title: Gene expression in hearts of mice fed a high fat diet

status: Public on May 11 2020

city: Suzhou

first: Tianqing

last: Peng

state: Jiangsu

address: No.199 Ren'ai Road

department: IBMS

institute: Soochow University

summary: To understand the impact of high fat diet on gene expression in mouse hearts, a micro-array analysis was carried out

overall_design: Male mice at age of 4 weeks were maintained on either a high fat diet (60% kcal from fat, HFD) or standard chow diet (normal diet, ND) for 4 months. Mice were sacrificed by the end of the 4th months with hearts harvested and subjected to micro-array analysis

first: Shengcun

last: Li

first: Lulu


title: Investigation of the contribution of Complex I to respiratory energy-coupling in glucose-grown cultures of the Crabtree-negative yeast Ogataea parapolymorpha

status: Public on May 12 2020

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

type: Expression profiling by high throughput sequencing

summary: The methylotrophic, thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host and exhibits a respiratory metabolism in the presence of oxygen. It possesses a branched respiratory chain with multiple entry points for NADH-derived electrons that differ in complexity and degree of energy conservation: proton-translocating respiratory Complex I and three putative alternative NADH dehydrogenases. To investigate the physiological importance of Complex I, wild type O. parapolymorpha and a Complex I-disrupted mutant were cultured in glucose-grown bioreactor experiments in batch, chemostat and retentostat cultivations which allowed quantitative characterization of the strains over a wide range of growth rates in the presence and absence of excess substrate.

overall_design: RNASeq-based transcriptome analysis was performed in biological duplicates on samples taken from aerobic, glucose-grown bioreactor cultivation. Conditions were batch cultivation (0.37h-1), chemostat cultivation at dilution rates of  0.1 and 0.025 h-1, as well as late-stage retentostat cultivation (approx. 0.001 h-1) for both wild type O. parapolymorpha (CBS11895) and a mutant devoid of functional repiratory Complex I (IMD010).

first: Hannes

last: Juergens

first: Xavier

last: Hakkaart

first: Jildau

last: Bras

first: Robert

last: Mans

first: Marcel

last: van den Broek


title: scRNA-seq of the developing human retina

status: Public on May 07 2020

city: St. Louis

first: Brian

middle: Stewart

last: Clark

state: Missouri

address: 660 S Euclid Ave; Campus Box #8096

department: Ophthalmology and Visual Sciences

country: USA

institute: Washington University

summary: Time-series of single-cell RNA-sequencing performed on stem cell derived retinal organoids or post-mortem tissue from the developing and adult retina

overall_design: 10x Genomics V2 single-cell RNA-sequencing

first: Seth

last: Blackshaw

middle: S

first: James

middle: T

last: Handa

first: Rod

last: Bremner

first: Donald

middle: J

last: Zack


title: A Unified View of the Sequence and Functional Organization of the Human RNA Polymerase II Promoter

status: Public on Apr 29 2020

city: Iowa City

first: David

last: Price

state: IA

address: 500 Newton Road, 260 EMRB

department: Biochemistry

institute: The University of Iowa

summary: To expand our understanding of the interplay of human RNA polymerase II (Pol II) promoter elements, promoter-proximal pausing, local chromatin structure, and co-transcriptional RNA processing, the 5′ and 3′ ends of nascent, capped transcripts and the locations of nearby nucleosomes were accurately identified. High-quality visualization tools revealed sequence elements defining the core promoter, sites of pausing, and nucleosome positioning across over 177,000 transcription start regions (TSRs). We found that cap methylation begins as transcripts attain a length of about 30 nt. A nuclear run-off assay was developed that utilizes the unique properties of the DNA fragmentation factor (DFF) to demonstrate the relative location of nucleosomes and paused Pol II. Our data support the conclusion that human Pol II promoters are functional TFIID binding sites with built-in downstream information directing ubiquitous promoter proximal pausing and the downstream nucleosome location.

overall_design: NasCap design: PRO-Seq protocol with a modified front end to rapidly isolate nuclei followed by selection of capped transcripts containing m7G or non methylated capped transcripts (NasCap). DFF-Seq design: The level of nucleosome occupancy across the human genome has been estimated using Micrococcal Nuclease (MNase) coupled with DNA sequencing. However, the nucleosome occupancy levels generated from the more selective nuclease, DNA Fragmentation Factor (DFF), has not been investigated.  We generated our own measure of nucleosome occupancy using DFF and subsequent DNA sequencing. HeLa cells were treated with the P-TEFb inhibitor flavopiridol prior to nuclei isolation and were then treated with DFF. Here, we have sequenced the undigested DNA from HeLa cells treated with 1 µM flavopiridol for 1 h and DFF for 30 minutes.

middle: H

first: Kyle

middle: A

last: Nilson

first: Donal

last: Luse

first: Benjamin

middle: M

last: Spector

first: Mrutyunjaya

last: Parida


title: CASCADE - Customizable high-throughput platform for profiling cofactor recruitment to DNA to characterize cis-regulatory elements and screen non-coding SNPs

status: Public on Apr 21 2020

city: Boston

first: Trevor

last: Siggers

state: MA

address: 24 Cummington Mall

department: Department of Biology

institute: Boston University

summary: Determining how DNA variants affect the binding of regulatory complexes to cis-regulatory elements (CREs) and non-coding single-nucleotide polymorphisms (ncSNPs) is a challenge in genomics. To address this challenge, we present CASCADE (Comprehensive ASsessment of Complex Assembly at DNA Elements) – a protein-binding microarray (PBM)-based approach to profile the recruitment of cofactors (COFs) to DNA sequence variants, and to infer the identity of the transcription factor-cofactor (TF-COF) complexes involved. We use CASCADE to characterize regulatory complexes binding to CREs and SNP quantitative trait loci (SNP-QTLs) in resting and stimulated human macrophages. By profiling p300 we correctly identify IRF3 and NF-κB as LPS-dependent regulators of the chemokine CXCL10, and by profiling a set of five functionally diverse COFs we identify a prevalence of ETS sites mediating COF recruitment at SNP-QTLs in macrophages. Our results demonstrate that CASCADE is a customizable, high-throughput platform to link DNA variants with the biophysical complexes that mediate functions such as chromatin modification or remodeling in a cell state-specific manner.

overall_design: LPS-responsive CXCL10 promoter segment CASCADE (ID: 085605) - These experiments profile recruitment of transcriptional cofactors (COFs: p300 and RBBP5) as well as transcription factors (TFs: IRF3, p65, IRF2, IRF8) to an LPS-responsive segment of the CXCL10 promoter using tiling probes and all single variant probes of each tile. Recruitment of COFs and TFs was profiled directly from nuclear extracts collected from untreated (UT) or LPS-stimulated (LPS45) PMA-treated THP-1 cells. Differential COF recruitment screen (ID: 085920) – These experiments profile recruitment of a panel of COFs (p300, SMARCA4, TBL1XR1, RBBP5, HDAC1, and GCN5) as well as TFs (PU.1 and p65) to pairs of reference and variant alleles from previously characterized myeloid SNP-QTLs. Technical replicates were performed (REP1 and REP2) for each COF/TF screened. SNP-QTL CASCADE (ID: 086248) – These experiments employ the full CASCADE approach to characterize recruitment motifs for significant differential COF recruitment and TF binding events measured using the previous COF recruitment screen. The same set of COFs/TFs were profiled in this series of experiments (p300, SMARCA4, TBL1XR1, GCN5, RBBP5, HDAC1, PU.1, and p65).

last: Bray

first: Heather

last: Hook


title: Skeletal Stem Cell Powering Cranial Suture Fate: An Answer to Craniosynostosis (bulk_RNA-seq)

status: Public on Apr 15 2020

city: Stanford

first: Natalina

last: Quarto

state: CA

address: 257 Campus Drive

department: Surgery - Plastic and Reconstructive Surgery

institute: Stanford University School of Medicine

summary:
Purpose: The cranial suture is a fibrous joint, and similar to the growth plates of the skeletal long bone, they serve as the major centers of calvarial vault morphogenesis. Our group’s identification of a skeletal stem cell isolated from the mouse tibial growth plate prompted us to investigate whether these skeletal stem cells are also resident in the mouse cranial sutures and if they govern postnatal suture patency or fusion.
Results: We preformed a spatio-temporal profiling of the mouse cranial sutures by flow cytometry, demonstrating a significant decrease in the temporal representation of skeletal stem cells in fusing versus patent sutures. Moreover, canonical Wnt signaling has a significant impact on skeletal stem cells proliferation and thus representation within the suture, dictating fate: fusion or patency. Breeding an Axin2+/-LacZ mouse, with enhanced activation of canonical Wnt signaling to a Twist1+/− mouse, harboring a coronal craniosynostosis enriched the skeletal stem cell pool in coronal sutures, thereby preventing Twist1+/− craniosynostosis.
Conclusions: Our findings suggest an imbalance and/or decrease in resident skeletal stem cells within the cranial sutures gives rise to craniosynostosis, however, restoring this representation by enriching skeletal stem cells within the suture can maintain patency.

overall_design: Total RNA profiles of SSCs isolated from post-natal day 3 posterior frontal (PF), sagittal (Sag), coronal (Cor) sutures and the growth plate (GP) of the tibial long bone from CD-1, wild-type mice were generated by sequencing using Illumina Hi-Seq 4000.

first: Siddharth

last: Menon

first: Ankit

last: Salhotra

first: Siny

last: Shailendra

first: Ryan

middle: C

last: Ransome

first: Charles

middle: K

last: Chan

first: Derrick

last: Wan

first: Michael

last: Longaker


title: Heterogeneity of mouse pro-epicardial cells

status: Public on Apr 04 2020

city: Houston

first: Mingfu

last: Wu

state: Texas

address: University of Houston College of Pharmacy, 4349 Martin Luther King Boulevard

department: Molecular and Cellular Physiology

institute: University of Houston

summary: Single cell RNA sequencing (scRNA-Seq) is applied to charaterize the pro-epicardial cells (proEpiCs) in mouse pro-epicardial organ (PEO).

overall_design: scRNA-Seq; 74 samples

first: Weihua

last: Huang


title: Skeletal Stem Cell Powering Cranial Suture Fate: An Answer to Craniosynostosis

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Skeletal Stem Cell Powering Cranial Suture Fate: An Answer to Craniosynostosis (scRNA-seq)

overall_design: Total single cell RNA profiles of SSCs isolated from post-natal day 3 posterior frontal (PF), sagittal (SAG), coronal (COR) sutures and the growth plate (GP) of the tibial long bone from CD-1, wild-type mice were generated by sequencing using Illumina Hi-seq 4000.


