Single-cell transcriptomics reveals widespread heterogeneity in gene regulation and RNA processing in stimulated immune cells
ABSTRACT: Here, we used single-cell RNA-Seq to discover extensive cell-to-cell variability – in transcript expression levels, cell state, circuit usage, and alternative splicing – between stimulated immune cells RNA seq libraries from 18 single cells, 3 populations of 10,000 cells, and 2 zero cells (negative controls) with SMARTer. Three additional single cell libraries with molecular barcodes (MB) using a modified SMARTer protocol.
Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5‑hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 3 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH) and syntetic hydrolysate with added lignotoxins (SynH_LT). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. 3 biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.
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:mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment. These data are total RNA-Seq data, from RNA sequencing of rRNA-depleted total cellular RNA -- except the LCL 4SU data, which derive from 4SU-labeled RNA. Please see the associated paper for more details.
Project description:Whole-genome DNA libraries were prepared from a population of just under 100 Col/Ler F1 backcrossed to Col. Low-coverage whole-genome sequencing was used to map meiotic crossovers in this population following the protocol described in Rowan et al., 2015, doi: 10.1534/g3.114.016501.
Project description: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:Both immunodeficient and wild type NOD mice exhibit defects in control of early T-cell development in the thymus. We show that Rag1-deficient NOD mice fail to enforce both the b-selection checkpoint and an earlier T-cell commitment checkpoint, based on genome-wide genetic and transcriptome analyses. A major QTL peak for the checkpoint breakthrough phenotype mapped to the diabetes susceptibility Idd9/11 region, as confirmed by congenic mouse analysis. Genome-wide RNA deep-sequencing revealed two classes of differences between NOD and B6 Rag1-deficient thymocytes: first, effects of genetic background prior to breakthrough, and second, effects of the breakthrough itself. These genotypes differentially express numerous signal transduction genes, prominently tyrosine kinase and actin-binding genes, some located within QTL regions. Emerging NOD breakthrough cells depart from the expected DN3 phenotype by expressing many stem cell-associated proto-oncogenes, such as Lmo2, Hhex, Lyl1, and Kit which are normally repressed earlier, and by illegitimate activation of post-b-selection genes like Cd2, Cd5, and Cd4. Co-expression of stem cell and T-cell genes persists in thymic lymphoma cells that emerge with high penetrance in these mice. These results imply that NOD thymocytes have defects that can collapse regulatory boundaries at two early T-cell checkpoints, which may predispose them to leukemia and autoimmunity. Genetic and transcriptome analyses of early T-cell checkpoint failure and leukemia initiation in Rag1-deficient NOD mice
Project description:We sequenced mRNA from 6 samples of FACsorted telencephalons from E14.5 Sip1|Nkx2-1 knockout and WT|Nkx2-1 control mouse embryos to find differentially expressed genes in the absence of the transcription factor Sip1. Examination of mRNA levels in 3 control and 3 Sip1|Nkx2-1 knockout samples
Project description:During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus ‘poising’ function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b activation, these inputs act in a stage specific manner, providing a multitiered mechanism for developmental gene regulation. Two sets of samples were generated from DN T-cell sub-populations derived from culture of bone marrow progenitors from mice containing a knock-in Bcl11b-YFP reporter
Project description:Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. By tandem affinity purification and mass spectrometry, we present a comprehensive characterisation of the MITF interactome comprising multiple novel cofactors involved in transcription, DNA replication and repair and chromatin organisation, including a BRG1 chromatin remodelling complex comprising CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. MITF, SOX10 and YY1 bind between two BRG1-occupied nucleosomes thus defining both a combinatorial signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of MAREs. Nevertheless, BRG1 silencing enhances MITF occupancy at MAREs showing that BRG1 acts to promote dynamic MITF interactions with chromatin. 19 samples corresponding to mRNA profiles of 501Mel and Hermes3A after MITF, BRG1 or control shRNA-mediated knockdown were generated by deep sequencing in triplicate (in duplicate for 501_shMITF and corresponding control 501_shSCR2), using HiSeq2500.
Project description:Mitochondrial heteroplasmy, the presence of more than one mtDNA variant in a cell or individual is not as uncommon as previously thought. It is mostly due to the high mutation rate of the mtDNA and limited repair mechanisms present in the mitochondrion. The phenomenon has been studied mostly in human samples and in medical contexts. Heteroplasmy has also been researched in other species in fields such as forensics or genetic foot printing, but these studies usually focused on contained families within closely related species. Here we describe a large cross-species evaluation of heteroplasmy in mammals. We employed a novel approach to detect mitochondrial heteroplasmy in both novel and previously reported ChIP-sequencing datasets, which include concomitant mitochondrial DNA sequenced in the experiment. Here, we report novel ChIP-seq experiments for H3K4me1 and CEBPA across mammals, as well as some H3K4me3, H3K27ac and total histone H3 experiments. Most of the reported CEBPA experiments are good quality pull-downs, however the quality of many of the other experiments reported here has not been interrogated in detail. Whereas this does not affect the investigation of mitochondrial DNA pollution for the purposes of this study, both H3K4me1 and total histone H3 ChIP-seq datasets were often sequenced to relatively low depth and showed low ChIP enrichment compared to the other antibodies.