Gene expression analysis of primary human trabecular meshwork cells cultured in fibroblast medium following selective laser trabeculoplasty treatment
ABSTRACT: Primary human trabecular meshwork cells cultured in fibroblast medium underwent selective laser trabeculoplasty treatment. RNA was extracted from a pool of cells 30 min after treatment while the remaining cells were further cultured and RNA was extracted respectively 2 and 6 hour after treatment. Control cells stored in incubator in absence of SLT treatment were used as reference samples. Gene expression was evaluated by hybridization on miRNA-microarray and laser scanner analysis. Time course experiment; HTM cells, ScienCell, San Diego, California: cat. n. 6590: isolated from juxtacanalicular and corneoscleral region of the human eye. TMC are cryopreserved on primary culture and delivered frozen.
Project description:T lymphocytes responding to microbial infection give rise to effector cells that mediate acute host defense and memory cells that provide long-lived immunity, but the fundamental question of when and how these cells arise remains unresolved. Here we combine single-cell gene expression analyses with machine-learning approaches to trace the transcriptional roadmap of individual CD8+ T lymphocytes throughout the course of an immune response in vivo. Gene expression signatures predictive of eventual fates could be discerned as early as the first T lymphocyte division and may be influenced by asymmetric partitioning of the interleukin-2 receptor during mitosis. These findings underscore the importance of single-cell analyses in understanding fate determination and provide new insights into the specification of divergent lymphocyte fates early during an immune response to microbial infection. The goal of the study was to profile the gene expression in single CD8+ T cells responding in vivo to a microbial infection over multiple timepoints. 5 x 103 CD8+ CD45.1+ T cells transgenic for the OT-1 T cell receptor (which recognizes Listeria monocytogenes expressing ovalbumin (Lm-ova)) were adoptively transferred into congenic wild-type CD45.2 C57/B6J recipients, followed by infection intravenously one day later with 5 x 103 colony-forming units (CFU) of Lm-OVA. Splenocytes were isolated from recipient mice at 5, 7, or 45 days post-infection. To isolate cells at 3 days post-infection, 2 x 104 OT-1 CD8+ T cells were adoptively transferred. To isolate cells that had undergone their first division, 2 x 106 OT-1 CD8+ T cells were first labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to adoptive transfer and recipient mice were sacrificed at 48 hours post-infection. Unactivated naïve OT-1 CD45.1+ CD8+ T cells were also included. Cells were stained with fluorochrome-labeled antibodies against CD8, CD44, CD4, CD11b, CD11c, and F4/80, and sorted on a MoFlo (Beckman Coulter) or FACS Aria II (BD Biosciences) flow cytometer. The Ct value for each gene analyzed in each individual cell at each time point is reported in the Matrix non-normalized. The 94 ABI TaqMan Assay IDs are listed on the left (A2-A96), along with the accession number (B2-B96) and corresponding gene name (C2-C96) of each gene studied. The Il2 gene expression assay was duplicated. Each heading (D1-BCV)) represents a single cell from each of 13 timepoints or T cell subset that were profiled. The timepoints/T cell subsets and number of each cells profiled from each timepoint/T cell subset were: unactivated naïve (149 cells), distal daughter (48), proximal daughter (83), division 1 (144), day 3 post-infection (143), day 5 post-infection memory precursor (Tmp) (90), day 5 post-infection short-lived effector (Tsle) (79), day 5 post-infection (154), day 7 post-infection memory precursor Tmp (62), day 7 post-infection short-lived effector Tsle (89), day 7 post-infection (134), central memory Tcm (138), and effector memory Tem (136).
Project description:In this experiment, we asked how the allelic distribution of the active and repressive chromatin marks in clonal cell lines relates to the transcriptional allelic bias. A multiplexed padlock probe approach (Zhang et al., 2009) enabled us to assess allelic bias in heterozygous exonic SNPs in two clones with GM12878 genotype, and four clones from GM13130 cells. We used this approach to assess allelic bias in H3K27me3 and H3K36me3 ChIP samples simultaneously with cDNA from the same cells, as well as ChIP input and genomic DNA controls. In order to pool data from two individuals, one of which (GM13130) lacked complete genotypes for parents, we assessed SNP bias as reference and alternative alleles (rather than maternal or paternal bias). SNPs in cDNA were assigned to one of three bins: reference allele bias; no bias; and alternative allele bias. For these groups, allelic bias in H3K27me3 (Fig.4B) and H3K36me3 (Fig.4C) was determined. In unbiased loci, both H3K27me3 and H3K36me3 were equally represented. In contrast, preferential expression of an allele was associated with elevated levels of H3K36me3 and decreased levels of H3K27me3 on that allele. Both effects were highly significant (p<2x10e-9). Genes predicted to have MAE were about four-fold over-represented among genes where SNPs showed significant bias (Fig.4D). SNPs with skewed H3K27me3 and H3K36me3 distribution were highly enriched in the genes predicted as MAE (p<10e-6 and p=0.01, respectively; two-tailed Fisher's exact test). This suggests that the asymmetric distribution of the histone modifications is to a large extent due to the genes that have the chromatin signature of monoallelic expression. Samples analyzed were A. polyclonal cell line GM12878 , and clones derived from it: DF1 and DF2, B. Polyclonal GM13130 (H0) and clones derived from it: H7, H14 and H16. gDNA, cDNA, ChIP material and input were used.
