Project description:A bead supsension and a solution of ERCC spike-ins at a concentration of ~100,000 molecules per droplet was used in Drop-Seq, a novel technology for high-throughput single cell mRNAseq An estimated 84 beads were selected for amplification.
Project description:A cell supsension containing an equal mix of HEK and 3T3 cells was used in Drop-Seq, a novel technology for high-throughput single cell mRNAseq. Cells were mixed at a final concentration of 12.5 cells per microliter in droplets. An estimated 570 STAMPs were selected for amplification.
Project description:Vsx2-GFP mouse retinas were dissected, FACS sorted for GFP+ cells and single-cell mRNAseq libraries generated with Drop-Seq Drop-Seq was performed on two different batches. Bipolar1-Bipolar4 are replicates from batch 1 and Bipolar5-6 are replicates from Batch 2
Project description:A cell suspension was prepared from wild-type P14 mouse retinas, and single-cell mRNAseq libraries were generated with Drop-Seq. Drop-Seq was performed on four separate days using the same age (P14) and strain (C57BL/6). On day 1, replicate 1 was obtained. On day 2, replicates 2 and 3 were obtained. On day 3, replicates 4-6 were obtained. On day 4, replicate 7 was obtained.
Project description:We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing, allowing discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method reveals novel associations between heterogeneous methylation of distal regulatory elements and transcriptional heterogeneity of key pluripotency genes. E14 ES cells were grown in either serum/LIF or 2i culture conditions and separated into single cells. RNA-Seq or Bisulfite-Seq libraries were prepared. This Series includes only the RNA-Seq data. The list of 61 samples that passed QC in both BS-seq and RNA-seq is included in "Supplementary Table 1" of the associated manuscript.
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:Single-cell expression profiling by RNA-Seq promises to exploit cell-to-cell variation in gene expression to reveal regulatory circuitry governing cell differentiation and other biological processes. Here, we describe Monocle, a novel unsupervised algorithm for ordering cells by progress through differentiation that dramatically increases temporal resolution of expression measurements. This reordering unmasks switch-like changes in expression of key regulatory factors, reveals sequentially organized waves of gene regulation, and exposes regulators of cell differentiation. A functional screen confirms that a number of these regulators dramatically alter the efficiency of myoblast differentiation, demonstrating that single-cell expression analysis with Monocle can uncover new regulators even in well-studied systems. We selected primary human myoblasts as a model system of cell differentiation to investigate whether ordering cells by progress revealed new regulators of the process. We sequenced RNA-Seq libraries from each of several hundred cells taken over a time-course of serum-induced differentiation. Please note that this dataset is a single-cell RNA-Seq data set, and each cell comes from a capture plate. Thus, each well of the plate was scored and flagged with several QC criteria prior to library construction, which are provided as sample characteristics; CONTROL indicates that this library is a off-chip tube control library constructed from RNA of approximately 250 cells and 'DEBRIS' indicates that the well contained visible debris (and may or may not include a cell). Libraries marked DEBRIS thus cannot be confirmed to come from a single cell.
Project description:Transcriptomes were determined for all blastomeres of 28 embryos at the 2- (n=8), 4- (n=16) and 8-cell (n=4) stages, and for individual cells taken from 16- (n=6) and 32- (n=6) cell stage embryos. We also carefully monitored the 2- to 4-cell divisions noting whether the cleavage plane was meridional (M, along the Animal-Vegetal (AV) axis marked by the second attached polar body) or equatorial (E, bisecting the AV axis) and the order in which such divisions occurred. This resulted in four groups of 4-cell stage embryos: ME, EM, MM and EE, which were all collected 10 hours after the first 2- to 4-cell division.
Project description:Th2 cells regulate helminth infections, allergic disorders, tumor immunity and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2-signaling molecules. While steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterised. We demonstrate production of the steroid pregnenolone by Th2 cells in vitro, and in vivo in a helminth infection model. To identify gene-expression identify of these steroidogenic-Th2 cells, we performed single-cell RNA-sequencing.
Project description:Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human acute myeloid leukemia (AML) cases with a 5-year overall survival of approximately 30%. In CN-AML with poorer prognosis, mutations in the de novo DNA methyltransferase (DNMT3A) and the FMS-like tyrosine kinase 3 (Flt3) commonly co-occur (1-3). We demonstrate that mice with Flt3-internal-tandem duplication (Flt3ITD) and inducible deletion of Dnmt3a spontaneously develop a rapidly-lethal, completely-penetrant, and transplantable AML of normal karyotype. These murine AML retain a single Dnmt3a floxed allele, revealing the oncogenic potential of Dnmt3a haploinsufficiency. FLT3-ITD/DNMT3A-mutant primary human and murine AML demonstrate a similar pattern of global DNA methylation. In the murine model, rescuing DNMT3A expression was accompanied by DNA re-methylation and loss of clonogenic potential, suggesting that Dnmt3a-mutant oncogenic effects are reversible. Differentially methylated genomic regions were associated with changes in the expression of nearby genes. Moreover, dissection of the cellular architecture of the AML model using single-cell RNA-Seq, flow cytometry and colony assays identified clonogenic subpopulations that differentially express genes that are sensitive to the methylation of nearby genomic loci and varied in response to Dnmt3a levels. Thus, Dnmt3a haploinsufficiency transforms Flt3ITD myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation. To identify the gene expression changes associated with Dnmt3a loss of function in human and murine Flt3-ITD and Dnmt3a-mutant AML (Single Cell RNA-Seq).