title: Systematic identification and analysis of light-responsive circular RNA and co-expression networks in lettuce (Lactuca sativa)

status: Public on Apr 14 2020

city: Yangling

first: yongjun

last: wu

address: No. 22 Xinong Road, Yangling Demonstration Zone

country: China

institute: Northwest A&F University

summary: Circular RNA (circRNA) is a covalently-closed single-stranded RNA molecule that plays an important role in transcriptional regulation of gene expression in a variety of species. Light intensity is a pivotal environmental factor affecting plant growth and development. However, little is known regarding photoresponsive plant circRNAs. Here, we aimed to investigate the expression and function of circRNAs in lettuce leaves in response to different light intensity treatments. We performed RNA sequencing (RNA-Seq) on leaves of lettuce (Lactuca sativa) to determine circRNA expression profiles and reverse-transcription polymerase chain reaction (PCR) to validate the candidate circRNA molecules. We then combined bioinformatics approach to explore the function of the parental genes of circRNA, including network, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. We identified 1650 circRNAs in lettuce, of which 1508 (86.40%) were derived from exons. Using real-time PCR, we characterized 10 validated differentially expressed circRNAs and their parental genes, all of which showed expression patterns consistent with RNA-Seq data. Interestingly, the expression of circRNA was, in some cases, inversely correlated with the expression of the parental gene. Furthermore, analysis of the circRNA–microRNA–mRNA network suggests that circRNAs may be involved in plant hormone signaling and chlorophyll metabolism during photoreactivity. These findings provide an essential reference basis for studying circRNAs’ biological mechanisms in light-treated plants.

overall_design: Lettuce mRNA profiles of 24-day-old 60±2 μmol·m–2·s–1 (Las_WL group. low light intensity), 175±2 μmol·m–2·s–1 (Las_ML group, medium light intensity) and 340±2 μmol·m–2·s–1 (Las_SL group, high light intensity) were generated by deep sequencing, in triplicate, using Illumina Hiseq 4000.

first: Zhenchao

last: Yang

first: Yongjun


title: Mechanism and Reversal of Nephrotoxicity on a Chip

status: Public on Mar 31 2020

city: Jerusalem

first: Avner

last: Ehrlich

address: Silberman 3-512, Edmond J. Safra Campus (Givat Ram)

department: Bioengineering

country: Israel

institute: The Hebrew University of Jerusalem

summary: The kidney plays a critical role in fluid homeostasis, glucose control, and drug excretion. Loss of kidney function due to drug-induced nephrotoxicity affects over 20% of the adult population. The kidney proximal tubule is a complex vascularized structure that particularly vulnerable to drug-induced nephrotoxicity. Here we introduce a model of vascularized human kidney organoids with integrated tissue embedded micro-sensors for oxygen, glucose, lactate and glutamine, providing real time assessment of cellular metabolism. Our model shows that both the immunosuppressive drug cyclosporin (Neoral®) and the anti-cancer drug cisplatin (Platinol®) disrupt proximal tubule polarity at sub-toxic concentrations, leading to glucose accumulation and lipotoxicity. Impeding glucose reabsorption using glucose transport inhibitors blocked cyclosporin and cisplatin toxicity by 1,000 to 10-folds, respectively. Retrospective study of 247 patients receiving cyclosporin or cisplatin in combination with the SGLT-2 inhibitor gliflozin showed significant reduction in creatinine and uric acid recognized markers of kidney damage. These results demonstrate the potential of sensor-integrated organoid-on-chip platforms to elucidate new mechanisms of action and rapidly reformulate effective therapeutic solutions, increasing drug safety and reducing the cost of clinical and commercial failures.

overall_design: Comparison between different proximal tubule in vitro models

first: Aaron

last: Cohen

first: Yaakov

last: Nahmias


title: Circulating miRNome expression profiling in Behçet's disease

status: Public on Apr 01 2020

city: Florence

first: Giacomo

last: Bagni

address: Viale GB Morgagni 50

department: Department of Experimental and Clinical Medicine

country: Italy

institute: Università degli studi di Firenze

summary: Circulating plasma miRNAs profiling in platelets-free plasma  samples from Behçet's disease patients compared with healthy subjects, aimed to both pathogenesis elucidation and candidate non-invasive biomarkers identification.

overall_design: Plasma microRNAs expression profile was assessed in Behçet's disease patients compared with healthy controls using Agilent miRNA microarray technology in order to perform a screening phase.

last: Emmi

first: Elena

last: Lastraioli

first: Francesca

last: di Patti

first: Angela

last: Guerriero

first: Annarosa

last: Arcangeli

first: Domenico

last: Prisco


title: Next Generation Sequencing Facilitates Quantitative Analysis of A549 Control and A549 infected with Zika virus Transcriptomes

status: Public on Mar 06 2020

city: Chengdu

first: Xinzhong

last: Liao

state: Sichuan

address: Institute of Blood Transfusion (IBT) Chinese Academy of Medical Sciences (CAMS)and Peking Union Medical College (PUMC)  Chengdu,Sichua

institute: Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College

summary:
Purpose:The goals of this study to compare NGS-derived  transcriptome profiling (RNA-seq) to examine differentially expressed genes berween  A549  infected with Zika virus and A549 Control .
Methods: A549 cells mRNA profiles of A549 cells-infected with Zika virus and aun-infected A549 cells were generated by deep sequencing, in triplicate, using Illumina Hiseq4000. The mapped reads of each sample were assembled by StringTie (v1.3.1) (Mihaela Pertea.et al. 2016) in a reference-based approach. StringTie uses a novel network flow algorithm as well as an optional de novo assembly step to assemble and quantitate full-length transcripts representing multiple splice variants for each gene locus.
Results: Using an optimized data analysis workflow, we mapped about identified 11124 transcripts in the A549 cells of control and  Zika virus with Cuffcompare workflow. RNA-seq data confirmed stable expression of 20 known housekeeping genes, and 10 of these were validated with qRT–PCR. Approximately 10% of the transcripts showed differential expression between the control and DV, with a fold change >1 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method.
Conclusions: Our study represents the first detailed analysis of A549 cells transcriptomes, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: A549 cells mRNA profiles of un-infected A549 cells (C) and Zika virus-infected A549 cells (ZV) were generated by deep sequencing, in triplicate, using Illumina HISEQ4000.

first: Limin

last: Chen

first: Xiaoqiong

last: Duan


title: Effect of depletion of KDM4D, NFIB, and MLL1 on gene expression during adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

status: Public on Feb 26 2020

city: Incheon

first: Hansol

last: Lee

address: 100, Inha-ro, Michuhol-gu

department: Biological science

country: South Korea

institute: Inha University

summary:
To investigate the cooperative function KDM4D/NFIB/MLL1 complex in the regulation of adipogenic differentiation, we established 10T1/2 cell lines in which each target gene has been knocked down by shRNA.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at two time points.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for C3H10T1/2 cells and its KD derivatives (shKDM4D, shNFIB, and shMLL1).

first: Janghyun

last: Choi


title: Lectin microarray analysis of  murine CTL EVs separated by ion exchange method

status: Public on Mar 12 2020

city: Tsu

first: Naohiro

last: Seo

state: Mie

address: 2-174 Edobashi

department: Immuno-Gene Therpy

country: Japan

institute: Mie University Graduate School of Medicine

summary:
EVs in culture supernatant can be concentrated with removing exsomeres and 97% free proteins by MWCO 750 kDa UF.
DEAE chromatography can be divided into bioactive EXO and other EVs as nucleic acid (DNA) cargo in UF-concentrated EVs.

overall_design: Recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (EVs). In this study, we propose a novel method for large scale preparation of high-performance extracellular vesicles focusing on membrane negative charge. Murine cytotoxic T lymphocyte (CTL) EVs in supernatant are concentrated more than 20 times at over 97% purity without leaking by 750 kDa MWCO ultrafiltration, replaced with PBS, and subjected to ion exchange DEAE column chromatography.

first: Hiroaki

last: Tateno


title: Characterization of True Germinal center follicular helper T cells

status: Public on Mar 17 2020

city: Durham

first: Chen-Hao

last: Yeh

state: NC

address: 207 Research Drive

department: Immunology

institute: Duke University

summary: Despite interest in T follicular helper (TFH) cell development and function, differences between TFH cells resident in germinal centers (GCs) and phenotypically similar, non-resident TFH cells are unclear. Both TFH subsets share the Bcl6+FoxP3−CXCR5hiPD-1hi phenotype but we show that only GC resident TFH cells express S1pr2 and lose CD90 expression. Whereas immunization elicits CD90neg/lo GCTFH and CD90hi GCTFH-like cells similarly, these TFH subsets have distinct properties. CD90neg/lo GCTFH cells require MHCII+ B cells to develop and once formed, do not proliferate. CD90hi GCTFH-like cells are generated in the absence of B cells and remain mitotically active. The Tcrβ repertoires of both TFH types are initially shared but with time, Tcrβ usage in CD90hi GCTFH-like cells substantially diverges by DC-driven proliferation. Transcriptome analysis shows CD90neg/lo GCTFH to be specialized for vesicle transport and exocytosis while gene expression in CD90hi GCTFH-like cells is linked to cell activation, proliferation and migration.

overall_design: RNAseq of GCTFH, TFH-like, TFR and eff non-TFH cell populations in the same draing LN 8, 12, 16 and 24 days post-immunizaiton.

first: Garnett

last: Kelsoe


title: miRNA microarray analysis of  murine CTL EVs separated by ion exchange method [miRNA]


title: Vaccination accelerates hepatic erythropoiesis induced by blood-stage malaria

status: Public on Feb 10 2020

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary: Increasing evidence suggests the liver as to be an effector against blood-stage malaria. Vaccination induces changes in the liver and survival of otherwise lethal blood-stage malaria of Plasmodium chabaudi which is associated with changes in the liver. Here, the time-course of expression of erythroid genes is investigated during infections with P. chabaudi in the liver of vaccination-protected and unprotected non-vaccinated mice.

overall_design:
Female Balb/c mice were vaccinated with a non-infectious vaccine at week 3 and week 1 before challenging with 106 P. chabaudi-parasitized erythrocytes. Gene expression microarrays and quantitative real-time PCR were used to compare mRNA expression of erythroid genes in the liver of vaccinated and non-vaccinated mice at different phases of infections on days 0, 1, 4 , 8, 11 p.i..