Project description:Retinal pigment epithelial cells are critical for eye function and loss of cell function is linked to age-related blindness. Relatively little is known about the transcriptional regulatory networks in these cells. The datasets presented here are ChIP-seq experiments for RNA polymerase II , transcription factors and histone modifications in human retinal pigment epithelial cells. ChIP-Seq for transcription factors, RNA polymerase, histone modifications and CTCF in retinal pigment epithelial cells
Project description:Peak oxygen consumption (VO2peak) strongly predicts morbidity and mortality better than other established risk factors, yet all mechanisms associated with its age-related decline are unknown. Our lab has shown that VO2peak first begins to decrease at the same age of 19 weeks old in Wistar in both sedentary and wheel-running, female Wistar rats. Here, we employed RNA-sequencing to determine the extent to which transcriptomic alterations in the left ventricle associate with age-related decreases in VO2peak. Transcriptomic comparisons were made within both sedentary (SED) and wheel-running (RUN) rats between 19 and 27 wks (n= 5-8/group).
Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by “bulk” assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) “Pre-MEP”, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) “E-MEP”, strongly biased towards erythroid differentiation, and (3) “MK-MEP”, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias. Multiplex RT-PCR gene expression profiling of 807 human megakaryocyte-erythroid progenitor cells (MEP) isolated from three healthy donors by apheresis following G-CSF treatment. Cells were excluded if more than 70 assays did not result in amplification or displayed Ct higer than 13 for B2M or higher than 15 for GAPDH. Furthermore cells with a mean non-dropout Ct value greater than 20 were removed. This resulted in a dataset of 681 cells, which were subsequently normalised to the mean of B2M and GAPDH expression.
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:We determined expression profiles of 667 miRNAs using TaqMan Low Density Arrays (TLDA-TaqMan Array Human MicroRNA Card Set v2.0, Applied Biosystems) in 8 samples of colorectal cancer tissues and 8 samples of paired non-tumoral colonic tissues.
Project description:MicroRNAs regulate various cellular processes. While several genes associated with replicative senescence have been described in endothelial cells, miRNAs that regulate these genes remain largely unknown. The present study was designed to identify miRNAs associated with replicative senescence and their target genes in HUVECs. We have employed Agilent Human MicroRNAs microarray platform to evaluate the expressions of 866 human miRNAs and 89 human viral miRNAs, based on Sanger miRNA database release 12.0 miRNA expression profiles were established for young and replicative senescent HUVECs
Project description:Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest. Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest.
Project description:In mammals, a key transition in spermatogenesis is the exit from spermatogonial differentiation and mitotic proliferation and the entry into spermatocyte differentiation and meiosis. Although several genes that regulate this transition have been identified, how it is controlled and coordinated remains poorly understood. Here we examine the role in male gametogenesis of the Doublesex-related gene Dmrt6 (Dmrtb1) and find that Dmrt6 plays a critical role in directing germ cells through the mitotic to meiotic germ cell transition. DMRT6 protein is expressed in late mitotic spermatogonia. In mice of the C57BL/6J strain a null mutation in Dmrt6 disrupts spermatogonial differentiation, causing expression in inappropriate cell types of spermatogonial differentiation factors including SOHLH1, SOHLH2 and DMRT1 and the meiotic initiation factor STRA8 and causing most late spermatogonia to undergo apoptosis. In mice of the 129Sv background, most Dmrt6 mutant spermatogonia can complete differentiation and enter meiosis, but they show defects in chromosome pairing, establishment of the XY body, and processing of recombination foci, and mainly arrest in mid-pachynema. mRNA profiling of Dmrt6 mutant testes together with DMRT6 ChIP-seq suggest that DMRT6 represses genes involved in spermatogonial differentiation and activates genes required for meiotic prophase. Our results indicate that Dmrt6 plays a key role in coordinating the transition in gametogenic programs from spermatogonial differentiation and mitosis to spermatocyte development and meiosis. Six samples for RNA-Seq with three biological replicates in each group. Two samples for ChIP-Seq (one input and one ChIP).