title: Human Feto-placental Arterial and Venous Endothelial Cells are Differentially Programmed by Gestational Diabetes Mellitus Resulting in Cell-specific Barrier Function

city: Graz

first: Silvija

last: Tokic

address: Auenbruggerplatz 14

institute: Medical University of Graz

summary:
We performed genome-wide methylation analysis of primary feto-placental arterial and venous endothelial cells from healthy (AEC and VEC) and GDM complicated pregnancies (dAEC and dVEC). Parallel transcriptome analysis identified variation in gene expression linked to GDM-associated DNA methylation, implying a direct functional link. Pathway analysis found that genes altered by exposure to GDM clustered to functions associated with ’Cell Morphology’ and ’Cellular Movement’ in both AEC and VEC. Further functional analysis demonstrated that GDM exposed cells have altered actin organization and barrier function.
Our data indicate that exposure to GDM programs atypical morphology and barrier function in feto-placental endothelial cells by DNA methylation and gene expression change. The effects differ between AEC and VEC, indicating a stringent cell-specific sensitivity to adverse exposures associated with developmental programming in utero.

overall_design: Primary human feto-placental endothelial cells form arteries (AEC) and veins (VEC) were isolated after healthy pregnancy (AEC, VEC) and after a pregnancy complicated by Gestational Diabetes Mellitus (dAEC, dVEC). Total DNA was isolated and subjected to methylation analysis.

last: Cvitic

first: Lavinia

last: Gordon

first: Boris

last: Novakovic

first: Christine

last: Ulz

last: Mühlberger

first: Francisca

middle: I

last: Diaz-Perez

first: JiHoon

last: Joo

first: Vendula

last: Svendova

first: Michael

middle: G

last: Schimek

first: Slave

last: Trajanoski

first: Richard

last: Saffery

first: Ursula

last: Hiden

first: Gernot

last: Desoye


title: Small sized mesenchymal stem-cells primed with hypoxia attenuate graft-versus-host disease [DNA methylome dataset]

status: Public on Aug 01 2018

city: Seoul

first: DONG-MYUNG

last: SHIN

address: Pungnap-2 dong, Songpa-gu

department: Department of Biomedical Sciences

country: South Korea

institute: University of Ulsan College of Medicine

type: Methylation profiling by genome tiling array

summary: Mesenchymal stem-cells (MSCs) are of particular interest for treating immune-related diseases due to their immunosuppressive capacities. Here, we show that Small sized MSCs primed with Hypoxia and Calcium ion (SHC-MSCs) exhibit the enhanced functions regarding stemness and immunomodulation for treating allogeneic conflicts. Compared with naïve cultured human umbilical cord-blood MSCs, SHC-MSCs were resistant to the passage dependent cellular senescence mediated by MCP-1 and p53/p21 cascade and highly secreted the pro-angiogenic and immune-modulatory factors, resulting in suppression of T-cell proliferation. Genome-wide DNA methylome and transcriptome analysis indicate that SHC-MSCs characteristically up-regulated immune-modulation, cell adhesion and cell-cycle related genes. As downstream factors, PLK1, ZNF143, DHRS3, and FOG2 proteins played a key role on the beneficial effects of SHC-MSCs, evidenced by the promoted self-renewal, migration, pro-angiogenic, anti-inflammatory, and T cell suppression capacities in their-over-expressing MSCs. Importantly, administration of SHC-MSCs or PLK1-over-expressing cells (PLK1-MSCs) significantly reduced the symptoms of graft-versus-host disease (GVHD) in a humanized mouse model which led to significantly improved survival, less weight loss, and less histopathologic injuries of GVHD target organs compared with naive MSC-infused mice. Collectively, our study suggests that small-sized MSCs primed with hypoxia could advance the therapeutic strategy for the clinical treatment of allogeneic conflicts including GVHD.

overall_design: Human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) were normally maintained (NT) or cultivated by SHC procedure (SHC) which enriched small sized cells and at the same time primed with hypoxia ( 3 % oxygen) and calcium ion (1.8 mM). Genomic DNA (gDNA) were isolated using the DNeasy Blood & Tissue Kit (Qiagen Inc, Valencia, CA, USA). 500 ng of gDNA were used in bisulfite conversion with Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA) and the DNA methylome of naïve and SHC mesenchymal stem cells (MSCs) was analyzed using on a Infinium HumanMethylation450K BeadChip assay (Illumina Inc, San Diego, CA), in accordance with the manufacturer’s instructions.

first: Dong-Myung

last: Shin

first: YongHwan

last: Kim

first: Hyein

last: Ju


title: Activated CARD11 accelerates germinal center kinetics, promoting mTORC1 and terminal differentiation.