30 samples were analyzed:
Nd0, Non-vaccinated day 0, 3 replicates
Nd1, Non-vaccinated day 1, 3 replicates
Nd4, Non-vaccinated day 4, 3 replicates
Nd8, Non-vaccinated day 8, 3 replicates
Nd11, Non-vaccinated day 11, 3 replicates
Vd0, Vaccinated day 0, 3 replicates
Vd1, Vaccinated day 1, 3 replicates
Vd4, Vaccinated day 4, 3 replicates
Vd8, Vaccinated day 8, 3 replicates
Vd11, Vaccinated day 11, 3 replicates

first: Denis

last: Delic

first: Frank

last: Wunderlich

first: Saleh

last: Al-Quraishy

first: Mohamed

last: Dkhil


title: Oocytes, a single cell and a tissue

status: Public on Feb 14 2020

city: Ashburn

first: Di

last: Wu

state: Virginia

address: 19700 Helix Drive

institute: HHMI Janelia

summary: Development of single cell sequencing allows detailing the transcriptome of individual oocytes. Here, we compare different RNA-seq datasets from single and pooled mouse oocytes and show higher reproducibility using single oocyte RNA-seq. We further demonstrate that UMI (unique molecular identifiers) based and other deduplication methods are limited in their ability to improve the precision of these datasets. Finally, for normalization of sample differences in cross-stage comparisons, we propose that external spike-in molecules are comparable to using the endogenous genes stably expressed during oocyte maturation. The ability to normalize data among single cells provides insight into the heterogeneity of mouse oocytes.

overall_design: To confirm the ERCC normalization, we performed mouse single oocyte RNA-seq with different amplification cycles during library preparation and also different fractions of GV oocytes (1/2, 1/4, 1/8).

first: Dean

last: Jurrien


title: Single cell RNA-seq data in the immune cells and endothelial cells isolated from spinal cord of EAE mice upon Ang2 blockade and overexpression

status: Public on Mar 10 2020

city: Helsinki

first: Zhilin

last: Li

address: Haartmaninkatu 8

country: Finland

institute: University of Helsinki

summary: Here we show that Ang2 blockade suppressed the EAE-induced activation of immune cells and endothelial cells in the spinal cord. In contrast, Ang2 overexpression promoted the EAE-induced activation of immune cells and endothelial cells. All the samples were analyzed using the Chromium Single-cell 3'RNA-sequencing system.

overall_design: We analyzed in total 4 samples, targeting cell number for all the samples are 10,000.

first: Emilia

last: Korhonen

first: Kari

last: Alitalo


title: A USP28-∆Np63 axis is a druggable vulnerability of squamous tumour formation and maintenance

status: Public on Mar 04 2020

city: Würzburg

first: Markus

last: Diefenbacher

address: Am Hubland

department: Chair of Biochemistry and Molecular Biology

institute: University Würzburg

summary: The oncoprotein ∆Np63 is an essential transcriptional master- and cell identity regulator in squamous cell carcinoma (SCC) of various origins, encompassing lung, head and neck, oesophagus, cervix and skin. While in non-transformed cells ∆Np63 protein abundance is tightly regulated by the ubiquitin proteasome system (UPS), in tumours E3-ligases ubiquitylating ∆Np63, such as FBXW7, are commonly mutated or lost, resulting in a hyper-stabilisation of the oncogenic driver. Targeting ∆Np63 protein abundance in SCC could present a possible therapeutic avenue. Here, we report that the deubiquitylase USP28 regulates ∆Np63 protein stability and abundance in SCC by counteracting the degradative UPS system. Interference with USP28 activity by pharmacological inhibition specifically affected human SCC cell lines and, finally, we were able to demonstrate in vivo using CRISPR/Cas9 mouse models that Usp28 is required for SCC induction and maintenance. Hence, targeting USP28 is a viable option to tackle ∆Np63 protein abundance in SCC tumours.

overall_design: RNA-seq analysis after knockdown or inhibition of USP28 and deltaNp63 in A431 cells (human squamous cell carcinoma cell line).

first: Cristian

last: Prieto-Garcia

first: Oliver

last: Hartmann

first: Michaela

last: Reissland

first: Fabian

last: Braun

first: Thomas

last: Fischer

first: Susanne

last: Walz

first: Annalena

first: Marco

last: Canale

first: Amir

last: Orian

first: Mathias

last: Rosenfeldt

first: Martin

last: Eilers

middle: E


title: H3K27ac in WT and Shh-/-, and Shh-/-;Gli3-/- E10.5 limb buds

status: Public on Jan 21 2020

city: Austin

first: Steven

last: Vokes

address: 2500 Speedway, Stop A4800

department: Department of Molecular Biosciences, Institute for Cellular & Molecular Biology

institute: The University of Texas at Austin

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers. While most Hh targets in the developing limb bud appear to be regulated by the activity of GLI-repressor, the role of GLI activator in this regulation is unclear. Here we assess the roles of GLI activator and repressor in regulating H3K27ac to activate their enhancers.
Methods:  ChIP-seq was performed in Embryonic day 10.5 mouse limb buds from WT, Shh-/- and Shh-/-Gli3-/- embryos
Results:  We found that most GLI binding regions can be activated in the absence of both GLI activator and repressor, while a small subset require GLI activator for activity. These indicate that most GLI enhancers in the limb are regulated by GLI repression.

overall_design: ChIP-sequencing of E10.5 forelimb buds from WT,  Shh-/-, and Shh-/-;Gli3-/- embryos. Two biological replicates and one input control were submitted per biological condition.

first: Rachel

last: Lex

first: Zhicheng

last: Ji

first: Hongkai


title: HDAC1 binding regions in mouse embryonic limbs

summary:
Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers and the majority of Hh targets in the developing limb bud are regulated solely by the activity of GLI-repressor.  Currently we do not have a comprehensive understanding of how GLI bound enhancers respond Hedgehog signaling.  The goal of this study is to identify how GLI bound enhancers are regulated by Hedgehog signaling and specifically by GLI-repressor.
Methods:  ChIP-seq was performed in Embryonic day 11.5 mouse forelimb and hidlimbs lfrom wildtype Swiss Webster mice.
Results:  We identified 15,347 HDAC1 binding sites in  mouse E11.5 limb buds

overall_design: ChIP-sequencing E11.5 forelimb and hindlimbs  from wildtype Swiss Webster mice. Four biological replicates and three input controls were submitted.


title: αKlotho and sTGFBR2 treatment promotes endogenous cartilage regeneration and prevents osteoarthritis progression

status: Public on Jan 22 2020

city: San Diego

first: April

middle: Elizabeth

last: Williams

state: California

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute for Biological Studies

summary: Osteoarthritis (OA) is the most prevalent musculoskeletal disorder and the most common form of arthritis among the elderly. Despite recent basic biology and clinical progress, the understanding and treatment of this  and other cartilage-associated diseases are still a challenge. By using an in vivo rat OA model  and in vitro human studies we report the improved homeostasis and repairing capacity of two soluble molecules, αKlotho and sTGFBR2, that in combination are able to prevent the progression of OA and promote cartilage regeneration.

overall_design: 2 WT, 3 disease, 3 disease untreated, and 2 disease treated samples were profiled by mRNA-seq to examine how global gene expression changes associated with disease progression and treatment effect.

first: Paloma

last: Redondo

first: Isabel

last: Guillen

first: Maxim

middle: N

last: Shokhirev

first: Ling

last: Huang

first: Juan

last: Izpisua-Belmonte


title: Transcriptome-wide Analysis of Intracranial Artery of Moyamoya Disease Showing Up-regulation of Inflammatory Response with Increased Expression of TLR2 and SYK, and Down-Regulation of Collagen Formation with Decreased Expression of COL15A1 [STA]

status: Public on Feb 29 2020

city: Showa-ku, Nagoya

first: Kinya

last: Yokoyama

state: Aichi

address: Tsurumai 65

department: Neurosurgery

institute: Nagoya University Hospital

summary: During superficial temporal artery (STA)-middle cerebral artery (MCA) anastomoses, micro-samples of both MCA and STA from the patients with MMD (n=4) and internal carotid artery aneurysm (IA) (n=4) were collected. Using microarray technique, transcriptome-wide analyses were performed. Comparing MCA gene expression profile between MMD and IA, we detected 39 up-regulated and 12 down-regulated genes in MMD, showing over 15-fold change with significant difference (P<0.05). Gene set enrichment analysis revealed positive correlation of inflammatory response and negative correlation of collagen formation with MCA of MMD. In addition, by quantitative polymerase chain reaction, significant increased gene expression of TLR2 (P=0.009) and SYK (P=0.04), and decreased gene expression of COL15A1 (P=0.0001) in MCA of MMD were confirmed. While in STA transcriptome analysis, no gene showed significant difference.

first: Humiaki

last: Kanamori


title: Viral integration transforms chromatin to drive oncogenesis

city: Toronto

first: Michael

middle: M.

last: Hoffman

state: ON

address: 101 College St PMCRT 11-311

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

summary: We report that the integration of the human papillomavirus into the host genome increases chromatin accessibility, transcription, and CTCF binding within 100 kbp of the integration site which promotes oncogenesis in some patients.

overall_design: We used 5 patient-derived HPV16-positive cell lines. Three biological replicates for CTCF ChIP-seq, 1 lane of sequencing for ATAC-seq of each sample, and 4 lanes of sequencing for RNA-seq of each sample.

first: Mehran

last: Karimzadeh

first: Christopher

last: Arlidge

first: Ariana

last: Rostami

first: Mathieu

last: Lupien

first: Scott

middle: V

last: Bratman


title: GLI3 binding regions in mouse embryonic forelimb buds

first: Steven

last: Vokes

summary:
Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers and the majority of Hh targets in the developing limb bud are regulated solely by the activity of GLI-repressor.  Currently we do not have a comprehensive understanding of how GLI bound enhancers respond Hedgehog signaling.  The goal of this study is to identify how GLI bound enhancers are regulated by Hedgehog signaling and specifically by GLI-repressor.
Methods:  ChIP-seq was performed in Embryonic day 10.5 mouse limb buds from mice with endogenously FLAG tagged Gli3.
Results:  We identified 7282 GLI3 binding regions in the E10.5 limb bud.

overall_design: ChIP-sequencing E10.5 forelimb buds from mice with enogenously FLAG-tagged Gli3. Two biological replicates and one input control were submitted.

first: Rachel


title: GLI transcriptional repression regulates enhancer activity and chromatin accessibility for Hedgehog target genes

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Genome-wide maps of chromatin accessibility in response to Hh signaling in mouse embryonic forelimb buds

city: Seattle

last: Lex

state: WA

address: 1100 Fairview Ave N

institute: Fred Hutchinson Cancer Center

summary:
Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers and the majority of Hh targets in the developing limb bud are regulated solely by the activity of GLI-repressor. Currently, we do not have a comprehensive understanding of how GLI bound enhancers respond to Hedgehog signaling. The goal of this study is to identify how GLI bound enhancers are regulated by Hedgehog signaling and specifically by GLI-repressor.
Methods: ATAC-seq was done in embryonic day 10.5 posterior limb buds from control and Shh-null embryos to identify GLI enhancers that change chromatin accessibility in response to Hedgehog signaling.
Results: We found that Hedgehog signaling regulates the chromatin accessibility of a subset of GLI binding regions. This subset of GLI bound enhancers has reduced accessibility in the absence of Hedgehog signaling and largely overlaps with GLI enhancers that also have reduced H3K27ac in the absence of Hedgehog signaling.

