Extra-coding RNAs regulate neuronal DNA methylation and long-term memory formation [RNA-seq]
ABSTRACT: This dataset contains whole-genome RNA sequencing results from cortical neuronal cultures and serves as the basis for characterization of extra-coding RNA species from neuronal systems. This experiment contains six biological samples, each of which underwent PolyA+ and PolyA- RNA-seq. Samples were either unstimulated (i.e., treated with media alone; samples V1 and V2), stimulated with 25mM potassium chloride for 1hr (K1, K2) or inactivated with tetrodotoxin for 1hr (T1, T2). Datasets were obtained using RNA-seq from PolyA+ fractions or PolyA- fractions of RNA. PolyA- fractions are denoted "ec". Thus, 12 samples are listed here due to the difference in RNA library preparation.
Project description:This dataset contains whole-genome MBD (methylbinding domain) sequencing results from cortical neuronal cultures and serves as the basis for characterization of DNA methylation profiles from neuronal systems. This experiment contains three sequencing datasets from 2 biological samples. Two datasets originate from samples that underwent MBD-capture prior to whole-genome sequencing. A third dataset contains non-MBD-captured genomic DNA as a control.
Project description:The aim of this experiment was to develop new methods to extract pure fractions of nuclear and cytoplasmic RNA. We compared the results of nuclear and cytoplasmic RNA-sequencing to results of the total and polyA+ RNA-sequencing. Cytoplasmic, nuclear, total and polyA+ RNA was extracted from two human brain samples and sequenced on the SOLiD system. We analyzed the data to look for differences in levels of nascent transcription and mature mRNAs between the different samples.
Project description:Purpose: The study focused on the RNA content of supportive (AFT024) and non supportive (BFC012) stromal lines and their respective exosomes to analyze the molecular complexity of the Hematopoietic Stem and Progenitor Cell niche Methods: All the samples from small RNA (SR) and polyA-RNA libraries were sequenced using 1×50 bp single reads high-throughput sequencing (RNA-Seq) in single lane on Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks and by Fisher's test for polyA-RNA, or with bowtie and parse tools for smallRNA (galaxy server https://lbcd41.snv.jussieu.fr/). Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10). We focused on differentially expressed transcripts between i) exosomes from supportive and non supportive stromal cells and ii) exosomes and their respective cells.Using a subtractive strategy, we identified 324 mRNAs and 23 miRNAs specific to the exosomes of the supportive stromal line AFT024. We used gene ontology annotation to study the biological functions associated to these specific gene sets. Transcripts involved in negative regulation of apoptosis were found enriched in AFT exosomes. Conclusions: Our RNA seq analysis combined with biological assays reveal that exosomes serve as an important and novel mediator for the HSPC supporting capacity of stromal cells. This unprecedented effort to resolve the molecular complexity of the HSPC-targeted exosomes, provide new candidat genes of the HSPC support and may help designing innovative stromal-free culture conditions to deliver specific molecules to HSPCs. Fetal liver derived stromal lines and their corresponding secreted exosomes were sequenced using Illumina HiSeq2500 technology (Fasteris, CH). Due to low amount of exosomal RNAs, several RNA extraction were pooled and split to perform 2 technical sequencing replicates that were merged during bionformatic analyses.
Project description:We compare global gene expression changes in the Min6 cell line in response to altered glucose flux and pharmacological manipulation of the O-GlcNAc postranslational protein modification. Min6 cells were treated for 1hr in the following conditions: Low glucose (LG), high glucose (HG) and LG+GlcNAcstatin (LG+GNS). After treatment, total RNA was extracted and used for sequencing.
Project description:Our data suggest that all SR proteins contribute to mRNA export via NXF1. To identify endogenous export targets we depleted all seven SR proteins individually from P19 WT cells prepared cytoplasmic fractions. We sequenced the cytoplasmic fraction and as a control whole celll RNA from the identical sample. Knockdown of seven SR Proteins plus control, total RNA and cytoplasmic RNA, polyA+ enriched, 2 biological replicates per condition, 2 technical replicates per condition
Project description:Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation. We present slncky, a computational lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we develop a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript. To study a comprehensive and comparable set of lncRNAs expressed in the pluripotent state, we analyzed RNA-Seq data from pluripotent cells derived from several strains and species, and grown in two physiological conditions. First we derived “naïve” ES cells (ESCs) from each of three different mice strains: 129SvEv, NOD, and Mus musculus castaneus (cast) mouse, a wild mouse subspecies originally from Thailand, as well as naïve induced pluripotent stem (iPS) cells from rat and human. Next, to facilitate comparisons across pluripotent cells from different species, we also cultured mouse and rat cells in “primed” epiblast stem cell (EpiSC) culture conditions, since human iPS cells in culture are much more similar molecularly and functionally to mouse primed EpiSCs rather than to a ground state naïve ESCs. We polyA selected RNA from each cell type and deeply sequenced on HiSeq2500
Project description:Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome—presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc. ChIP-Seq experiments were performed on wild type and mutant cells of Bacillus subtilis 1A700.
