Single-cell RNA sequencing (scRNA-seq) allows researchers to collect large catalogues detailing the transcriptomes of individual cells. Unsupervised clustering is of central importance for the analysis of these data, as it is used to identify putative cell types. However, there are many challenges involved. We discuss why clustering is a challenging problem from a computational point of view and what aspects of the data make it challenging. We also consider the difficulties related to the biological interpretation and annotation of the identified clusters.

Challenges in unsupervised clustering of single-cell RNA-seq data.

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

/pdf/cells-of-the-adult-human-heart-2nt3kofuaj.pdf

Cells of the adult human heart.

The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. This study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types. We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific. In conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.

/pdf/scrna-seq-assessment-of-the-human-lung-spleen-and-esophagus-1xa1gjwepd.pdf

scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation

Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual.

https://www.science.org/doi/pdf/10.1126/science.abl4896?download=true

The Tabula Sapiens: a multiple organ single cell transcriptomic atlas of humans

https://www.cell.com/cell/pdf/S0092-8674(20)31078-3.pdf

A Quantitative Proteome Map of the Human Body.

Post-mortem tissues samples are a key resource for investigating patterns of gene expression. However, the processes triggered by death and the post-mortem interval (PMI) can significantly alter physiologically normal RNA levels. We investigate the impact of PMI on gene expression using data from multiple tissues of post-mortem donors obtained from the GTEx project. We find that many genes change expression over relatively short PMIs in a tissue-specific manner, but this potentially confounding effect in a biological analysis can be minimized by taking into account appropriate covariates. By comparing ante- and post-mortem blood samples, we identify the cascade of transcriptional events triggered by death of the organism. These events do not appear to simply reflect stochastic variation resulting from mRNA degradation, but active and ongoing regulation of transcription. Finally, we develop a model to predict the time since death from the analysis of the transcriptome of a few readily accessible tissues.

/pdf/the-effects-of-death-and-post-mortem-cold-ischemia-on-human-3hoqz1jq27.pdf

The effects of death and post-mortem cold ischemia on human tissue transcriptomes

Circadian and circannual cycles trigger physiological changes whose reflection on human transcriptomes remains largely uncharted. We used the time and season of death of 932 individuals from GTEx to jointly investigate transcriptomic changes associated with those cycles across multiple tissues. Overall, most variation across tissues during day-night and among seasons was unique to each cycle. Although all tissues remodeled their transcriptomes, brain and gonadal tissues exhibited the highest seasonality, whereas those in the thoracic cavity showed stronger day-night regulation. Core clock genes displayed marked day-night differences across multiple tissues, which were largely conserved in baboon and mouse, but adapted to their nocturnal or diurnal habits. Seasonal variation of expression affected multiple pathways, and it was enriched among genes associated with the immune response, consistent with the seasonality of viral infections. Furthermore, they unveiled cytoarchitectural changes in brain regions. Altogether, our results provide the first combined atlas of how transcriptomes from human tissues adapt to major cycling environmental conditions. This atlas may have multiple applications; for example, drug targets with day-night or seasonal variation in gene expression may benefit from temporally adjusted doses.

/pdf/day-night-and-seasonal-variation-of-human-gene-expression-1d3daolj.pdf

Day-night and seasonal variation of human gene expression across tissues

Circadian and circannual cycles trigger physiological changes whose reflection on human transcriptomes remains largely uncharted. We used the time and season of death of 932 individuals from GTEx to jointly investigate transcriptomic changes associated with those cycles across multiple tissues. For most tissues, we found little overlap between genes changing expression during day-night and among seasons. Although all tissues remodeled their transcriptomes, brain and gonadal tissues exhibited the highest seasonality, whereas those in the thoracic cavity showed stronger day-night regulation. Core clock genes displayed marked day-night differences across multiple tissues, which were largely conserved in baboon and mouse, but adapted to their nocturnal or diurnal habits. Seasonal variation of expression affected multiple pathways and were enriched among genes associated with SARS-CoV-2 infection. Furthermore, they unveiled cytoarchitectural changes in brain subregions. Altogether, our results provide the first combined atlas of how transcriptomes from human tissues adapt to major cycling environmental conditions.

/pdf/day-night-and-seasonal-variation-of-human-gene-expression-28d1yvo98f.pdf

Over the last decade, the increasing interest in long non-coding RNAs (lncRNAs) has led to the discovery of these transcripts in multiple organisms. LncRNAs tend to be specifically, and often lowly, expressed in certain tissues, cell types and biological contexts. Although lncRNAs participate in the regulation of a wide variety of biological processes, including development and disease, most of their functions and mechanisms of action remain unknown. Poor conservation of the DNA sequences encoding for these transcripts makes the identification of lncRNAs orthologues among different species very challenging, especially between evolutionarily distant species such as flies and humans or mice. However, the functions of lncRNAs are unexpectedly preserved among different species supporting the idea that conservation occurs beyond DNA sequences and reinforcing the potential of characterising lncRNAs in animal models. In this review, we describe the features and roles of lncRNAs in the fruit fly Drosophila melanogaster, focusing on genomic and functional comparisons with human and mouse lncRNAs. We also discuss the current state of advances and limitations in the study of lncRNA conservation and future perspectives. 

/pdf/genomic-and-functional-conservation-of-lncrnas-lessons-from-e7koqaht.pdf

Raziel Amador

Papers

The effects of death and post-mortem cold ischemia on human tissue transcriptomes

Day-night and seasonal variation of human gene expression across tissues

Day-night and seasonal variation of human gene expression across tissues

Genomic and functional conservation of lncRNAs: lessons from flies