Single-cell transcriptome analysis of secretagogin mRNA expressing cells from the mouse hypothalamic paraventricular nucleus
ABSTRACT: The molecular mechanism regulating phasic corticotropin-releasing hormone (CRH) release from parvocellular neurons (PVN) remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin’s Ca2+ sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. single cells from the PVN region juvenile (21-28 days) mice were dissected and subject to whole transcriptome 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: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:5069 transcriptomes of single oligodendrocyte cells from spinal cord, substantia nigra-ventral tegmental area, striatum, amygdala, hypothalamic nuclei, zona incerta, hippocampus, and somatosensory cortex of male and female mice between post-natal day 21 and 90. The study aimed at identifying diverse populations of oligodendrocytes, and revealing dynamics of oligodendrocyte maturation. 5069 individual cells were sampled from CNS regions of mice of various strains as detailed in the protocols section
Project description:Three libraries from 100 HEK293 cells each were prepared using a Smartseq based custom library preparation approach with unique molecular identifiers. Libraries were sequenced on a Illumina NextSeq 500 HEK293 cell (100 cells) 5' selective RNAseq profiling, N4H4 unique molecular identifiers, 3 replicates
Project description:Five libraries from 100 HEK293 cells each were prepared using a Smartseq based custom library preparation approach with unique molecular identifiers. One batch of 2 replicates (A) and one batch of 3 replicates (B) were prepared from different cell cultures. Libraries were sequenced on an Ion Proton HEK293 cell (100 cells) 5' selective RNAseq profiling, N4H4 unique molecular identifiers, 2 replicates (A) and 3 replicates (B)
Project description:The control of cell identity is orchestrated by transcriptional and chromatin regulators in the context of specific chromosome structures. With the recent isolation of human naive embryonic stem cells (ESCs) representative of the ground state of pluripotency, it is possible to deduce this regulatory landscape in one of the earliest stages of human development. Here we generate cohesin ChIA-PET chromatin interaction data in naive and primed human ESCs and use it to reconstruct and compare the 3D regulatory landscapes of these two stages of early human development. The results reveal shared and stage-specific regulatory landscapes of topological domains and their subdomains, which consist of CTCF-CTCF/cohesin loops and enhancer-promoter/cohesin loops. The enhancer-promoter loop data reveal that genes with key roles in pluripotency are nearly always regulated by one or more super-enhancers, and show that these genes tend to occur in insulated neighborhoods. Our results reveal the key features of the 3D regulatory landscape of early human cells that form the foundation for embryonic development. ChIA_PET data against SMC1 from naive and primed human embroynic stem cells.
Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for DSBs after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 1 h after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, and R 1h, respectively. The strains from which the samples were collected are indicated following the time point, e.g. G1_MEC1 or R 1h_mec1. The experiment with mec1 was done twice (Experiments A and B) and that with MEC1 was done once (Experiment C). In addition, a control experiment of in vitro digestion with BamHI using the G1_mec1 sample (G1_BamHI) was performed.