted in rodents, much less is known about the mechanisms of sensory perception in primates. We generated a transcriptome atlas for mouse, guinea pig, cynomolgus monkey, and human DRGs using a framework that implements a common laboratory workflow (i.e sequencing technologies, laboratory protocols, and sample archival methods) and multiple data-integration approaches to generate accurate cross-species mappings of sensory neuron subtypes. Overall design: Fresh or frozen DRG tissues from mouse, guinea pig, cynomologus monkey and human were collected and nuclei were isolated with FACS or density-gradient centrifugation and analyzed with snRNA-seq.

Single Cell RNA Sequencing of the Adult Drosophila Eye Reveals Distinct Clusters and Novel Marker Genes for All Major Cell Types

10.1038/s42003-022-04337-1

Although the adult Drosophila eye is an important model system for phototransduction and human disease research, high quality single cell resolution transcriptome data is lacking for this tissue. To address this, we performed single cell RNA-sequencing on wild-type Canton-S adult eyes from 1 day old male and female, 3-day and 7-day old male flies. These data sets will serve as an valuable resource for future research using the adult eye. Overall design: Adult eyes of Canton-S flies were dissected and dissociated into single cell solution with collagenase. Single cells were analyzed with 10x Chromium based scRNA-seq