overall_design: ATAC-sequencing in Shh-/- and WT control posterior E10.5 forelimb buds. Two biological replicates were done for each ATAC experiment.

first: Weiqiang

last: Zhou

first: Hongkai


title: Expression profiling of mitochondria-associated microRNAs during osteogenic differentiation of human MSCs

status: Public on Jan 01 2020

city: St. Louis

first: Jinsheng

last: Yu

state: MO

address: 660 S. Euclid Ave.

department: Genetics

institute: Washington University School of Medicine

summary: The goal of this study was to determine expression profiles of mitochondria-associated microRNAs (mitomiR) in human bone marrow-derived mesenchymal stem/stromal cells (MSCs) as well as in MSCs during osteogenic differentiation. MicroRNAs are epigenetic regulators that commonly function by targeting specific mRNAs resulting in suppression of protein expression. In addition to their location in the cytosol, microRNAs have also been found in other sub-cellular compartments including the mitochondria. While some studies suggest that mitomiRs may affect mitochondrial function, research on mitomiRs is still in its infancy. To date, there is no information on mitomiR expression in MSCs or osteoblasts. A standard in vitro osteogenesis assay system was used to differentiate MSCs toward the osteoblast lineage. Purified mitochondrial extracts were obtained from MSCs or from MSCs at specific time points of osteogenic induction. RNA was isolated from mitochondrial extracts and then biotin-labeled in preparation for microRNA array (Affymetrix miRNA array 4.0). Array data was analyzed to generate information on most abundantly-expressed mitomiRs in non-induced MSCs as well as in MSCs at set time points (day 3, 7, or 14) of osteogenic induction. Information on significantly differentially-expressed mitomiRs during osteogenesis (comparing day 0 with either day 3, 7 or 14) was also obtained.

overall_design: In this study, 3 independent MSC cell lines were used. Each MSC cell line was differentiated toward the osteoblast lineage and mitochondrial RNA was isolated from MSCs at day 3, 7, or 14 of differentiation. As a control, mitochondrial RNA from non-induced MSCs (day 0) was also collected, Therefore, 12 samples in total were analyzed.

first: Audrey

last: McAlinden

first: Hongjun

last: Zheng


title: Genome wide maps of chromatin modification in response to Hh signaling in mouse embryonic forelimb buds

summary:
Purpose: The response to Hedgehog signaling in the limb is driven by GLI bound enhancers and the majority of Hh targets in the developing limb bud are regulated solely by the activity of GLI-repressor.  Currently we do not have a comprehensive understanding of how GLI bound enhancers respond Hedgehog signaling.  The goal of this study is to identify how GLI bound enhancers are regulated by Hedgehog signaling and specifically by GLI-repressor.
Methods:  Histone modification ChIP-sequencing was done in Embryonic day 10.5 limb buds from control and Shh null embryos to identify histone modifications that change in response to Hedgehog signaling.  We also did a comparative analysis of histone modificaiton response to Hedgehog stimulation in NIH3T3 cells to assess tissue specificity of GLI bound enhancer response to Hedgehog signaling.
Results:  We found that Hedgehog signaling specifically regulates the acetylation status of Histone 3 lysine 27 at a subset of GLI binding regions.  These regions correlate with both known Hedgehog target genes in the limb bud and with characterized enhancers in the limb.  This set of Hedgehog responsive GLI bound enhancers demonstrate enhanced tissue specificity.

overall_design: ChIP-sequencing of 3 different histone modifications (H3K27ac, H3K4me2, and H3K27me3) in Shh-/- and WT control E10.5 forelimb buds.  Additional ChIP-seq of H3K27ac in NIH3T3 cells with and without Hedgehog pathway stimulation.  Two biological replicates were done for each ChIP experiment.  Enrichment values were determined using input control samples from one or both replicates for each ChIP experiment.

first: Kristin

last: Falkenstein

first: Zhicheng

last: Ji


title: The role of NUP93 in breast cancer

status: Public on Jan 18 2020

first: April

last: Williams

summary: NUP93 expression inversely correlates with the survival of triple negative breast cancer patients. However, the role of NUP93 in tumor propagation has not yet been fully clarified. In this project we combined high-resolution imaging, migration assays and genetic analyses to demonstrate that NUP93 modulates actin cytoskeleton remodeling, epithelial-to-mesenchymal transition and invasion of triple negative breast cancer cells through direct interaction with the genome.

overall_design: Human triple negative breast cancer cells MDA231 were silenced using validated siRNA for NUP93 (Ibarra et al., Genes and Development, 2016). Global gene expression changes were measured by mRNA-Seq, while epigenetic profiling was performed using the Cut&Run technique.

first: Simone

last: Bersini

first: Nikki

last: Lytle

first: Roberta

last: Schulte

first: Geoff

last: Wahl

first: Martin

middle: W

last: Hetzer

first: Maxim

last: Shokhirev

first: Ling

last: Huang


title: An RNA Polymerase Ribozyme that Synthesizes its Own Ancestor

status: Public on Dec 17 2019

summary: As part of an examination of a newly-evolved RNA polymerase ribozyme, 38-6, products of primer extension experiments using an RNA template encoding the hammerhead RNA endonuclease ribozyme, using 38-6 and its less active ancestor 24-3. These products were analyzed by next-generation sequencing to determine the rates of substitution, deletion, and insertion mutations for both polymerases.

overall_design: RNA primer extension products using the 38-6 and 24-3 polymerase ribozymes were converted into cDNA libraries, sequenced on an Illumina Miniseq, and analyzed to determine the rates of mutation across the length of the sequence synthesized by the polymerases.

first: Katrina

middle: F

last: Tjhung

first: David

middle: P

last: Horning

first: Gerald

last: Joyce


title: RNA-Seq analysis of gene expression in 25 cases of canine lymphoma undergoing CHOP chemotherapy

first: Miles

last: Mee

address: 661 University Ave Suite 510

institute: Ontario Institute for Cancer Research

summary: Canine lymphoma is the most common hematological cancer in dogs and shares many molecular and clinical characteristics with human Non-Hodgkin lymphoma (NHL). The standard treatment for canine lymphoma is the “CHOP” sequential multiagent chemotherapy protocol consisting of Cyclophosphamide, Doxorubicin (Hydroxydaunorubicin), Vincristine (Oncovin™), and Prednisone. Approximately 70 - 85% of patients treated with CHOP achieve clinical remission. However, duration of remission varies and the majority of dogs eventually relapse. To identify possible biomarkers for patients failing to achieve remission in response to CHOP, we performed RNA-Seq analysis on 25 cases of canine lymphoma taken at the start of their CHOP therapy regime, and determined patient progression free survival (PFS).

overall_design: Tumor samples from 25 dogs with lymphoma. Samples were collected prior to treatment with CHOP. There were no replicate or control samples.

first: Brenda

last: Coomber


title: Tumor exosomal miR-3473b promotes the metastatic colonization of lung cancer cells by activating the nuclear factor-κB signaling pathway of local fibroblasts

status: Public on Dec 25 2019

city: Zhengzhou

first: Can

middle: can

last: Du

address: Daxue Road

institute: The First Affiliated Hospital of Zhengzhou University

summary: Tumor-derived exosomes are key factors that mediate the communication between tumor cells and stromal cells. In this study, we demonstrated that there was an uptake of cancer-derived exosomes by lung fibroblasts both in vivo and in vitro, leading to upregulation of inflammatory genes in fibroblasts, such as Il-6, Ccl1, Ccl2, Ccl5 and Cxcl2. Further analysis indicated that exosomal miR-3473b-mediated suppression of Nfkbid caused the inflammatory cytokine secertion in fibroblasts and further resulted in B cells accumulation in lungs. Inhibition of miR-3473b blocked inflammatory cytokine genes such as IL-6 expression in lung fibroblasts and B cell accumulation in lung. We further demonstrated that the lung metastasis areas and numbers were significantly decreased by using B cell neutralizing antibodies, suggesting B cell may play an important role in establishment of tumor promoting microenvironment for metastatic cancers cells colonization in the lung. In the present study, we showed that intercellular crosstalk occurred between cancer cells and fibroblasts via cancer-derived exosomes. Moreover, inhibition of the NF-kB activation in fibroblasts and B cells accumulation may become a potential target for the prevention and treatment of lung cancer.

overall_design: LLC cell supernatant was collected for exosomes isolation and microarray analysis were performed to generate miRNA profiles of LLC exosomes. Two independent experiments were performed.

first: Cancan

first: Ming

last: Wang

first: Zhihai

last: Qin


title: Fine-mapping within eQTL Credible Intervals by E-CROPseq

status: Public on Nov 28 2019

city: Atlanta

first: Greg

last: Gibson

state: Georgia

address: 950 Atlantic Drive

institute: Georgia Institute of Technology

summary: Precise identification of causal variants within credible intervals of eQTL associations is needed to identify regulatory GWAS variants. We show that CROPseq, namely multiplex CRISPR-Cas9 genome editing combined with single cell RNAseq, is a viable strategy for fine mapping regulatory SNPs.  Mutations were induced nearby 67 SNPs in three genes, two of which, rs2251039 and rs17523802, significantly altered CISD1 and PARK7 expression, respectively, and overlap with chromatin accessibility peaks.

overall_design: Single cell RNA-seq profiles of HL60/S4 infected by 67 gRNA CROP-seq lenti-viral libraries of two biological replicates. Each gRNA targets to candidate eSNPs for comprehensive eQTL screening.

first: Yidan

last: Pan

first: Ruoyu

last: Tian

first: Ciaran

last: Lee

first: Gang

last: Bao


title: Pathophysiological role of MIG-6 and ERBB2 in progesterone resistance and endometriosis-related infertility.

status: Public on Nov 30 2019

city: Columbia

first: Jae-Wook

last: Jeong

address: 1030 Hitt Street

department: Obstetrics, Gynecology and Women's Health

institute: University of Missouri

summary:
MIG-6 loss caused progesterone resistance through ERBB2 overexpression in non-receptive endometrium of endometriosis-related infertility.
We used microarrays to understand the molecular mechanisms of progesterone resistance in infertility

overall_design: Uteri of control (Mig-6 f/f and Mig-6 f/f Erbb2 f/f), Mig-6 d/d, Mig-6 d/d Erbb2 d/d mice were selected at GD3.5 for RNA extraction and hybridization on Affymetrix microarrays.

