Multi-platform single cell transcriptomic profiling as a benchmarking resource
Ontology highlight
ABSTRACT: In this study, we perform comparative analysis of Illumina HiSeq and BGISEQ-500 sequencing platforms for single-cell transcriptomics data. We performed scRNA-seq on a homogenous population of mouse embryonic stem cells along with two kinds of control spike-in molecules and sequenced across both sequencing platforms. The matched Illumina platform datasets can be found with accession numbers (E-MTAB-5483, E-MTAB-5484, E-MTAB-5485). The additional data comparison performed in the study can be found (BioProject# PRJNA430491, SRA# SRP132313 and CNBG# CNP0000075).
Project description:Endovascular biopsy and fluorescence activated cell sorting was used to enrich for viable endothelial cells (ECs) from a vertebrobasilar aneurysm and the femoral artery. scRNAseq was then performed on 24 aneurysmal endothelial cells and 23 patient-matched non-aneurysmal femoral artery endothelial cells. cDNA libraries were prepared using the Smart-seq2 protocol on a Fluidigm C1 system (Fluidigm, South San Francisco, California) and sequenced on a HiSeq2500 machine (Illumina, San Diego, California).
Project description:Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. Using novel computational approaches we define highly variable gene clusters; and reveal their distinct epigenetic characteristics. We show that bivalent genes are prone to expression variation. At the same time, we identify an ESC priming pathway that initiates the exit from the naïve ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum free culture reduces cellular heterogeneity and transcriptome variation in ESCs. Single cell mRNA-seq analysis of ES cells cultured with serum
Project description:For unbiased, whole-organism wide cell type profiling, we randomly sampled cells from dissociated Platynereis larvae. To generate the single-cell mRNA-sequencing data, P. dumerilii larvae were dissociated, followed by cell capture, cDNA synthesis and amplification on the C1 Single-Cell Auto Prep IFCs for 5-10 um or 10-17 um cells (Fluidigm). Sequencing libraries were produced using Nexera XT DNA kit (Illumina). In total, we sequenced 596 samples, of which 373 correspond to single, alive cells that passed the quality check criteria. Part of this dataset was previously published (ArrayExpress accession number E-MTAB-2865). Here, we publish additional 383 sequenced cells.
Project description:Pluripotent stem cells (PSCs) are capable of dynamic interconversion between distinct substates, but the regulatory circuits specifying these states and enabling transitions between them are not well understood. We set out to address this issue and map the landscape of gene expression variability in PSCs by single-cell expression profiling of PSCs under different chemical and genetic perturbations. We find that signaling factors and developmental regulators show highly variable expression in PSCs, with expression states for some variable genes heritable through multiple cell divisions. Expression variability and population heterogeneity can be influenced by perturbation of signaling pathways and chromatin regulators. Strikingly, either removal of mature miRNAs or pharmacologic blockage of external signaling pathways drives PSCs into a low-noise ground state characterized by a reconfigured pluripotency regulatory network, increased self-renewal efficiency, and a distinct chromatin state, an effect mediated by the action of opposing miRNA families on the c-myc / Lin28 / let-7 axis. These findings illuminate the causes of transcriptional heterogeneity in PSCs and their consequences for cellular decision-making. Single-cell RNA-Seq on 183 individual v6.5 mouse embryonic stem cells (mESCs) cultured in serum+LIF media, 94 v6.5 mESCs cultured in 2i+LIF media ('ground state' conditions), and 84 Dgcr8 -/- mESCs (constructed in a v6.5 background), that lack mature miRNAs due to knockout of a miRNA processing factor, cultured in serum+LIF. ChIP-Seq for RNA polymerase II, H3K4me3, H3K27me3, H3K27ac, H3K9me3, and H3K36me3 on the three populations of mESCs profiled by single-cell RNA-Seq. Single-cell RNA-Seq on 54 individual nestin-positive neural precursor cells derived from v6.5 mESCs.
Project description:Improved Smart-Seq for sensitive full-length transcriptome profiling in single cells. Cells of four different origins were profiled using commercial SMARTer and compared to five variants of an improved protocol (Smart-Seq2).