Project description:This experiment seeks to ascertain the transcriptional changes in the adult mouse hippocampus (CA1 subregion) that occur following viral knockdown of the histone variant H2A.Z. We are especially interested in understanding the role of this histone variant in memory formation and memory maintenance in the adult central nervous system. This experiment includes 3 groups, each with 3 biological replicates. Samples S108, S109, and S110 are from controls infected with an AAV expressing a scrambled shRNA control. Samples A100, A101, A102, A104, A106, and A107 were infected with an AAV expressing an shRNA against H2A.Z. Samples A100, A101, and A102 were naive animals, whereas samples A104, A106, and A107 were trained in contextual fear conditioning.
Project description:The C2H2 zinc finger is the most prevalent DNA-binding motif in the mammalian proteome, with DNA-binding domains usually containing more tandem fingers than are needed for stable sequence-specific DNA recognition. To examine the reason for the frequent presence of multiple zinc fingers, we generated mice lacking finger 1 or finger 4 of the 4-finger DNA-binding domain of Ikaros, a critical regulator of lymphopoiesis and leukemogenesis. Each mutant strain exhibited a specific subset of the phenotypes observed with Ikaros null mice. Of particular relevance, fingers 1 and 4 contributed to distinct stages of B- and T-cell development and finger 4 was selectively required for tumor suppression in thymocytes and in a new model of BCR-ABL+ acute lymphoblastic leukemia. These results, combined with transcriptome profiling (this GEO submission: RNA-Seg of whole thymus from wt and the two ZnF mutants), reveal that different subsets of fingers within multi-finger transcription factors can regulate distinct target genes and biological functions, and they demonstrate that selective mutagenesis can facilitate efforts to elucidate the functions and mechanisms of action of this prevalent class of factors. RNA-Seq from BCR-ABL+ transformed cells at day 21 and day 28 of in vitro cell culture comparing wt, Ikaros-ZnF1-/- mutant and Ikaros-ZnF4-/- mutant.
Project description:The Caenorhabditis elegans somatic gonad was the first organ to have its cell lineage determined, and the gonadal lineages of the two sexes differ greatly in their pattern of cell divisions, cell migration and cell types. Despite much study, the genetic pathways that direct early gonadal development and establish its sexual dimorphism remain largely unknown, with just a handful of regulatory genes identified from genetic screens. To help define the genetic networks that regulate gonadal development, we employed cell-specific RNA-seq. We identified transcripts present in Z1/Z4 or Z1/Z4 daughter cells in each sex at the onset of somatic gonadal sexual differentiation. For comparison, transcripts were identified in whole animals at both time points. Pairwise comparisons of samples identified several hundred gonad-enriched transcripts, including most known Z1/Z4-enriched mRNAs, and reporter analysis confirmed the effectiveness of this approach. Prior to the Z1/Z4 division few sex-biased Z1/Z4 transcripts were detectable, but less than six hours later, we identified more than 250 sex-biased transcripts in the Z1/Z4 daughters, of which about a third were enriched in the somatic gonad cells compared to cells from whole animals. This indicates that a robust sex-biased developmental program, some of it gonad-specific, initiates in these cells around the time of the first Z1/Z4 division. Cell-specific analysis also identified approximately 70 previously unannotated mRNA isoforms that are enriched in Z1/Z4 or their daughters. Our data suggest that early sex differentiation in the gonad is controlled by a relatively small suite of differentially expressed genes, even after dimorphism has become apparent. 20 total sample: two time points, two sexes, and gonadal cells or whole animals. The earlier time point was collected in triplicate and was harvested 9.5 hours after starved, hatched L1s were fed. The later time point was collected in duplicate and was harvested 15 hour after starved, hatched L1 were fed. Replicates of either dissociated whole animals or gonadal cells (Z1/Z4 or Z1/Z4 daughter) from both male and hermaphrodites were harvested for each time point.