first: Tae

last: Kim


title: EXOSC10 sculpts the transcriptome during oocyte growth-to-maturation transition

first: Di

last: Wu

summary: Growing mammalian oocytes accumulate substantial amounts of RNA, most of which are degraded during the subsequent maturation stage. The growth-to-maturation transition begins with germinal vesicle breakdown (GVBD, envisioned as nuclear envelope breakdown) and is critical for oocyte quality. However, the concomitant changes in the transcriptome during GVBD as well as the underlying machinery remained unclear. Here, we report that an RNA exosome-associated RNase, EXOSC10, sculpts the transcriptome at multiple level to facilitate the oocyte growth-to-maturation transition. We establish an oocyte-specific knockout of Exosc10 in mice using CRISPR/Cas9 and find female subfertility due to failed GVBD. By performing single oocyte RNA-seq in different ways, we document dysregulated transcriptomes, unsuccessfully processed rRNAs in mutant oocytes, and many up-regulated RNAs that encode proteins important for endomembrane trafficking, meiotic cell cycle and RNA metabolism. EXOSC10-depleted oocytes have impaired endomembrane components including endosome, lysosome, ER and Golgi. In addition, CDK1 fails to be activated possibly due to persistent WEE1 activity, which blocked lamina phosphorylation and disassembly in mutant oocytes. Collectively, we propose that EXOSC10 promotes the growth-to-maturation transition in mouse oocytes by degrading mRNAs that encode growth-phase factors and sculpting the transcriptome to support the maturation phase of oogenesis.

overall_design: There are 64 single oocyte polyA-RNA libraries generated with poly(A)-based enrichment and reverse transcription, including 22 at GV stage, 19 at GV+3hr stage, and 23 at MII stage. In addition, there are 22 single single oocyte RiboMinus libraries generated independent of poly(A), including 15 at GV stage and 7 at MII stage. Morever, there are 22 single oocyte rRNA libraries generated by total RNA extraction without ribosome depletion. Each stage contains control oocytes and Exosc10 oocyte-specific knockout oocytes (cKO). The information of the stage, genotype, female ID and oocyte ID is in the name of each file: stage_genotype_femaleID_oocyteID. In each oocyte, equal amounts of ERCC spike-in mix was added.

first: Jurrien

last: Dean


title: The biochemical basis of microRNA targeting efficacy [1]

status: Public on Nov 27 2019

city: Cambridge

first: Sean

middle: E.

last: McGeary

address: 455 Main St.

department: Biology

institute: Whitehead Institute / MIT

type: Non-coding RNA profiling by high throughput sequencing

summary: MicroRNAs (miRNAs) act within Argonaute proteins to guide repression of mRNA targets. Although various approaches have provided insight into target recognition, the sparsity of miRNA–target affinity measurements has limited understanding and prediction of targeting efficacy. Here, we adapted RNA bind-n-seq to enable measurement of relative binding affinities between Argonaute–miRNA complexes and all ≤12-nucleotide sequences. This approach revealed noncanonical target sites unique to each miRNA, miRNA-specific differences in canonical target-site affinities, and a 100-fold impact of dinucleotides flanking each site. These data enabled construction of a biochemical model of miRNA-mediated repression, which was extended to all miRNA sequences using a convolutional neural network. This model substantially improved prediction of cellular repression, thereby providing a biochemical basis for quantitatively integrating miRNAs into gene-regulatory networks.

overall_design: small RNA-seq of AGO2–miRNA overexpressedin HEK293T, loaded with either a synthetic miR-1 or miR-155 duplex in lysate, and purified as described according to the methods. This study consists of 2 libraries.

first: Kathy

middle: S

last: Lin

first: David

last: Bartel


title: The biochemical basis of microRNA targeting efficacy [3]

overall_design: mRNA sequencing of HeLa cells serially transfected with synthetic miRNA duplexes and a massively parallel reporter library. This study consists of 12 libraries with 6 different miRNA transfected in replicate.


title: The biochemical basis of microRNA targeting efficacy [5]

overall_design: mRNA expression profiling by RNA-seq of HEK293FT cells transfected with synthetic miRNA duplexes. This study consists of 24 libraries, with 12 distinct miRNA transfections performed in replicate over 3 batches.


title: Whole transcriptome analysis of brain hippocampal tissue from SAMP8 mice and rat primary neurons treated with the Alzheimer's disease drug candidates CMS121 and J147

status: Public on Nov 26 2019

first: April

last: Williams

summary: Alzheimer's disease (AD) drug discovery has rarely been addressed in the context of aging even though sporadic AD accounts for 99% of the cases. Phenotypic screens based upon old age-associated brain toxicities were used to develop the potent AD drug candidates CMS121 and J147. The aim of this project was to investigate whether these two different AD drug candidates prevented the progression of dementia in SAMP8 mice when administered at advanced stages of disease, and whether they shared common modes of action. These transcriptomic data are part of an integrative multi-omics approach that also investigated protein expression, metabolite levels as well as cognition. In addition, in order to further investigate the effect of the drugs in in vitro neuronal cultures, rat primary neurons were treated with the compounds and the transcriptome sequenced.

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: Six nine-month old female SAMP8 mice per group were fed with vehicle diet and with CMS121 (400ppm) and J147 (200ppm) diets. Diet treatment lasted for four months until mice reached thirteen months of age. Mice were then sacrificed and RNA extracted from the brain hippocampus. Five eight-month old male SAMP8 mice were used as the baseline control group. For the in vitro experiments, Primary cortical neurons were prepared from day seventeen rat embryos and allowed to differentiate in culture. At seven days in vitro, neurons were treated with 1  M of each compound for 24h, and RNA purified

first: Antonio

last: Currais

first: Ling

last: Huang


title: The biochemical basis of microRNA targeting efficacy [4]

overall_design: mRNA sequencing of HeLa cells trasnfected with synthetic miRNA duplexes. This study consists of 34 libraries with 17 distinct miRNA transfections performed in replicate over 5 batches.


title: Transcriptome profiling of the microdissected breast tissue compartments

status: Public on Dec 12 2019

city: Indianapolis

first: Natascia

last: Marino

state: IN

address: 980 W. Walnut St

department: Medicine

institute: Indiana University

summary: In addition to the accumulation of pro-oncogenic mutations in the epithelial cells, the tumorigenic process involves the dysregulation of the interactions between the epithelial cells and their microenvironment, as well as alterations within the microenvironment itself. Therefore, investigating the causes of the loss of the normal tissue homeostasis in the earliest stages of breast cancer carcinogenesis is the necessary underpinning of an effective breast cancer prevention strategy.  In order to comprehensively capture the transcriptomic alterations occurring in the breast in a precancerous stage, we examined a unique resource present in our institute including breast tissue biopsies donated by women two to six years prior to the clinical manifestation of breast cancer (labeled “susceptible normal tissue”) as well as specimens from healthy women. Donors in the two cohorts were at a premenopausal status and were matched according to age, racial background and menstrual phase. Both the susceptible and healthy breast tissues appear histologically normal. Upon microdissection of the three breast tissue compartments including epithelial, stromal and adipose tissue, transcriptomic analysis was performed. Upregulation of genes involved in lipid metabolism and fatty acid transport was observed also in the susceptible stroma and adipose tissue compartments, respectively.

overall_design: The study cohorts included 16 healthy women and 7 additional individuals, who were clinically healthy at the time of tissue donation but later had a diagnosis of breast cancer (labeled “Susceptible”). The subjects in these two cohorts were matched to the cases according to age, racial background, body mass index and follow-up time. To comprehensively capture the transcriptomic alterations occurring in the breast cancerization field, we used laser microdissection microscopy to isolate the epithelial (or terminal duct luminal unit)-, stromal- and adipose tissue compartments separately from each of 23 fresh-frozen breast biopsies. However, while all the 23 breast tissue cores included enough epithelial and stromal area, 7 samples lacked of sufficient adipose tissue for the following step.  RNA was isolated from the captured cells and interrogated with next generation RNA sequencing. Moreover, breast epithelial compartment from paired susceptible, adjacent normal and contralateral normal breasts donated by two women was also analyzed through RNA-sequencing.

first: Rana

last: German

first: Xi

last: Rao

first: Ed

last: Simpson

first: Shang

last: Liu

first: Jun

last: Wan

first: Yunlong

first: Anna

last: Storniolo


city: bronx

first: Nicholas

last: Baker

state: NY

address: 1300 Morris Park Avenue

department: genetics

institute: albert einstein college of medicine

summary:
mRNA Seq analysis of Drosophila wing imaginal discs from Rp mutants and controls in the presence and absence of RpS12 mutations RpS12 is a regulatory gene required for Xrp1-mediated control of translation and growth in mutants for other ribosomal protein genes
Loss of function mutations in Ribosomal Protein (Rp) genes are generally lethal, but heterozygous mutant phenotypes increasingly reveal previously cryptic functions of Rps in growth control and tumor suppression.  Here we report that RpS12 in particular is a regulatory gene that controls its own expression levels and also signals to control hundreds of gene expression changes in mutants for haploinsufficient Rp genes, acting through the bZip protein Xrp1.  Through Xrp1, RpS12 reduces the bulk translation rate of Rp mutant cells and increases the developmental rate of Rp mutant genotypes.  RpS12 also has an Xrp1-independent, positive contribution to normal development, so that the effects of RpS12 mutations in different genetic backgrounds reflects the sum of the Xrp1-dependent and Xrp1-independent effects.  Our findings outline a regulatory response to mutations affecting essential Rp genes that controls overall translation and growth.

overall_design:
9 samples comprising Xrp1+/- in triplicate (m2-73_w_1-3), rpS12G97D/G97D in triplicate (ny1_ny1_1-3), and RpS3+/- rpS12 G97/G97D in triplicate (ny1_ny1M95_1-3)
The raw data for some of these samples (frt_w, M95_w, M67_w, and m2-73_M95) was deposited previously (GSE112864)

first: Marianthi

last: Kiparaki

first: Jorge

last: Blanco

first: Virginia

last: Folgado

first: Zhejun

last: Ji

first: Amit

last: Kumar

first: Gerard

last: Rimesso


title: The biochemical basis of microRNA targeting efficacy

first: Sean

last: McGeary

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Refer to individual Series


title: Solo: doublet identification via semi-supervised deep learning

status: Public on Nov 13 2019

city: South San Francisco

last: Bernstein

address: 1130 Veterans Blvd

institute: Calico labs

summary: We developed a semi-supervised deep learning framework for the identification of doublets in scRNA-seq analysis called Solo. To validate our method, we used MULTI-seq, cholesterol modified oligos (CMOs), to experimentally identify doublets in a solid tissue with diverse cell types, mouse kidney, and showed Solo recapitulated experimentally identified doublets.

overall_design:
To better understand the ability to computationally identify doublets we ran two wells on the 10X v3 platform to generate single cell gene expression profiles and cholesterol-modified oligo count profiles for two mouse kidneys from one mouse.
CMO_groups.csv: How CMO barcodes were pooled together. To ensure redundancy during MULTI-seq we used three CMOs per sample for identification. We used the combined counts to demultiplex cells and identify doublets.
feature_barcodes.csv: CMO barcodes used and their names.