status: Public on Jul 31 2018

city: Seattle

first: Raghav

last: Chawla

state: WA

address: 3720 15th Ave NE

department: Department of Genome Sciences

country: USA

institute: University of Washington

type: Expression profiling by high throughput sequencing

summary: This scRNA-seq experiment is an integral part of a manuscript with the above title. Our analysis of the scRNA-seq data suggests that activated CARD11 promotes immunoglobulin class-switching in germinal center B cells and generation of IgG1-secreting plasma cells.

overall_design: Single-cell suspensions were prepared from spleens harvested from mice 5 days post immunization with sheep red blood cells. B cells were enriched using an immunomagnetic negative selection kit. scRNA-seq was performed using the Chromium product suite by 10x Genomics.

first: R

first: M

middle: N

last: Wray-Dutra

first: C

last: Trapnell

last: James

first: D

middle: J

last: Rawlings


title: ABCA1 haplodeficiency affects the brain transcriptome following traumatic brain injury in mice expressing human APOE isoforms

city: Pittsburgh

first: Iliya

last: Lefterov

state: PA

address: 130 De Soto Street

department: Enviromantal and Occupational health

institute: University of Pittsburgh

summary: We examined the impact of Abca1 deficiency and APOE isoform expression on the response to TBI using 3-months-old, human APOE3+/+ (E3/Abca1+/+) and APOE4+/+ (E4/Abca1+/+) targeted replacement mice, and APOE3+/+ and APOE4+/+ mice with only one functional copy of the Abca1 gene (E3/Abca1+/-; E4/Abca1+/-). TBI-treated mice received a craniotomy followed by a controlled cortical impact (CCI) brain injury in the left hemisphere; sham-treated mice received the same surgical procedure without the impact. We performed RNA-seq using samples from cortices and hippocampi collected at 14 days post-injury, followed by genome-wide differential gene expression analysis.

overall_design: We used 3-months-old, human APOE3+/+ (E3/Abca1+/+) and APOE4+/+ (E4/Abca1+/+) targeted replacement mice, and APOE3+/+ and APOE4+/+ mice with only one functional copy of the Abca1 gene (E3/Abca1+/-; E4/Abca1+/-). Groups consisted of 6-8 animals of both genders. TBI-treated mice received a craniotomy followed by a controlled cortical impact (CCI) brain injury in the left hemisphere; sham-treated mice received the same surgical procedure without the impact. We performed RNA-seq using samples from cortices and hippocampi collected at 14 days post-injury from 58 samples, followed by genome-wide differential gene expression analysis.

first: Emilie

middle: L

last: Castranio

first: Cody

middle: M

last: Wolfe

first: Kyong

last: Nam

first: Florent

last: Letronne

first: Nicholas

middle: F

last: Fitz

first: Radosveta

last: Koldamov


title: P-TEFb function in ncRNA transcription

first: Jongbum

address: 254, Beotkkot-ro, Geumcheon-gu

institute: Macrogen, Inc.

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Many ncRNAs serve as regulatory molecules in various physiological pathways. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. Transcriptional regulation in ncRNA genes is thus important to be understood and is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA Pol II promoter-proximal pausing and in a tissue-specific manner. In this study, human stimulus-inducible ncRNA genes were monitored to assess the function of P-TEFb, a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb inhibition in serum responsive ncRNA genes was observed: S2 phospho-Pol II was largely increased in the TSS (–300 to +300) whereas overall, it was decreased in the gene body (> +350) upon chemical inhibition of P-TEFb. In addition, the three representative ncRNAs, MALAT1, NEAT1, and XIST, were further analyzed for determining P-TEFb association. Taken together, we propose the transcriptional activation of many human ncRNAs utilizing pausing and releasing Pol II and the regulatory mechanism of transcriptional elongation of these genes requiring the function of P-TEFb.

overall_design: A total of 16 ChIP-seq samples were included in the study. Starved (serum 0)  and serum-induced (Serum 15) ones with DMSO or flavopiridol treated. Input chromatin DNAs per each condition were sequenced. Replicates for each condition.

first: Heeyoun

last: Bunch

first: Stuart

middle: S

last: Levine


title: Integrated transcriptomic and proteomic analysis of human eccrine sweat glands

city: Baltimore

first: Garry

middle: R

last: Cutting

state: MD

address: 700 N. Broadway, Suite 551

department: Institute of Genetic Medicine

institute: Johns Hopkins University

summary: Eccrine sweat gland is an exocrine gland that is involved in the secretion of sweat for control of temperature. Malfunction of the sweat glands can result in disorders such as miliaria, hyperhidrosis and bromhidrosis. In addition, lack of reabsorption of Cl- ions from reabsorptive duct of eccrine sweat gland is a major feature of cystic fibrosis. Understanding the proteome of eccrine sweat glands is important for understanding the physiology of sweat formation. In spite of this, no systematic transcriptome or proteome analysis of eccrine sweat glands has yet been reported. To this end, we isolated eccrine sweat glands by microdissecting them from human skin and performed both RNA-seq and proteome analysis. In total, ~138,000 transcripts and ~6,100 proteins were identified. The proteome data showed the enrichment in protein digestion/absorption and salivary secretion, while the transcriptome data did not show any enrichment for a specific pathway. This study also enabled us to confirm 2 missing proteins. Integrating RNA-seq and proteomic data allowed us to identify 7 peptides from 5 novel genes. Most of the novel proteins were from short open reading frames (sORFs) suggesting that many sORFs still remain to be annotated in the human genome. The peptides mapping to the missing or novel proteins were validated by analyzing synthetic peptides. This study provides the first integrated analysis of the transcriptome and proteome of the human eccrine sweat gland and should become an invaluable resource to biomedical research community for studying sweat glands in physiology and disease.

