RNA-seq of IMR-5/75 neuroblastoma cell line treated with JQ1, THZ1 or combination against untreated controls
ABSTRACT: RNA-seq upon JQ1 (1 uM), THZ1 (35 nM), UT (untreated) or combination treatment in the neuroblastoma cell line IMR-5/75. Analysis was performed 10h upon treament. Four biological replicates per condition.
Project description:RNA-seq upon JQ1 (1 uM), THZ1 (35 nM), UT (untreated) or combination treatment in the neuroblastoma cell line IMR-5/75. Analysis was performed 10h upon treament. Four biological replicates per condition.
Project description:RNA-seq upon TBX2 knockdown in the neuroblastoma cell line CLB-GA. Cells were transduced with two different shRNAs (sh#2 and sh#4) targeting TBX2 and a non-targeting control (NTC), and selected with puromycin. Analysis was performed seven days upon TBX2 knockdown, including three biological replicates per condition.
Project description:RNA-seq upon TBX2, MYCN or combination of TBX2 and MYCN knockdown in the neuroblastoma cell line IMR-5/75. Cells were transduced with two different shRNAs (shTBX2_2 and shTBX2_4) targeting TBX2 and a non-targeting control (NTC), and selected with puromycin. Cells were treated with doxycycline for shMYCN induction (with DOX or not). Analysis was performed three days upon TBX2 knockdown and two days upon MYCN knockdown, including six biological replicates per condition.
Project description:Whole-exome sequencing was performed on DNA samples extracted from eight patient-derived melanoma cell lines grown in vitro in serum-free EGF/bFGF-containing medium. The aim of the experiment was to search for genetic alterations responsible for phenotypic diversity of melanoma cell lines reported at the level of cell morphology, activity of signaling pathways essential for melanoma development and progression, and response to drugs.
Project description:Background: Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET enzymes are able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. Furthermore, these modified bases are stable and detectable in the genome, raising the possibility that they could have epigenetic functions in their own right. To date, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture based systems, while its in vivo profile, in particular during development, remains unknown. Results: Here we describe the first analysis of the in vivo genome-wide profile of 5fC, across a range of dissected tissues from both wild type and Tdg-deficient E11.5 mouse embryos. Changes in the formylation profile of cytosine upon depletion of TDG suggest TET/TDG-mediated active demethylation occurs preferentially at intron-exon boundaries, and reveals a major role for TDG in shaping 5fC distribution at CpG islands. Moreover, we find enhancer regions exhibit high levels of 5fC, which accumulates at tissue-specific enhancers implicating a role in embryonic development. Conclusions: The tissue-specific distribution of 5fC can be regulated by the collective contribution of TET-mediated oxidation and excision by TDG. We show that the in vivo profile of 5fC during embryonic development resembles that of embryonic stem cells, sharing key features including enrichment of 5fC in enhancer and intragenic regions. Additionally, by investigating 5fC profiles in a tissue-specific manner from mouse embryos, we identified a targeted enrichment at active enhancers involved in tissue development. 5-formylcytosine has been mapped genomewide by pull-down and sequencing in mouse hindbrain, heart, carcass and liver. Each tissue was replicated in two different mice except for hindbrain which was replicated in four different mice.
Project description:CD4 T cell deficient for the Elp3 gene have a delayed cell cycle entry. We explored by RNAseq by which molecular pathway the absence of Elp3 delayed the cell cycle entry. Naive T cell from CD4-cre Elp3 fl/fl mice and WT littermate were purified and activated in vitro with CD3 and CD28 antibodies for 18h.
Project description:We differentiated the human embryonic stem cell line H9 into retinal pigment epithelium (RPE) cells, to assess their transcriptomic profiles over time in culture. In-depth molecular analysis was performed by single cell RNA-Sequencing to access the molecular signature of RPE cells grown and harvested at two time points in culture (30 days and 369 days post passaging). We performed high-resolution comparisons at subpopulation and single-cell levels, to assess gene expression pathway signatures in RPE cells and upon aging in vitro. The hESC line H9 was grown to 70-80% confluence, transitioned to E6 medium for 2 days, with supplementation of N2 from day 2 until day 33. On day 33, medium was switched to RPEM (alpha-MEM, N1 supplement, 5% FBS, NEAA, Pen Strep Glutamine, taurine-hydrocortisone-triiodo-thyronin (THT)) for an additional 32 days. Cells were enzymatically passaged using 0.25% Trypsin EDTA and plated at 75,000 cells/cm2 on Matrigel growth factor reduced pre-coated plates (P1). Cells were subsequently harvested at day 30 (sample \"YOUNG\") and day 369 (sample \"AGED\"). Both samples were sorted for live cells using PI on a BD FACS Aria. Cells were centrifuged at 300g for 5 min and resuspended in PBS containing 0.04% BSA to a concentration of ~800-1000 cells/ µl. Approximately 17,400 cells were loaded onto a 10X chip single Cell 3′ Chips along with the reverse transcription master mix as per the manufacturer's protocol for the Chromium Single Cell 3′ v2 Library for a target recovery of 10,000 cells. Samples were then processed for sc Seq.
Project description:Loss of Elp3 in CD4 T cells hindered the development of an optimal T follicular helper T cell (TFH) response. To understand the molecular pathway by which Elp3 is necessary for TFH responses, mice deficient for Elp3 in T cells and WT littermate were immunized with ovalbumin emulsified with complete Freund Adjuvant. 8 days later, TFH were sorted from the draining lymph nodes and submitted for RNAseq
Project description:Distinct shaping of the upper versus lower facial skeleton is essential for function of the vertebrate jaw and middle ear, yet the cellular mechanisms by which this occurs have remained unclear. Here, we show that Endothelin1 (Edn1) signaling accelerates mesenchymal condensation and subsequent cartilage formation in the lower face through antagonism of Jagged-Notch signaling and Prrx1 transcription factors. A genomic analysis of facial skeletal precursors in mutants and overexpression embryos reveals that Jagged-Notch signaling represses genes that are strongly induced as pharyngeal arch neural crest-derived cells begin skeletal differentiation. In wild types, initial Jagged-Notch repression dorsally ensures that barx1+ condensations and cartilage differentiation occur first in ventral-intermediate zones of the pharyngeal arches. Reduced Jagged-Notch signaling results in an expansion of pre-cartilage condensations in the upper face, with loss of barx1 partially restoring dorsal cartilage shapes in jag1b mutants. Further, by studying new mutants for zebrafish prrx1a and prrx1b, we find that Prrx1 genes function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Consistently, combined losses of jag1b and prrx1a/b robustly rescue ventral barx1+ condensations and lower facial cartilage development in edn1 mutants. Together, our work suggests that Edn1 works through parallel inhibition of Jagged-Notch and Prrx1 pathways to promote an earlier and more extensive establishment of cartilage condensations in the lower face. We performed RNAseq on FACS-sorted neural crest-derived pharyngeal arch cells (fli1a:GFP; sox10:DsRed double positive) from wild-type embryos at 3 different stages (20, 28, and 36 hours post fertilization) and embryos with altered levels of Edn1 and Notch signaling (edn1 mutants and hsp70I:Gal4; UAS:Edn1 transgenics; jag1b mutants, dibenzazepine-treated embryos, and hsp70I:Gal4; UAS:NICD transgenics. We also sequenced RNA from heat-shocked UAS:Edn1+ and hsp70I:Gal4+ transgenics and jag1b+/+ controls.