first: Nicole

last: Fong

first: Margaret

last: Roy

first: Irene

last: Lam

first: David

last: Hendrickson

last: Kelley


title: Single myofiber RNA-seq of young and old mice

status: Public on Nov 20 2019

city: Montreal

first: Vahab

last: Soleimani

state: QC

address: 3640 rue University

department: Human Genetics

institute: McGill University

summary: We developed a method of combining single myofiber isolation with SMART-seq to analyze the whole transcriptome of single myofibers.  Here we apply this technique to analyze the variation in the transcriptome of young (4 weeks) and old (19 months) mice.  We see the deregulation of a variety of genes that have been reported, or may cause, the deterioration of the muscle in aging.  This confirms the applicability of our novel technique for use in analyzing the transcriptome of individual myofibers under various different conditions.

overall_design: In this study we use single myofiber RNA sequencing to analyze the whole transcriptome of single myofibers extracted from young and old mice


title: Transcriptional response of Mycobacterium tuberculosis in mouse peritoneal neutrophils

status: Public on Nov 09 2019

city: Moscow

first: Artem

last: Grigorov

address: Ulitsa Miklukho-Maklaya, 16/10

department: Genomics and Postgenomic Technologies

country: Russia

institute: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry

summary: This study was conducted to descripe transcriptomic changes of M.tuberculosis during neutrophil infection

overall_design: Six samples, representing two groups: in vivo neutrophil infection (mice of the I/St inbred strain) by Mycobacterium tuberculosis H37Rv and control (Mycobacterium tuberculosis H37Rv cells were incubated in Dubos broth). In each group, three independent biological samples were sequenced.

first: Taiana

last: Kondratieva

first: Konstantin

last: Majorov

first: Tatyana

last: Azhikina

first: Alexander

last: Apt


title: RNA sequencing to identify NPR-8-regulated genes in C. elegans defense against pathogen infection

status: Public on Oct 15 2019

city: Spokane

first: Yiyong

last: Liu

address: 412 E Spokane Falls Blvd

institute: Washington State University

summary: To gain molecular insights on how NPR-8 regulates C. elegans defense against pathogen infection, we used RNA sequencing to profile gene expression in npr-8(ok1439) animals relative to wild-type animals with or without P. aeruginosa infection.  We found that NPR-8 suppresses the expression of genes related to cuticle structure activity including collagen genes, and lack of NPR-8-mediated suppression in npr-8(ok1439) animals contributes to their improved survival against P. aeruginosa infection.

overall_design: Five replicates of four groups of RNA samples (npr-8(ok1439) and wild-type animals with or without exposure to P. aeruginosa for 24 hrs) were sequenced.  Gene expression was compared between groups to identify NPR-8-regulated genes under uninfection or infection conditions.

first: Durai

last: Sellegounder

first: Jingru

last: Sun


title: Characterization of QTL and eQTL linked with early Fusarium graminearum infection and deoxynivalenol levels in a Wuhan -1 x Nyubai doubled-haploid wheat population at two days post inoculation

city: Ottawa

first: Ziying

address: 1200 Montreal Road

institute: National Research Council, Canada

summary: In this study, early infection by F. graminearum was characterized in a doubled-haploid population derived from the cross of the moderately FHB resistant wheat genotypes Wuhan 1 and Nyubai. Three significant QTL were identified: 1AL was associated with reduced deoxynivalenol levels, and 4BS and 5A were associated with reduced F. graminearum levels at 2 days after infection. The early resistance alleles for 1AL and 4BS were inherited from Wuhan 1, and those for the 5A QTL were inherited from Nyubai. The LOD support interval for the 5A QTL was in the same region as FHB5, a QTL previously associated with field resistance to FHB. Cis and trans eQTL were identified using RNA sequencing data from infected head samples. In total, 240 cis eQTL and 17,401 trans eQTL were identified within LOD support intervals for the three QTL. Hotspots for trans eQTL were identified within LOD support intervals for the 1AL and 4BS QTL. Differential gene expression analysis revealed twenty genes with cis eQTL within one of the 3 QTL support intervals, that had higher expression associated with FHB early resistance, and 17 genes with higher expression associated with FHB early susceptibility. Among those, genes with a cis eQTL peak coinciding with QTL peaks were enriched in functions related to signaling and protein-protein interaction, including NBS-LRR, BAH, LRR/F-box and TPR domain-containing proteins. Several genes located within one of the three QTL support intervals also had a trans eQTL with a peak at the same position as QTL peaks on other chromosomes, suggesting interactions between the QTL.

overall_design: In this study, we have analyzed RNA-seq and genotype (SRR, SNP) data in two parent genotypes with moderate resistance to F. graminearum and 81 DH lines obtained by crossing the two parents at 2 dpi

first: François

last: Fauteux

first: Yunli

last: Wang

first: Hélène

last: Rocheleau

first: Youlian

last: Pan

first: George

last: Fedak

first: Curt

last: McCartney

first: Thérèse

last: Ouellet


title: The biochemical basis of microRNA targeting efficacy [2]

first: Sean

last: McGeary

overall_design: small RNA sequencing of AGO-RBNS binding reactions and input libraries. This study comprises 81 samples spanning six miRNAs and a variable number of purifications of each.

first: Kathy

last: Lin

first: David

last: Bartel


title: Transcription-induced formation of extrachromosomal DNA during yeast ageing

status: Public on Oct 18 2019

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

country: United Kingdom

institute: Altos Labs

summary: Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation, and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper resistance gene CUP1. Transcription of CUP1 causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2 / Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant we find that CUP1 eccDNA does not replicate efficiently, and high copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

overall_design: Aged cell samples analysed in pairs of -/+ Cu, for both wt and various mutants. 3 replicates of the 3xCUP1 experiment are included

first: Ryan

last: Hull

first: Michelle

last: King

first: Jonathan

last: Houseley


title: ARID1A and PI3-Kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion [Mouse_RNA-seq]

status: Public on Jul 01 2019

city: Grand Rapids

first: Ronald

middle: L.

last: Chandler

state: MI

department: Obstetrics, Gynecology and Reproductive Biology

institute: Michigan State University

summary: ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. Here we show that monoallelic loss of ARID1A in the mouse endometrial epithelium is sufficient for vaginal bleeding when combined with PI3K activation. Sorted mutant epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT). We further show that ARID1A is bound to promoters with open chromatin, but ARID1A loss leads to increased promoter chromatin accessibility and the expression of EMT genes. PI3K activation partially rescues the mesenchymal phenotypes driven by ARID1A loss through antagonism of ARID1A target gene expression, resulting in partial EMT and invasion. We propose that ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates, and that coexistent ARID1A and PI3K mutations promote epithelial transdifferentiation and collective invasion. Broadly, our findings support a role for collective epithelial invasion in the spread of abnormal endometrial tissue.

overall_design: EPCAM+ endometrial epithelial cells from hyperplastic LtfCre0/+; (Gt)Rosa26Pik3ca*H1047R; Arid1afl/fl and healthy control wild-type CD-1 mice were purified and assayed via RNA-seq. Mature female mice were euthanized prior to vaginal bleeding.

first: Mike

middle: R

last: Wilson

first: Jake

middle: J

last: Reske

middle: L


title: H3K36me2 recruits DNMT3A and shapes intergenic DNA methylation landscapes

status: Public on Aug 30 2019

city: Santiago de Compostela

first: Carmen

last: Rivas

address: Avda Barcelona

department: CIMUS

country: Spain

institute: Universidad de Santiago de Compostela

type: Methylation profiling by high throughput sequencing

summary: Enzymes catalyzing CpG methylation in DNA, including DNMT1 and DNMT3A/B, are indispensable for mammalian tissue development and homeostasis. They are also implicated in human developmental disorders and cancers, supporting a critical role of DNA methylation during cell fate specification and maintenance. Recent studies suggest that histone post-translational modifications (PTMs) are involved in specifying patterns of DNMT localization and DNA methylation at promoters and actively transcribed gene bodies. However, mechanisms governing the establishment and maintenance of intergenic DNA methylation remain poorly understood. Germline mutations in DNMT3A lead to a childhood overgrowth syndrome that is phenotypically overlapping with Sotos syndrome caused by haploinsufficiency of NSD1, a histone methyltransferase catalyzing di-methylation on H3K36 (H3K36me2), pointing to a potential mechanistic link between the two disorders. Here we report that NSD1-mediated H3K36me2 is required for recruitment of DNMT3A and maintenance of DNA methylation at intergenic regions. Genome-wide analysis shows binding and activity of DNMT3A are co-localized with H3K36me2 at non-coding regions of euchromatin. Genetic ablation of NSD1 and its paralogue NSD2 in mouse and human cells redistributes DNMT3A to H3K36me3-marked gene bodies and reduces intergenic DNA methylation. NSD1 mutant tumors and Sotos patient samples are also associated with intergenic DNA hypomethylation. Consistently, PWWP-domain of DNMT3A shows dual recognition of H3K36me2/3 in vitro with a higher binding affinity towards H3K36me2, which is abrogated by overgrowth syndrome-derived missense mutations. Taken together, our study uncovers a trans-chromatin regulatory pathway that, when perturbed, promotes neoplastic and developmental overgrowth.

sample_organism:
Drosophila melanogaster
Homo sapiens
Mus musculus

overall_design: WGBS, RNA-seq, and ChIP-seq for DNMT proteins and histone H3 post-translational modifications in C3H10T1/2 cells. WGBS and ChIP-seq for H3K36me2 in human head and neck squamous cell carcinoma cell lines and mouse embryonic stem cells.

first: Daniel

last: Weinberg

first: Simon

last: Papillon-Cavanagh

first: Haifen

last: Chen

first: Xiao

first: Kartik

last: Rajagopalan

first: Cynthia

last: Horth

first: Yuan

last: Yue

first: Hamid

last: Nikbakht

first: Dylan

middle: M

last: Marchione

first: Ashot

last: Harutyunyan

first: Nada

last: Jabado

first: Benjamin

middle: A

last: Garcia

first: Haitao

last: Li

first: C

middle: D

last: Allis

first: Jacek

last: Majewski

first: Chao

last: Lu


title: ARID1A and PI3-Kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion [Mouse_ATAC-seq]

first: Ronald

last: Chandler

overall_design: EPCAM+ endometrial epithelial cells from hyperplastic LtfCre0/+; (Gt)Rosa26Pik3ca*H1047R; Arid1afl/fl, LtfCre0/+; (Gt)Rosa26Pik3ca*H1047R; Arid1afl/+, LtfCre0/+; (Gt)Rosa26Pik3ca*H1047R; Arid1afl/V1068G, and healthy control wild-type CD-1 mice were purified and assayed for genome-wide chromatin accessibility via ATAC. Mature female mice were euthanized prior to vaginal bleeding.

first: Mike

last: Wilson

first: Jake

last: Reske


title: ARID1A and PI3-Kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion [12Z_RNA-seq]

overall_design: 12Z endometriotic epithelial cells transfected with either siARID1A or non-targeting siRNA control were assayed 48 hours post-transfection via RNA-seq.