overall_design: One set of RNA sample purified from dissected sweat glands isolated from the skin dermis by microscopic dissection

first: Pandey

last: Akhilesh


title: Expression data from mouse CD19-targeted CAR (chimeric antigen receptor) T cells after antigen stimulation

city: Tampa

first: Gongbo

last: Li

state: Florida

address: 3011 W Holly Dr. MRC 2068

institute: Moffitt Cancer Center

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

overall_design: Refer to individual Series


title: Expression data from mouse CD19-targeted CAR (chimeric antigen receptor) T cells after antigen stimulation [RNA-seq]

summary: Second-generation CD19-targeted chimeric antigen receptors (CAR) have an antigen-binding domain fused to transmembrane, co-stimulatory, and CD3ζ domains. The two CARs with regulatory approval include a CD28 or 4-1BB co-stimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences between the two endodomains have become apparent but not completely understood. The objective is to evaluate gene expression in different mouse CD19-targeted CAR T cells, including m19z, m1928z and m19-humBBz.

overall_design: Different CAR T cells were stimulated with antigen presenting cells overnight for RNA extraction and sequence analysis. Nine samples were analyzed.

first: Marco

last: Davila


title: Identification of genes involved in GABAergic Wiring

city: London

first: Antonio Jesus

last: Hinojosa Garcia

state: London

address: Room 4.4 New Hunt's House, Guy's Campus

department: Centre for Developmenta Neurobiology

country: United Kingdom

institute: King's College London

summary:
How neurons are wired to form precise circuits is crucial to understand the development of cortical functions. Glutamatergic pyramidal cell and GABAergic interneuron wire up the cortex through differentiated cellular events. However, little is known about the molecular mechanisms that underlie the unique features of interneuron wiring.
We performed a high-throughput genomic screen for genes upregulated specifically during GABAergic wiring.

overall_design: We used fluorescence-activated cell sorting (FACS) to isolate pyramidal cells and interneurons using mice in which these populations of cells are labeled with GFP (NexCre;RCE and Nkx2-1Cre;RCE, respectively) at two different postnatal stages (P0 and P12 for pyramidal cells and P0 and P10 for interneurons), and subsequently carried out transcriptome analyses

first: Antonio

last: Hinojosa

first: Beatriz

last: Rico


title: Region-specific alteration of histone modification by LSD1 inhibitor conjugated with pyrrole-imidazole polyamide

city: Chiba

first: Masaki

last: Fukuyo

address: 1-8-1 Inohana, Chuo-ku

department: Department of Molecular Oncology

country: Japan

institute: Chiba University

summary: We conjugated NCD38 with two different Py-Im polyamides and analyzed the regions epigenetically altered by parental NCD38 and the two conjugates.

overall_design: Human colorectal cancer cells, RKO, were treated with conjugated NCD38 with two different Py-Im polyamides (P4_NCD38 and PIPP_NCD38) for 4 days or 30 days followed by total RNA extraction and ChIP (H3K4me2, H3K4me3 and H3K27Ac). For control, RKO cells were treated with Py-Im polyamides(P4 and PIPP) for 30 days followed by ChIP. ChIP-seq and RNA-seq were performed using NexSeq and HiSeq.

first: Kokiladevi

last: Alagarswamy

first: Ken-ichi

last: Shinohara

first: Atsushi

last: Okabe

last: Kaneda


title: Expression data for human peripheral blood mononuclear cells (PBMCs) transfected with EBV Bart 6-3p mimics.

city: Changsha

first: Jian

last: Ma

state: Hunan

address: 110  Xiangya  Road

department: Cancer  Research Institute

country: China

institute: Central South University

summary: Epstein-Barr virus (EBV) is highly successful virus infecting the majority of the human population worldwide. During the latent infection period, there are only a few of EBV-encoded proteins can be detected, whereas EBV-encoded non-coding RNAs are highly activated, especially microRNAs. Recent studies found that those BART microRNAs not only disturb EBV genes expression, but also interfere many host genes expression, thus modulating cellular proliferation and immune regulation. In the present study, we investigate the effect of EBV BART6-3p on gene expression profile of the human PBMCs.

