RNA-seq of human ES cell lines HUES4 and H1, differentiated for 5 or 7 days towards pancreatic endoderm
ABSTRACT: The experiment was designed to unravel how endogenous signaling in differentiating hESCs is affected by exogenous factors. RNA-seq of human ES cell lines HUES4 and H1, differentiated for 5 or 7 days towards pancreatic endoderm. The cells were treated with IWP-L6, TGFb1 or both from day 3 and onwards. Vehicle controls were done with 0.1% DMSO.
Project description:To better understand the mechanism by which HES1 regulates pancreas development we took advantage of recent developments in directed differentiation of human embryonic stem cells (hESCs) to the pancreatic endocrine lineage via a series of progenitor stages (Figure 1 and (Rezania et al., 2014). Using the iCRISPR platform (González et al., 2014), we introduced indels in the HES1 and NEUROG3 genes, either singly or in combination in iCRISPR H1 cells. Using previously described gRNAs (Zhu et al., 2016) to target exon2 of the NEUROG3 gene and exon2 of the HES1 gene we detected by Sanger and RNA-sequencing a 2 bp insertion and a T insertion, respectively, resulting in premature STOP codons in two/multiple clonal cell lines carrying the introduced mutation and the loss of NEUROG3 by immunostaining We then subjected two or more clonal lines of H1-iCRISPR (wildtype), HES1−/−, NEUROG3−/− and HES1−/−NEUROG3−/− (abbreviated H1N3-dKO or H1−/−N3−/−) to differentiation to β-like cells using a modified version of the protocol from Rezania et al. (2014). Marker analysis showed that all cell lines maintained pluripotency (OCT4+) as hESC and were able to differentiate to FOXA2+ and SOX17+ co-positive definitive endoderm (DE, Day 3), PDX1+ cells at the posterior foregut stage (PF, Day 7), to bipotent progenitors marked by PDX1+ and NKX6-1+ at the Pancreatic Endoderm stage (PE, Day 10), and the Endocrine Precursor stage (EP, Day 13)
Project description:A 2-hour heat-shock at 37C was used to activate hs-FLP and an actin5C-FRT-stop-FRT-GAL4 transgene in larvae carrying any possible combination of the genetic elements UAS-Myc, UAS-Atu-IR, Max-/-. 48 hours later 16-27 wing imaginal discs were isolated from wandering L3 larvae and polyA-RNA was processed for sequencing.
Project description:Glioblastoma (GBM) is a highly heterogeneous malignant brain tumour. We took multi-region spatially separated samples from tumours and isolated invasive GBM cells using FACS based on 5ALA of near normal brain parenchyma. RNAseq was then performed to compare expression profiles for tumour cells from different microenvironment.
Project description:In this study,we demonstrated the transcription factor EGR1 is activated by TCM YYJD and such activation mediated YYJD-induced apoptosis in lung cancer cells and provided a novel insight to understand the anti-tumor mechanism of Chinese herb YYJD.
Project description:The purpose of the study was to determine what genes in DN2 pro-T cells are immediately regulated by the transcription factor GATA-3, either as activation targets or as repression targets. To do this, two pairs of Gata3-floxed and control pro-T cells were generated and analyzed by RNA-seq within the first day of deletion of the Gata3 gene. Pro-T cells were generated by differentiation in vitro on OP9-DL1 monolayers of fetal liver-derive precursors from wildtype or Gata3-floxed mice, and the Gata3 gene was acutely deleted by transduction with Cre retroviral vector. Within 20 hr after transduction, samples of acutely Gata3-deleted and control DN2 cells were sorted and RNA prepared for RNA-seq analysis. High-throughput sequencing of the samples was carried out. Experimental Gata3 deleted samples in both cases were Gata3-floxed, ROSA26R-EYFP samples infected with Cre retrovirus and sorted for EYFP+ (Cre-activated) DN2 phenotype. Control for experiment 1: wildtype (C57BL/6) DN2 pro-T cells generated in parallel, also treated with Cre retrovirus but sorted only for DN2 phenotype. Control for experiment 2: same genotype as experimental, but infected with a GFP+ empty retroviral vector and sorted for GFP+ DN2 phenotype. Two pairs of RNA-seq samples of DN2 pro-T cells were generated for comparison, each pair consisting of a Gata3-deleted sample plus a stage-matched control.
