Single cell RNA-seq of sorted epitope-specific autoreactive B cells from healthy donor buffy coats
Ontology highlight
ABSTRACT: The aim of the experiment is to identify candidate genes to phenotype epitope-specific autoreactive B cells. These genes could serve as markers to delineate further populations of autoreactive B cells that were shown to be key players in regulating autoimmunity and in explaining B cell selection.
Project description:We have applied a recently developed, highly accurate and sensitive single-cell RNA-seq method (STRT/C1) to perform a molecular census of two regions of the mouse cerebral cortex: the somatosensory cortex and hippocampus CA1. We isolated cells fresh from somatosensory cortex (S1) and hippocampus CA1 area of juvenile (P22 - P32) CD1 mice, 33 males and 34 females. Cells were collected without selection, except that 116 cells were obtained by FACS from 5HT3a-BACEGFP transgenic mice. A total of 76 Fluidigm C1 runs were performed, each attempting 96 cell captures and resulting in 3005 high-quality single-cell cDNAs, containing Unique Molecular Identifiers allowing counting of individual mRNA molecules, even after PCR amplification.
Project description:We report dynamics of X-chromosome upregulation (XCU) along X-chromosome inactivation (XCI) in mESCs as they differentiate into EpiSCs. F1 hybrid C57BL6/J × CAST/EiJ male and female mESCs were adapted to 2i/LIF and female cells grown in serum/LIF conditions were differentiated using Fgf2 and Activin A for 1, 2, 4 and 7 days to induce random XCI. scRNA-seq was performed using the Smart-seq3 protocol, providing full-length coverage together with molecular counting using UMIs. Allelic resolution is achieved using strain-specific SNPs in the data. We reveal dynamic balancing of X alleles as cells undergo XCI to compensate dosage imbalances between sexes as well as between X and autosomes. Furthermore, we reveal that female naïve mESCs with two active X chromosomes lack XCU on both alleles which has major implications for reprogramming studies. Finally, we estimate allelic transcriptional burst kinetics from the data and find that progressively increased burst frequencies underlies the XCU process.
Project description:We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained from epileptic patients during temporal lobectomy for medically refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. Examination of cell types in healthy human brain samples.
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.
Project description:We report dynamics of X-chromosome upregulation (XCU) along X-chromosome inactivation (XCI) in mESCs as they differentiate into EpiSCs. F1 hybrid C57BL6/J × CAST/EiJ male and female mESCs were grown in serum/LIF conditions were differentiated using Fgf2 and Activin A for 1, 2, 4 and 7 days to induce random XCI in female cells. Multi-modal single-cell sequencing was performed using scATAC on nuclei and Smart-seq3 to assay chromatin accessibility and poly-A+ RNA expression, respectively. Allelic resolution is achieved using strain-specific SNPs in the data. We reveal dynamic balancing of X alleles as cells undergo XCI to compensate dosage imbalances between sexes as well as between X and autosomes. Furthermore, we reveal that female naïve mESCs with two active X chromosomes lack XCU on both alleles which has major implications for reprogramming studies. Finally, we estimate allelic transcriptional burst kinetics from the data and find that progressively increased burst frequencies underlies the XCU process.
Project description:Human cytomegalovirus infection (CMV) can stimulate robust human leukocyte antigen (HLA)-E restricted CD8 T cell responses. These T cells recognize a peptide from UL40, which differs by as little as a single methyl group from self-peptides that also bind HLA-E, challenging their capacity to avoid self-reactivity. We showed in one donor 2 distinct populations of UL40/HLA-E T cells, with vastly different T cell receptor (TCR) affinities for the UL40/HLA-E complex. However, paradoxically, lower cytokine responses were observed from UL40/HLA-E T cells bearing TCRs with high affinity for HLA-E. To identify why these T cells bearing high affinity T cell receptors were less responsive to antigens, we performed single cell RNAseq analysis. These 2 distinct populations of T cells were single-cell sorted into 96-well plates prior to single cell RNA-seq analysis.
Project description:Purpose: We applied cDNA molecule counting using unique molecular identifiers combined with high-throughput sequencing to study the transcriptome of individual mouse embryonic stem cells, with spike-in controls to monitor technical performance. We further examined transcriptional noise in the embryonic stem cells. One 96-well plate of single-stranded cDNA libraries generated from 96 single R1 mouse embryonic stem cells sequenced on two lanes, and one 96-well plate of the same libraries further amplified by 9 PCR cycles sequenced on one lane.
Project description:Plasmodium-specific CD4+ T cells from mice infected with Plasmodium chabaudi chabaudi AS parasites were recovered at Days 0, 7, 10, 14, 17, 21, 28 to undergo processing and generate scRNA-seq dataset. From Day 10 onwards, mice were administered with either saline or artesunate (intermittent artesunate therapy - IAT). scRNA-seq dataset was analysed to investigate transcriptome dynamics of CD4+ T cells from effector to memory states.
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:Human embryonic stem cells with one allele of the NEUROG3 gene genetically modified into a fusion gene NEUROG3-LINKER-TAGRFPT-P2A-EGFP-NLS were differentiated into the pancreatic lineage until Stage 4 Day 1 of the in vitro differentiation protocol (Rezania et al., 2014, as modified in Petersen et al., 2017). GFP positive and negative cells were FAC-sorted according to their cell cycle state (G0/G1 or G2M), and the global transcriptomes of these four groups were compared using bulk RNA-seq.