overall_design: Human PBMCs from a health donor were transfected with a control mimics or EBV BART6-3p mimics (150 μM) for 36 hours.  Total RNA were extracted and detected by Affymetrix Human Genome U133 Plus 2.0 Array.

first: Zailong

last: Qin

first: Yuanjun

last: Lu

first: Guiyuan


title: Induction of Myelinating Oligodendrocytes in Human Cortical Spheroids

status: Public on Jul 30 2018

city: Cleveland

first: Paul

last: Tesar

state: Ohio

address: 10900 Euclid Ave.

department: Genetics & Genome Sciences

institute: Case Western Reserve University

summary: Organoid technologies provide an accessible system in which to examine the generation, self-organization,and 3-dimensional cellular interactions during development of the human cerebral cortex. However, oligodendrocytes, the myelinating glia of the central nervous system and third major neural cell type, are conspicuously absent from current protocols. Here we reproducibly generate human oligodendrocytes and myelin in pluripotent stem cell-derived cortical spheroids. Transcriptional and immunohistochemical analysis of the spheroids demonstrates molecular features consistent with maturing human oligodendrocytes within 14 weeks of culture, including expression of MyRF, PLP1, and MBP proteins. Histological analysis by electron microscopy shows initial wrapping of human neuronal axons with myelin by 20 weeks and maturation to compact myelin by 30 weeks in culture. Treatment of spheroids with previously identified promyelinating drugs enhances the rate and extent of human oligodendrocyte generation and myelination. Furthermore, generation of spheroids from patients with a severe genetic myelin disorder, Pelizaeus-Merzbacher disease, demonstrates the ability to recapitulate human disease phenotypes, which were in turn improved with both pharmacologic and CRISPR-based approaches. Collectively, these 3-dimensional, multi-lineage cortical spheroids provide a versatile platform to observe and perturb the complex cellular interactions   that occur during developmental myelination of the brain and offer new opportunities for disease modeling and therapeutic development in human tissue.

overall_design: RNAseq profiles comparing neuro-cortical spheroids and oligo-cortical spheroids

first: Mayur

middle: C

last: Madhavan


title: CDK12 mediated transcriptional regulation in U2OS cells

city: Cambridge

first: Kejie

state: Massachusetts

address: 225 Binney Street

institute: Biogen

summary: While activation of canonical NF-κB signaling through the IKK complex is well studied, few regulators of NIK-dependent non-canonical p52 nuclear translocation have been identified. We discovered a novel role for cyclin dependent kinase 12 (CDK12) in transcriptionally regulating the non-canonical NF-κB pathway. High-content phenotypic screening identified a novel compound, 919278, which inhibits lymphotoxin β receptor (LTβR)- and FN14-dependent p52 nuclear translocation, but not TNFα receptor (TNFR)-mediated, canonical NF-κB p65 nuclear translocation. Chemoproteomics identified cyclin dependent kinase 12 (CDK12) as the target of 919278. CDK12 inhibition by 919278, THZ1, or siRNA knock down all affect similar global transcriptional changes and prevent LTβR and FN14-dependent MAP3K14 (NIK) mRNA induction and subsequent protein accumulation. In addition, 919278 and THZ1 treatment reduce RNA Pol II CTD phosphorylation. This powerful approach of coupling a phenotypic screen with chemoproteomics revealed a novel regulatory pathway of the non-canonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

overall_design: There are TWEAK stimulated and unstimulated conditions, 4hr and 24hr time points. 7 treatments (DMSO, BIO0702697, BIO0919278, BIO032202, NTsiRNA, siRNAs523626, siRNAs523629) in duplicates. In total, 56 sample were sequenced and analyzed.

first: Kate

last: Henry

first: Debra

last: Kellner

first: Bekim

last: Bajrami

first: John

last: Anderson

first: Mercedes

last: Beyna

first: Govinda

last: Bhisetti

first: Tom

last: Cameron

first: Andrew

last: Capacci

first: Andrea

last: Bertolotti-Ciarlet

first: Jun

last: Feng

first: Benbo

last: Gao

first: Brian

last: Hopkins

first: Tracy

last: Jenkins

first: Tricia

last: May-Dracka

first: Paramasivam

last: Murugan

first: Ru

last: Wei

first: Weike

last: Zeng

first: Norm

last: Allaire

first: Alan

last: Buckler

first: Christine

last: Loh

first: Peter

last: Juhasz

last: Lucas

first: Katelin

middle: A

last: Ennis

first: Elisabeth

last: Vollman

first: Ellen

last: Cahir-McFarland

first: Erik

last: Hett

first: Michelle

last: Ols


title: RNA-Seq of murine CD4 positive T cells isolated from Nur77-deficient mice and their Nur77-competent counterparts

city: Münster

first: Luisa

last: Klotz

address: Albert-Schweitzer-Campus 1, A1

country: Germany

institute: Universitätsklinikum Münster

summary: Endogenous changes in the nuclear encoded genes were analysed using RNA-Seq in order to compare the genetic profile of murine CD4 positive T cells isolated from Nur77-deficient mice and their Nur77-competent counterparts.