Project description:Polycomb repressive complexes are important histone modifiers, which silence gene expression, yet there exists a subset of polycomb-bound genes actively transcribed by RNA polymerase II. To investigate the switching between polycomb-repressed and active states, we sequence mRNA from OS25 mouse embryonic stem cells cultured in serum/LIF. To validate our finding that polycomb modulates stochastic gene expression and transcriptional bursting, we perform knockout experiments and we sequence mRNA from Ring1A knockout (untreated) and Ring1A/B double knockout cells with constitutive Ring1A knockout and tamoxifen-inducible conditional Ring1B knockout.
Project description:Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell-type in terms of their origins and functional effects in vivo. Methods: Here we generate Postn (periostin) gene-targeted mice containing a tamoxifen inducible Cre for cellular lineage tracing analysis. This Postn allele identifies essentially all myofibroblasts within the heart and multiple other tissues. Results: Lineage tracing with 4 additional Cre-expressing mouse lines shows that periostin-expressing myofibroblasts in the heart derive from tissue-resident fibroblasts of the Tcf21 lineage, but not endothelial, immune/myeloid or smooth muscle cells. Deletion of periostin+ myofibroblasts reduces collagen production and scar formation after myocardial infarction. Periostin-traced myofibroblasts also revert back to a less activated state upon injury resolution. Conclusions: Our results define the myofibroblast as a periostin-expressing cell-type necessary for adaptive healing and fibrosis in the heart, which arises from Tcf21+ tissue-resident fibroblasts. Fluidigm C1 whole genome transcriptome analysis of lineage mapped cardiac myofibroblasts
Project description:The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed 10 differentiated distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous novel marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSN) which have specific markers and which overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states. We measured the transcriptome of 1208 single striatal cells using two complementary approaches; microfluidic single-cell RNAseq (Mic-scRNAseq) and single cell isolation by FACS (FACS-scRNAseq) (Table S1). We sampled cells either randomly or enriched specifically for MSNs or astrocytes using FACS from D1- tdTomato (tdTom)/D2-GFP or Aldhl1-GFP mice, respectively
Project description:We used micro-dissection with FACS sorting techniques to isolate single cells from the metanephric mesenchyme of the E11.5 developing kidney. A subset of these single cell populations is analysed individually via Fluidigm single cell analysis. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database. Kidneys are harvested from Tg(Crym-EGFP)GF82Gsat mice. Single cells are extracted from E11.5 metanephric mesenchyme using manual micro-dissection techniques. A subset of these cells is analyzed individually via Fluidigm single cell analysis. The long term goal is to generate a transcriptional atlas of the developing kidney.
Project description:A subset of adipocytes residing within the inguinal white adipose tissue (ingWAT) of mice exhibit thermogenic activity in response to various external stimuli, including cold exposure. The inducible nature of this thermogenic response, coupled with its robust energy-depleting capacity have prompted investigation into the adipose precursor cells (APCs) from which thermogenic adipocytes derive. To this end, we performed single-cell transcriptomics on cells derived from ingWAT, interscapular brown adipose tissue (iBAT) and epididymal WAT. A subset of single cells collected from ingWAT and epiWAT of mice were chronically (4 days) treated with CL-316,243 (dose at 1 mg kg -1). Tissues of n=28, 10 week-old mice were digested and stromal cells were subsequently purified via differential centrifugation. Single-cell RNA extraction and mRNA amplification were performed on the C1™ Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) following the protocol (PN 100-7168, http://www.fluidigm.com/). Following centrifugation and removal of the medium, cells were resuspended at a concentration of 150–500 cells/μL. This cell suspension was mixed with C1 Cell Suspension Reagent (Fluidigm, Cat # 634833) at the recommended ratio of 3:2 immediately before loading 5 μL of this final mix on the C1 IFC. We obtained, on average, 2.5 million mapped reads per one single cell and successfully reconstructed single-cell expression of ~9,000 genes. Our results revealed a unique cluster of cells that exhibited enriched expression of canonical thermogenic and adipogenic gene markers. Notably, we identified tetraspanin CD81 as a discretely expressed membrane-bound protein conserved specifically within this population. All experiments were performed at our facility at the University of California, San Francisco.