Project description:We have previously generated ABIN1[D485N] knock-in mice that appear relatively normal for 2 months after birth, but develop large spleen at 3 months, which is followed by the appearance of high levels of antibodies against self DNA and self-nuclear antigens and severe inflammation of the kidney and other tissues after 4-5 months, hall marks of lupus like autoimmunity. Autoimmunity in ABIN1[D485N] mice appears to be caused by hyper activation of pathogen sensing Toll-like Receptor (TLR) pathway, because the phenotype of the ABIN1[D485N] mice is completely suppressed by crossing them to mice that are either lacking or expressing functionally defective proteins, which play key role in TLR signalling. MyD88 KO, IRAK4 or IRAK1 kinase-inactive knock-in mice. However, cell type(s) required to drive lupus, and how this contributes to the development of the pathology, is unknown. Recently, we have identified atypical myeloid (CD11b+ve) populations in the blood, lungs liver, kidney and spleen of ABIN1[D485N] mice. Importantly, these cells are detectable at four weeks, at least two months before auto-antibodies are detected in the serum. More detailed flow cytometry analysis of these cells revealed that, in the blood, the majority were CD115+ve and these cells could be further subdivided into Ly6C+ve and Ly6C-ve, which are characteristics of the inflammatory and patrolling monocytes, respectively. While in the lungs and spleen of WT mice the Ly6C-ve CD115+ve (patrolling monocytes) are almost absent, in the ABIN1[D485N] mice this population is more than 20-fold increased. There is also increase in the number of neutrophil population (CD11b+ GR-1hi) in the spleen of the ABIN1[D485N] mice. Although we have shown that the formation of these atypical myeloid cells proceeds the development of auto-antibodies and organ inflammation in the ABIN1[D485] mice, the signalling pathway leading to formation of these cells and their role in autoimmunity is unknown. We therefore undertook RNAseq analysis to characterise these cells in more detail. The atypical CD11b+ve cells present in spleen of ABIN1[D485N] mice are heterogeneous with three major populations, neutrophils (CD11b+ GR-1hi), Ly6C+veCD115+ve cells and Ly6C-veCD115+ve. How these cell populations in ABIN1[D485N] mice relate to one another, and whether they share a common precursor is unknown. In order to profile the cells, the neutrophils, Ly6C+veCD115+ve cells and Ly6C-veCD115+ve populations were purified by cell sorting and RNAseq analysis carried out. The RNA seq analysis was carried out with at least four replicate samples from each cell populations from ABIN1[D485N] and WT controls.
Project description:One of the major hurdles for the early detection of cancer is our poor understanding of tumour initiating events. Historically, cancer research has focused on histological and molecular characterisation of established tumours, which has led to the identification of hundreds of putative driver mutations. It is currently unclear how these genetic aberrations impact the cell state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) for example has been shown to arise from luminal progenitor cells yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that the perturbation of Brca1/p53 in luminal progenitors induces an aberrant alveolar differentiation pre-malignancy. Unlike alveolar differentiation occurring during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Our experimental approach has allowed us to further identify responses in the stromal and immune cell compartments during the early steps of tumourigenesis. The data in this repository contains the human bulk RNA-sequencing that was presented as part of this study.
Project description:PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here we studied the function of PAX5-ETV6 and PAX5- FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested Blymphopoiesis at the pro-B-to-pre-B cell transition and, contrary to their proposed dominantnegative role, did not interfere with the expression of most Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressor in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-BCR signaling and migration/adhesion, which could contribute to the proliferation, survival and tissue infiltration of leukemic B-cells. Together with similar observations made in human PAX5-ETV6+ B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein. 36 samples in total: A) 24 RNA-Seq samples in 5 cell types: pro-B (5 genotypes, 2-4 replicates) large pre-B (2 genotypes, 2 replicates each) small pre-B (1 genotype, 2 replicates) lymph node (1 genotype, 3 replicates) bone marrow (1 genotype, 2 replicates) B) 12 ChIP-Seq samples in 2 cell types: pro-B (H3K27me3, H3K9ac, H3K4me2, H3K4me3, H3K27ac, 1 replicate each; Pax5Etv6 ChIP, Prd ChIP, 2 replicates each; Pax5 ChIP 1 replicate) lymph node (1 genotype, 2 replicates).
Project description:Using RNA-seq we aimed to characterise the transcriptome of patients with PSC-associated IBD relative to patients with ulcerative colitis or healthy. This was performed across multiple tissue locations in order to define tissue-dependent associations.