overall_design: Total RNA from each sample was isolated prior changes in gene expression using RNA-Seq were analysed.

first: Martin

last: Schädlich

first: Marie

last: Liebmann


title: Dual RNA-Seq Analysis of Trichophyton rubrum and HaCat Keratinocyte Co-Culture Highlights Important Genes for Fungal-Host Interaction

city: Ribeirão Preto

first: Ana

middle: Lucia

last: Fachin

state: SP

address: Av. Costabile Romano, 2201

department: Biotechonology  Unit

country: Brazil

institute: Unaerp

summary:
Purpose: Evaluate the transcriptional profile of genes involved in fungus-host interactions using RNA sequencing
Methods: T. rubrum and HaCat cells line were co-cultured in RPMI medium supplemented with 5% of fetal bovine serum and were incubated for 24 h  at 37ºC in a humidified atmosphere containing 5% CO2  , followed RNA by extraction of both organisms, libraries construction and sequence.
Results: Our data demonstrated the induction of specific genes that may improve the assimilation of nutrients and fungal survival in the host and genes encoding keratinolytic proteases that are important for T. rubrum virulence during infection. In HaCat cell lines it were induced genes encoding antimicrobial activity , cell migration and epithelial barrier repair. Futhermore, the genes KRT1 and FLG involved in epithelial barrier integrity were repressed
Conclusions: This mixed transcriptome analysis showed the modulation of important genes involved in the mechanism of T. rubrum infection and in the defense and maintenance of cell homeostasis of keratinocytes, which could represent potential antifungal targets for new therapeutic approaches to the treatment of dermatophytoses

sample_organism:
Trichophyton rubrum
Homo sapiens

platform_organism:
Trichophyton rubrum
Homo sapiens

overall_design: The transcriptional profile of T. rubrum and keratinocytes co-culture was assessed using the RNA-sequencing technique in order to provide new insights about fungal-host interaction.

first: Monise

last: Petrucelli

first: Kamila

last: Peronni

first: Pablo

last: Sanches

first: Tatiana

middle: T

last: Komoto

first: Josie

middle: B

last: Matsuda

first: Wilson

last: da Silva Junior

first: Rene

middle: O

last: Beleboni

first: Nilce

last: Martinez-Rossi

first: Mozart

last: Marins


title: NMuMG cell response to TGF-beta and TGF-beta +2APB treatments

city: Grand Forks

first: Archana

last: Dhasarathy

state: North Dakota

address: 501 N Columbia Rd.

institute: University of North Dakota

summary: Recent studies demonstrate that Ca2+ signaling has an important role in EMT. Use of Ca2+ blockers such as 2APB can inhibit cell migration induced by TGF-β. Interestingly, we see an unexpected increase in Snail expression upon Ca2+ blocker treatment of both MCF10A and NMuMG cells; this increase is not observed with 2APB treatment alone. Therefore, we believe that 2APB plays a synergistic role with TGF-β in Snail induction. We propose to investigate the gene networks that change following 2APB +TGF-β treatment.

overall_design: mRNA profiles of NMuMG cells treated with TGFbeta, TGFBeta and 2APB, and DMSO control  were generated using deep sequencing, in duplicate

first: Humaira

last: Qureshi

first: Janani

last: Kumar

first: Atrayee

last: Bhattacharya

first: Kole

last: Hermanson

first: Adam

last: Scheidegger

first: Brij

last: Singh

first: Danielle

last: Perley


title: Single cell profiling of peripheral blood mononuclear cells from healthy human donor

status: Public on Jul 28 2018

city: Parkville

first: Saskia

last: Freytag

state: Victoria

address: 1G Royal Parade

department: Population Health and Immunity

country: Australia

institute: WALTER AND ELIZA HALL INST MEDICAL RES

summary: Healthy human peripheral blood mononuclear cells (PBMCs) investigated with 10x Genomics single cell RNA-sequencing.

overall_design: Peripheral blood mononuclear cells (PBMCs) from one healthy human donor were processed on the Chromium 3' single cell platform (10x Genomics) and sequenced on an Illumina HiSeq 2500. FASTQ data were preprocessed using CellRanger.

first: Ingrid

last: Lonnstedt

first: Milica

last: Ng

first: Melanie

last: Bahlo


title: Transcriptome analysis of Casz1 conditional mutant progenitors at postnatal day 2

city: Ottawa

first: Pierre

last: Mattar

state: ON

address: CCW6109, Box 511, 501 Smyth Rd.