title: ARID1A and PI3-Kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion [12Z_ATAC-seq]

overall_design: 12Z endometriotic epithelial cells transfected with either siARID1A or non-targeting siRNA control were assayed 48 hours post-transfection for genome-wide chromatin accessibility via ATAC.


title: ARID1A and PI3-Kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion [12Z_ChIP-seq]

overall_design: Wild-type 12Z cells were assayed by chromatin immunoprecipitation (ChIP) for ARID1A followed by sequencing.


title: Gene expression changes by exogenous expression of epithelial-to-mesenchymal (EMT)-inducing transcription factors (Snail and Zeb1) and mesenchymal-to-epithelial transition (MET)-inducing transcription factor (Ovol2) in MCF10A cells

city: Yokohama

first: Kazuhide

last: Watanabe

state: Kanagawa

address: 1-7-22 Suehiro-cho Tsurumi-ku

department: Center for Integrative Medical Sciences

institute: Riken

summary: Multiple EMT-promoting/inhibiting transcription factors have been identified including Snail, Zeb1 and Ovol2. To understand differential roles of these factors, we performed gene expression profiling by RNA-seq upon Snail-/Zeb1-induced EMT or Ovol2-induced MET in human mammary epithelial MCF10A cells.

overall_design: Examination of 4 samples.

first: Xing

last: Dai


title: Benchmarking NF-kB inhibitors

status: Public on Aug 29 2019

city: Beaverton

first: Charles

last: Keller

state: OR

address: 12655 SW Beaverdam Rd

institute: Children's Cancer Therapy Development Institute

summary: We report the presence of the C11orf95-RELA fusion in cells isolated from a patient presenting with anaplastic ependymoma

overall_design: Metastatic tumor was resected from right temporal lobe and a primary culture was made. Cells were cultured in Optimem with 15%FBS and 1% PenStrep. Cells were expanded to passage 8 for RNA sequencing

first: Megan

last: Cleary


title: Radiomic and gEnomic approaches for the enhanced DIagnosis of REnal Cancer (REDIRECt): A translational pilot study

status: Public on Jun 28 2019

city: Milan

first: Francesco

last: Cianflone

address: via Olgettina 60

department: Department of Urology

institute: IRCCS Ospedale San Raffaele

summary:
Introduction: The aim of this pilot study is to establish a radiogenomic characterisation of a clear-cell renal cell carcinoma (ccRCC) subpopulation, focusing on the transcriptomic underpinnings of radiomic features.
Materials & Methods: To establish the viability of conducting a combined analysis of both radiomic and genomic data, a pilot cohort of 6 patients with <5cm G2 unilateral non-metastatic T1a-b ccRCC, who underwent surgery, was evaluated. Transcriptomic analysis was conducted through RNA-seq on tumor samples, while radiomic data was extracted from pre-operative 4 phase contrast-enhanced multidetector CT scans. Genomic heterogeneity was assessed with principal component analysis run on unrestricted data, on a clear-cell renal cell carcinoma associated gene list with zero-centered Reads Per Kilobase of transcript, per million mapped reads values. The underlying pathways and gene ontologies were established with enrichment analysis. In addition, Pearson’s correlation between radiomic data and the transcription of significant genes was fitted, and dendrogram and heatmap plots were drawn.
Results: Even in a clinically homogeneous population, the employed analyses have demonstrated that RCC should be regarded as an intrinsically heterogeneous disease. The analysis of the radiomic features and gene expression correlation using heatmap and dendrogram showed four distinct radiogenomic correlation patterns: with one including 5 radiomic features, and the other three including 2 features each.
Conclusion: The current pilot study is the first investigation demonstrating an innovative radiogenomic characterisation of clear-cell RCC. Based on such observations, further investigation into the radiomic and genomic approaches for the enhanced diagnosis of RCC is warranted.

overall_design: 6 ccRCC tumor samples collected from patients who underwent surgery for RCC were collected and analysed.

first: Dejan

last: Lazarevic

first: Marco

last: Morelli

first: Giulia

last: Scotti

first: Giovanni

last: Tonon

first: Umberto

last: Capitanio


title: ARID1A and PI3-Kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion

summary: This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: ARID1A and PI3-Kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion [12Z_1A_PI3K_RNA-seq]

overall_design: 12Z endometriotic epithelial cells transfected with a combinaton of siARID1A or non-targeting siRNA control and pBabe-PIK3CA*H1047R or empty vector were assayed 72 hours post-transfection via RNA-seq.


title: Genome-wide analysis of eIF4G-RNA interaction in CDK12-depleted cells (Ribo-Seq)

status: Public on Jun 12 2019

city: San Diego

first: April

middle: Elizabeth

last: Williams

address: 10010 N Torrey Pines Rd

institute: Salk Institute for Biological Studies

summary: Methods: U2OS mRNA profiles of control siRNA(siControl) and siCDK12 were generated by deep sequencing, in triplicate, using Illumina. The sequence reads that passed quality filters were analyzed at the transcript isoform level. Total RNA-Seq was used for normalzation of RIP-Seq signals.Results: We report the application of RNA-immunoprecipitation-based sequencing for high-throughput profiling of mRNA abundence in mammalian cells. We generated genome-wide views of translation rate in CDK12-depleted U2OS cells in comparion to mTOR inhibitor, rapamycin treatment. We find that significant overlap and high corelation of change in eIF4G-associated mRNAs between two condition, rapamycin treatment and siCDK12 transfected cells. This study provides a role of CDK12 in translation control of  mammalian cell.

overall_design: Ribosome footprinting followed by sequencing (Ribo-Seq) in 2 different treatments in U2OS cells.

first: Seung

middle: H

last: Choi

first: Maxim

last: Shokhirev

first: Thomas

last: Martinez

first: Kathrine

last: Jones


title: Elafibranor restricts lipogenic and inflammatory responses in a human skin stem cell-derived model of NASH

status: Public on Jun 01 2019

city: Brussels

first: Joost

last: Boeckmans

address: Laarbeeklaan 103

department: In Vitro Toxicology & Dermato-Cosmetology (IVTD)

country: Belgium

institute: Vrije Universiteit Brussel (VUB)

summary: Non-alcoholic steatohepatitis (NASH) is characterized by hepatocellular steatosis with concomitant hepatic inflammation. Currently, no tools exist that can adequately recapitulate the molecular mechanisms of NASH and assess the efficacy of potential anti-NASH drug candidates.  The present study describes the development and application of a new preclinical model for NASH using hepatic cells generated from human skin-derived precursors (hSKP-HPC). Exposing these cells to lipogenic (insulin, glucose, fatty acids) and pro-inflammatory factors (IL-1β, TNF-α, TGF-β) resulted in a characteristic NASH response, including intracellular lipid accumulation, modulation of NASH-specific gene expression, increased caspase-3/7 activity and the secretion of inflammatory markers, such as CCL2, CCL5, CCL7, CCL8, CXCL5, CXCL8, IL1a, IL6 and IL11.  The human relevance of the proposed NASH model was verified by transcriptomics analyses that revealed commonly modulated genes and identified the same gene classes between the in vitro system and patients suffering from NASH.   The application potential of this in vitro model was demonstrated by testing elafibranor, a promising anti-NASH compound currently under clinical phase III trial evaluation. Elafibranor attenuated in vitro key features of NASH, and dramatically lowered lipid load as well as the expression and secretion of inflammatory chemokines, which in vivo are responsible for the recruitment of immune cells. This reduction in inflammatory response is NFκB-mediated. In summary, this human-relevant, sensitive in vitro system can be a valuable testing tool for the investigation of novel anti-NASH compounds.

overall_design: hSKP-HPC were exposed for 24h to lipogenic (insulin, glucose, fatty acids) and pro-inflammatory factors (IL-1β, TNF-α, TGF-β) in the abscence or presence of elafibranor, a PPAR-α/δ agonist. All experiments were conducted using three biological replicates: 'hSKP-HPC' control samples (n=3), 'hSKP-HPC NASH' samples (n=3), 'hSKP-HPC NASH' + elafibanor 10 µM samples (n=3) and 'hSKP-HPC NASH' + elafibanor 30 µM samples (n=3).

first: Karolien

last: Buyl

first: Alessandra

last: Natale

first: Valerie

last: Vandenbempt

first: Steven

last: Branson

first: Veerle

last: De Boe

first: Vera

last: Rogiers

first: Joery

last: De Kock

first: Robim

last: Rodrigues

first: Tamara

last: Vanhaecke


title: Deregulated high expression of NEAT1 lncRNA in multiple myeloma is unrelated to molecular features and clinical outcome

status: Public on Jun 18 2019

city: MILAN

first: Luca

last: Agnelli

address: Venezian 1

department: Department of Advanced Diagnostics

institute: IRCCS Istituto Nazionale dei Tumori

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: Multiple myeloma (MM) is a malignant proliferation of bone marrow plasma cells, whose pathogenesis remains largely unknown. Long ncRNAs (lncRNAs) are a large class of non-protein-coding RNA, involved in many physiological cellular and genomic processes as well as in carcinogenesis, cancer metastasis and invasion. The biological role and therapeutic potential of lncRNAs in MM are still to be explored. Herein, we investigated the nuclear paraspeckle assembly transcript 1 (NEAT1) in the context of plasma cell dyscrasia, in a cohort of 50 MM and 15 plasma cell leukemia samples. Array expression data indicated that NEAT1 was upregulated in tumor samples compared to four healthy controls. Moreover, in MM patients, NEAT1 was globally overexpressed irrespectively of molecular characteristic, as further supported by Q-RT-PCR validation and by RNA sequencing data in a representative subgroup of cases. The functional annotation of genes and the lncRNAs transcriptional signature associated with NEAT1 expression indicated the modulation of DNA repair and metabolism, dynein interaction and unfolded protein response pathways. We tested NEAT1 clinical relevance in a retrospective proprietary dataset including 55 MM and in the large TT2/TT3 trials cohort from the University of Arkansas encompassing more than 550 patients; in both cases, NEAT1 overexpression was not correlated with patient’s prognosis.

overall_design:
Total RNA samples from highly purified plasma cells of 50 MM cases at onset, 15 plasma cell leukemia and 4 normal controls (purchased from  Voden, Medical Instruments IT).
30 MM and 4 N samples in common with GSE109116.

first: Elisa

last: Taiana

first: Domenica

last: Ronchetti

first: Vanessa

last: Favasuli

first: Katia

last: Todoerti

first: Martina

last: Manzoni

first: Antonino

last: Neri


title: Transcription profile analysis of wild type and Irf9-/- human monocytic THP1 cells in response to type I interferons

status: Public on May 28 2019

city: Vienna

first: Ekaterini

last: Platanitis

address: Dr.-Bohr-Gasse 9

department: Department of Microbiology, Immunobiology and Genetics

country: Austria

institute: University of Vienna

summary: Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity.   Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Our results show how mRNA expression changes upon IFNb treatment in wild type and Irf9-/- THP1 cells.