country: Canada

institute: OHRI

summary: Transcriptome analysis of Casz1 was performed using litters of postnatal day 2 retinas from crosses between Casz1Flox/Flox; R26-Stop-EYFP and Casz1Flox/Flox; IKCre-A; R26-Stop-EYFP. The IK-A Cre driver is described in Tarchini et al., Dev Dyn 241(12):1973-85. EYFP marks Cre expressing (and therefore cKO) cells. Cells were incubated for 30 min in RGM serum-free medium described in Cayouette et al., Neuron 40(5):897, supplemented with Hoechst 33342 (Invitrogen), and 4N cells were sorted using a MOFLO cytometer (Beckman) based on Hoechst, GFP expression, and propidium iodide exclusion.

overall_design: Cells were sorted directly into lysis buffer on ice and RNA was immediately extracted using RNeasy mini columns (Qiagen). Two independent experiments were performed, and the RNA samples were processed in parallel. RNA amplification was performed using Truseq PE Cluster kit v3 PE50 (Illumina). Deep sequencing was performed using Truseq stranded mRNA (Illumina) with mRNA enrichment and strand-specific parameters, using a Hiseq 2000 instrument (Illumina).

first: Michel

last: Cayouette

first: Alexis

last: Blanchet-Cohen


title: Expression data from cultivated Human Thymic Epithelial Cells (hTECs) in comparison with cultivated Human Epidermal derived Keratinocytes (hEKs)

city: Lausanne

first: Melissa

last: Maggioni

address: Station 15

department: IBI

country: Switzerland

institute: EPFL

summary:
The thymic epithelium and the epidermis display many common features despite their different structure, function and embryonic origin.
We used microarrays to uncover differences and similarities in the gene expression profile of hTECs and hEKs in vitro and investigate their significance for thymic structure and function.

overall_design: Single-cell derived hTECs colonies with different colony morphology and single hEKs colonies were selected for RNA extraction and hybridization on Affymetrix microarrays. Four biological replicates were selected for each condition.

first: Yann

last: Barrandon


title: Primary human monocytes either mock treated, infected with A/California/07/2009 and A/Victoria/361/2001

city: Stanford

first: Lisa

last: Kronstad

state: CA

address: S131 Grant Building

department: Medicine

institute: Stanford  University

summary: 241 genes differentially expressed between two viruses

overall_design: Primary human monocytes were infected for 1 hour then primary autologous NK cells were added for a 6 or 23 hr co-coculture. Post co-culture, the monocytes and NK cells were re-isolated by magnetic separation, monocyte RNA was harvested via RNA bee and sent for RNA-sequencing.

first: Catherine

last: Blish


title: Chromatin accessibility at Polycomb target genes is independent of PRC1 and PRC2 catalytic core complexes (MEF ATAC-seq)

first: Hamish

middle: W

last: King

address: 4 Newark St

department: Blizard Institute

institute: Queen Mary University of London

summary: Polycomb repressive complexes 1 and 2 (PRC1/2) act to limit transcriptional activity at gene promoters. One of the mechanisms by which they are proposed to achieve this is through the compaction of nucleosomal chromatin. However, this model is based largely upon in vitro studies and has yet to be explored sufficiently in living cells. We therefore characterised the chromatin accessibility of Polycomb-occupied gene promoters using assay for transposase accessible chromatin (ATAC-seq). Although Polycomb-occupied promoters exist in a less accessible state compared to Polycomb-free promoters, we were surprised to observe that deletion of either PRC1, PRC2 or both PRC1/2 did not result in any increases in chromatin accessibility. This was in contrast to widespread increases in the transcriptional activity of Polycomb target genes. Together with experiments examining chromatin accessibility in the context of RNA polymerase II inhibition, we therefore demonstrate an apparent uncoupling of the chromatin accessibility and transcriptional activity of gene promoters.

overall_design: Ring1a-/-;Ring1bfl/fl mouse embryonic fibroblasts were profiled for chromatin accessibility using ATAC-seq with and without tamoxifen treatment for 96 hours.

first: Robert

last: Klose


title: Genome-wide mRNA and miRNA expression data from different populations of human thymic epithelial cells (hTECs) compared to human epidermal derived keratinocytes

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


title: Chromatin accessibility at Polycomb target genes is independent of PRC1 and PRC2 catalytic core complexes (ESC Mnase-seq)

overall_design: Ring1a-/-;Ring1bfl/fl mouse embryonic stem cells were profiled for nucleosome occupancy and positioning using MNase-seq.


title: Expression data of LUAD cell line PC9 treated with 2 uM HCI-2509 for 48 hours

status: Public on Jul 27 2018

city: Cologne

first: Iris

middle: Felicitas

last: Macheleidt

address: Kerpenerstr. 63

department: Institute for Pathology

institute: University Hopsital Cologne

summary:
The epigenetic regulator LSD1 is overepxpressed in lung adenocarcinoma (LUAD). HCI-2509 a LSD1 inhibitor leads to growth arrest in in vitro tumor models.
To identify the underlying molecular mechanims behind LSD1 overexpression, we examined the gene expression patterns using microarray in the LUAD cell line PC9 after HCI-2509 treatment

overall_design: PC9 cells were treated with 2 µM HCI-2509 for 48 h. RNA was exctraced using the automated isolation kit AS-1280 (Promega) in the Maxwell-16 (Promega) with subsequent microarray hybridization.

first: Priya

last: Dalvi

first: Margarete

last: Odenthal


title: An atypical parvovirus driving chronic tubulointerstitial nephropathy and kidney fibrosis

city: Newtown

first: Ben

last: Roediger

state: NSW

address: Locked Bag No. 6

institute: Centenary Institute

summary: We have performed RNA sequencing on kidneys from inclusion body nephropathy-affected mice and compared the data to healthy, uninfected controls. Using a metagenomics approach, we report the identification of the disease causing agent as an atypical virus, mouse kidney parvovirus (MKPV), belonging to a divergent genus of the Parvoviridae. The RNA sequencing also enabled us to assess the host response to MKPV-infection.

overall_design: Examination of whole kidney transcriptomes from 2 sick and 2 healthy mice. BioProject ID: PRJNA482006. SRA accession no: SRP154484

first: Shweta

last: Tikoo

first: Babak

last: Shaban


title: Circular DNA tumor viruses make circular RNAs

city: Pittsburgh

first: Tuna

last: Toptan

address: 5117 Centre Ave, Research Pav. Suite 1.8

department: Cancer Virology Program

institute: Hillman Cancer Center

summary: Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) cause ~2% of all  human cancers. RNase R-resistant RNA sequencing revealed that both  gammaherpesviruses encode multiple, uniquely stable, circular RNAs (circRNA). EBV  abundantly expressed both exon-only and exon-intron circRNAs from the BART locus  (circBARTs) formed from a spliced BART transcript and excluding the EBV miRNA  region. CircBARTs were expressed in all verified EBV latency types, including EBV-  positive post-transplant lymphoproliferative disease (PTLD), Burkitt lymphoma,  nasopharyngeal carcinoma, and AIDS-associated lymphoma tissues and cell lines. Only  cells infected with the B95-8 EBV strain, with a 12-kb BART locus deletion, were  negative for EBV circBARTs. Less abundant levels of EBV circRNAs originating from  LMP2 and BHLF1-encoding genes were also identified. CircRNA sequencing of KSHV-  infected PEL cells revealed a KSHV-encoded circRNA from the vIRF4 locus (circvIRF4)  that was constitutively expressed. In addition, KSHV polyadenylated nuclear (PAN) RNA  locus generated a swarm (>100) of multiply backspliced, low-abundance RNase R-  resistant circRNAs originating in both sense and antisense directions consistent with a  novel hyper-backsplicing mechanism. In EBV and KSHV co-infected cells, exon-only  EBV circBARTS were located more in the cytoplasm, whereas the intron-retaining  circBARTs were found in the nuclear fraction. KSHV circvIRF4 and circPANs were  detected in both nuclear and cytoplasmic fractions. Among viral circRNAs tested, none  were found in polysome fractions from KSHV-EBV co-infected BC1 cells although low  abundance protein translation from viral circRNAs could not be excluded. CircRNAs are  a new class of viral transcripts expressed in gammaherpesvirus-related tumors that  might contribute to viral oncogenesis.

overall_design: Ribodepleted, RnaseR treated RNA from EBV/KSHV positive or negative tissue or cell lines was used for RNA sequencing

first: Patrick

last: Moore


title: Chemotherapy recurrence colon cancer spheroid L145

city: Amsterdam

first: Richard

last: Volckmann

state: Noord-Holland

address: van der Boechorststraat 6a

department: Laboratory of Experimental Oncology and Radiobiology

country: Netherlands

institute: msterdam UMC and University of Amsterdam

summary: Gene expression changes in colon cancer spheroids induced by chemotherapy.

overall_design: Patient-derived colonospheres were exposed to six cycles of 5-fluorouracil (5-FU). RNA was isolated from colonospheres prior to treatment (pre-treatment, n=5), from the surviving tumor cells after the last cycle (post-treatment, n=5) and from cells recovered after therapy (recurrence, n=5).

first: K

last: Trumpi

first: I

last: Ubink

first: O

last: Kranenburg


title: Single cell transcriptomic profiling provides insight into maturational and developmental status of pluripotent stem cell-derived erythroblasts

city: Boston

first: George

last: Murphy

state: MA

address: 670 Albany Street

institute: Center for Regenerative Medicine of Boston University and Boston Medical Center

summary: Robust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) would increase the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line through the insertion of a GFP cassette after the endogenous β-globin promoter, allowing for the mapping of β-globin expression throughout erythroid development in real time at single cell resolution. Sorting live GFP+ and GFP- cells at the most mature stage of erythroid differentiation, followed by single cell RNA sequencing (scRNAseq), identifies features that distinguish GFP- from GFP+ β-globin expressing cells and allows for the dissection of the developmental and maturational status of iPSC-derived erythroid cells. Co-expression of embryonic, fetal and adult globins in individual cells indicates a yolk sac erythro-myeloid progenitor (EMP) stage of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identifies a gradient of erythroid maturation with GFP+ β-globin expressing cells showing increased maturation. In addition, scRNAseq analysis reveals that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.