overall_design: Methods: mRNA of untreated and IFNb treated wild-type (WT) and Irf9 knock out (IRF9-/-) human monocytic THP1 cells were analyzed by deep sequencing, in triplicate, using Illumina sequencing.

first: Thomas

last: Decker


title: STAT1, STAT2 and IRF9 transcription factor binding analysis in wild type human monocytic THP1 cells in response to type I interferons

summary: Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity.   Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Mechanisms to meet this demand on the relevant molecular machinery include remodeling of chromatin but also changes in transcription factor interaction prior and during the IFN response.  Our results show how transcription factors STAT1, STAT2 and IRF9 change binding patterns upon IFNb treatment in wild type human monocytic THP1 cells.

overall_design: Methods: Genome wide binding of transcription factors STAT1, STAT2 and IRF9 in untreated and 90 min IFNb treated wild-type (WT)  human monocytic THP1 cells. Samples were analyzed by using Illumina sequencing.


title: Transcription profile analysis of wild type and Irf9-/- mouse embryonic fibroblasts (MEF) in response to type I interferons

summary: Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity.   Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Our results show how mRNA expression changes upon IFNb treatment in wild type and Irf9-/- mouse embryonic fibroblasts.

overall_design: Methods: Mouse embryonic fibroblast (MEF) mRNA of wild-type (WT) and Irf9 knock out mice (IRF9-/-) untreated, as well as 2h IFNb  treated were generated by deep sequencing, in triplicate, using Illumina sequencing.


title: Anteroposterior Wnt-RA Gradient define adhesion and migration properties of neural progenitors in developing spinal cord

status: Public on Jun 04 2019

city: St Lucia

first: Mohammed

last: Shaker

state: QLD

address: Building 75,Cnr College Rd & Cooper Rd,, Brisbane

department: AIBN

country: Australia

institute: The University of Queensland

summary:
NPCs adhesion affinity along the AP axis is under the control of Wnt/RA molecular networks
Caudal NPCs express a high amount of ECM genes compare to rostral-NPCs.

overall_design: Examination of the tail and brachial embryonic mouse NPCs from the E12.5 embryonic stage.

first: Woong

last: Sun


title: Comprehensive determination of transcription start sites derived from all RNA polymerases using ReCappable-seq

status: Public on Jun 14 2019

city: Ipswich

first: Bo

last: Yan

state: MA

address: County Road

institute: New England Biolabs

summary:
We develop a method Re-Cappable-seq for determining eukaryotic transcription start sites derived from all RNA polymerases at nucleotide resolution. In particular, this method identifies the Pol-I and Pol-III TSSs, which are missing by CAGE.
Applied to human A549 cell line, our method results in the identification of 33,468 and 5,269 high confidence Pol-II and non-Pol-II TSS respectively. Re-Cappable-seq identifies Pol-II TSS with higher specificity than CAGE.

overall_design: We isolated total RNA from human A549 cancer cell line. First we used yDcpS to decap the canonical G-cap structure from Pol-II transcripts leaving  a di-phosphorylated 5'end. Then we used VCE to specifically add a biotin cap to these di-phosphorylated 5'ends as well as the tri-phosphorylated 5'ends from the non-Pol-II transcripts. Finally we isolated these biotin capped RNA using streptavidin beads and sequenced using illumina. Here the RNA without streptavidin enrichment served as control. In addition, we also performed CIP treatment before decapping, which was used to distinguish the Pol-II and non-Pol-II transcripts.

first: George

last: Tzertzinis

first: Ira

last: Schildkraut

first: Laurence

last: Ettwiller


title: STAT1, STAT2 and IRF9 transcription factor binding analysis in wild type mouse embryonic fibroblasts (MEF) in response to type I interferons

summary: Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity.   Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Mechanisms to meet this demand on the relevant molecular machinery include remodeling of chromatin but also changes in transcription factor interaction prior and during the IFN response.  Our results show how transcription factors STAT1, STAT2 and IRF9 change binding patterns upon IFNb treatment in wild type mouse embryonic fibroblasts.

overall_design: Methods: Genome wide binding of transcription factors STAT1, STAT2 and IRF9 in untreated and 90 min IFNb treated wild-type (WT)  mouse embryonic fibroblasts. Samples were analyzed by using Illumina sequencing.


title: Genome-wide analysis of eIF4G-RNA interaction in CDK12-depleted cells (RIP-Seq)

first: April

last: Williams

department: IGC

summary:
Methods: U2OS mRNA profiles of control siRNA(siControl) and siCDK12 were generated by deep sequencing, in triplicate, using Illumina. The sequence reads that passed quality filters were analyzed at the transcript isoform level. Total RNA-Seq was used for normalzation of RIP-Seq signals.
Results: We report the application of RNA-immunoprecipitation-based sequencing for high-throughput profiling of mRNA abundence in mammalian cells. We generated genome-wide views of translation rate in CDK12-depleted U2OS cells in comparion to mTOR inhibitor, rapamycin treatment. We find that significant overlap and high corelation of change in eIF4G-associated mRNAs between two condition, rapamycin treatment and siCDK12 transfected cells. This study provides a role of CDK12 in translation control of  mammalian cell.

overall_design: Examination of quantification of mRNA in eIF4G complex in 2 different treatments in U2OS cells.

first: Seung

last: Choi

first: Maxim

middle: N

last: Shokhirev

last: Martinez

first: Kathrine

last: Jones


title: Genome-wide analysis of eIF4G-RNA interaction in CDK12-depleted cells


title: Transcription profile analysis of wild type and Irf9-/- bone marrow derived macrophages in response to type I and type II interferons

summary: Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity.   Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Our results show how mRNA expression changes upon IFNb or IFNg treatment in wild typ and Irf9-/- bone marrow derived macrophages.

overall_design: Methods: Bone marrow derived macrophage mRNA of wild-type (WT) and Irf9 knock out mice (IRF9-/-) untreated, as well as 2h IFNb and IFNg treated were generated by deep sequencing, in triplicate, using Illumina sequencing.


title: Irf9 function in immunity in mouse


title: STAT1, STAT2 and IRF9 transcription factor binding analysis in wild type and Irf9-/- bone marrow derived macrophages in response to type I and type II interferons

summary: Host defense by the innate immune system requires the establishment of antimicrobial states allowing cells to cope with microorganisms before the onset of the adaptive immune response. Interferons (IFN) are of vital importance in the establishment of cell-autonomous antimicrobial immunity.   Speed is therefore an important attribute of the cellular response to IFN. With much of the antimicrobial response being installed de novo, this pertains foremost to gene expression, the rapid switch between resting-state and active-state transcription of host defense genes. Mechanisms to meet this demand on the relevant molecular machinery include remodeling of chromatin but also changes in transcription factor interaction prior and during the IFN response.  Our results show how transcription factors STAT1, STAT2 and IRF9 change binding patterns upon IFNb or IFNg treatment in wild type and Irf9-/- bone marrow derived macrophages.

overall_design: Methods: Genome wide binding of transcription factors STAT1, STAT2 and IRF9 in untreated, 90 min IFNb and 90 min IFNg treated wild-type (WT) and Irf9 knock out (IRF9-/-) bone marrow derived macrophages. Biological duplicates were analyzed by using Illumina sequencing.


title: Control of FoxO1 mRNA expression by microRNAs

status: Public on Apr 24 2019

city: Kagoshima

first: Daichi

last: Ijiri

address: Korimoto 1-21-24

institute: Kagoshima University

summary: To identify miRNAs, a microarray analysis were differentially expressed between C2C12 myotubes stimulated with 1 μM clenbuterol for 1 h and their control myotubes.

overall_design:
RNA was isolated using the mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA)　in accordance with the manufacturer’s instructions. All samples were diluted to a final concentration of 3 ng/mL.
miRNA was hybridized to Affymetrix GeneChip microRNA 4.0 arrays (Affymetrix, CA, USA). miRNA array processing was performed according to the manufacturer's protocol.

first: Saki

last: Shimamoto


title: Loss of Nuclear TDP-43 Is Associated with Decondensation of LINE Retrotransposons [ATAC-Seq]

status: Public on Mar 15 2019

city: Philadelphia

first: Edward

middle: B

last: Lee

state: Pennsylvania

address: 422 Curie Blvd

institute: Perelman School of Medicine at the University of Pennsylvania

summary: Loss of the nuclear RNA binding protein TAR DNA binding protein-43 (TDP-43) into cytoplasmic aggregates is the strongest correlate to neurodegeneration in amyotrophic lateral sclerosis and frontotemporal degeneration. The molecular changes associated with the loss of nuclear TDP-43 in human tissues are not entirely known. Using a novel subcellular fractionation and fluorescent activated cell sorting method to enrich for diseased neuronal nuclei without TDP-43 from post-mortem FTD-ALS human brain, we characterized the effects of TDP-43 loss on the transcriptome and chromatin accessibility. Nuclear TDP-43 loss is associated with gene expression changes that affect RNA processing, nucleocytoplasmic transport, histone processing and DNA damage. Loss of nuclear TDP-43 was also associated with chromatin decondensation around long interspersed nuclear elements (LINEs) and increased LINE1 DNA content. Moreover, loss of TDP-43 leads to increased retrotransposition that can be inhibited with antiretroviral drugs, suggesting that TDP-43 neuropathology is associated with altered chromatin structure including decondensation of LINEs.

overall_design: Examination of gene expression profiles and chromatin accessibility in neuronal nuclei, which have either retained or lossed TDP-43, extracted from FTD/ALS patient post-mortem frontal cortex

first: Elaine

middle: Y

last: Liu

first: Jenny

last: Russ

first: Christopher

last: Cali

first: Jessica

last: Phan

first: Alexandre

last: Amlie-Wolf


title: Loss of Nuclear TDP-43 Is Associated with Decondensation of LINE Retrotransposons [RNA-Seq]


title: Loss of Nuclear TDP-43 Is Associated with Decondensation of LINE Retrotransposons


title: Single-cell sequencing of JMJD1C Jumonji domain mutated MLL-AF9 Leukemia cells

status: Public on Apr 12 2019

city: Milwaukee

last: Zhu

state: WI

address: 8733 W Watertown Plank Rd

institute: Versiti Blood Research Institute Milwaukee

summary: We performed single-cell sequencing on mouse MLL-AF9-cas9 leukemia cells 7 days after transduction with sgRNA against Renilla or JMJD1C JmjC domain. We revealed heterogeneity within each population.

overall_design: We performed single-cell sequencing on mouse MLL-AF9 cells harboring JMJD1C sgRNA targeting jumonji domain or renilla control sgRNA.

first: Robert

last: Burns