overall_design: Single cell transcriptomic profiling of d29 iPSC-derived erythroid cells sorted for β-globin expression (GFP+ versus GFP-).  The β-globin reporter iPSC line has an insertion of a GFP cassette after the endogenous β-globin promoter.

first: Kim

last: Vanuytsel

first: Taylor

last: Matte


title: White blood cells from rheumatoid arthritis patients and matched healthy donors

first: CSL

last: Research

state: VIC

address: 30 Flemington Rd

institute: CSL Limited / bio21 Institute

summary: The experiment was designed to assess gene expression differences between rheumatoid arthritis patient and healthy donor white blood cells.

overall_design: White blood cells were collected from peripheral blood (PB) of 50 RA donors and 50 age, gender and ethinicity matched healthy donors to compare whole blood transcriptomes from RA patients to healthy individuals

first: Gabrielle

last: Goldberg

first: Milica

last: Ng

first: Simon

last: Chatfield

first: Jane

first: Damian

last: D’Silva

first: Ee

last: Pang

first: Yunshun

last: Chen

first: Monther

last: Alhamdoosh

first: Nicholas

last: Wilson

first: Ian

last: Wicks


title: JAG2 signaling induces differentiation of CD14+ monocytes into Langerhans Cell Histiocytosis-like cells

status: Public on Jul 29 2018

city: Vienna

first: Maximilian

last: Kauer

address: Zimmermannplatz 10

department: Molecular Biology

country: Austria

institute: CCRI, St.Anna Children Cancer Research Institute

summary: This study aims to identify the cell of origin of Langerhans Cell Histiocytosis and the molecular mechanisms of its pathogenesis.

overall_design:
Primary cells were isolated from peripheral blood (CD14 monocytes, CD1c DCs) and treated with JAG2, IgG or IL4
6 different conditions: CD14 and CD1c treated with IgG, JAG2 or IL4

first: Raphaela

last: Schwentner

first: Gunhild

last: Jug

first: Thomas

last: Schnöller

first: Petra

last: Waidhofer-Söllner

first: Wolfgang

last: Holter

first: Caroline

last: Hutter


title: miRNA-Seq identifies a serum miRNA panel which can detect and monitor liver disease in pediatric cystic fibrosis

city: Brisbane

first: Diego

last: Calvopina

state: QLD

address: 300 Herston Rd

department: Hepatic Fibrosis

institute: QIMR-Berghofer

type: Non-coding RNA profiling by high throughput sequencing

summary:
Purpose: Cystic fibrosis (CF)-associated liver disease (CFLD) is a hepatobiliary complication of CF. Current diagnostic modalities rely on non-specific assessments, while liver biopsy is the gold standard to assess severity of disease. Using miRNA-sequencing we investigated the use of serum miRNA to diagnose and monitor CFLD progression in children with CF.
Methods: This was a cross-sectional cohort study of the circulatory miRNA signature of 90 children grouped by clinical, biochemical and imaging assessments as follows: healthy controls (n=30), CF patients with no evidence of liver disease (CFnoLD, n=30) and CFLD (n=30). Serum miRNAs were analysed by miRNA-sequencing. Following total circulatory RNA extraction, RNA samples were pooled together to create three libraries (i.e., 10 patients/library) per study cohort.  Libraries were prepared using the Ion Total RNA-Seq Kit v2 protocol (Thermo Fisher, Revision E) and sequenced on the Ion Proton System, using the Ion PI Chip v3 following the Ion PI Hi-Q Sequencing 200 Kit protocol (Thermo Fisher, Revision C).  Selected differentially expressed serum miRNA candidates were further validated in 124 individual children serum samples by qRT-PCR and statistical analysis performed to evaluate utility to predict CFLD and hepatic fibrosis severity validated by liver biopsy.
Results: The nine libraries  sequenced by the Ion Proton System produced 64,658,545 total reads. During processing of the sequencing data, we noticed significant low counts mapped from the CFnoLD library 3. Following principal component analysis this library was confirmed to be an outlier and excluded from the analysis. For the remaining libraries, initial threshold filtering was performed by discarding miRNAs with 10 or less counts and normalized for compositional bias in sequenced libraries and library size. After threshold filtering, 659 miRNAs were identified with a minimum average count of 10.

overall_design: Serum miRNA profiles of healthy controls (n=30), CF no liver disease (n=30) and CF liver disease (n=30) children were generated by miRNA-sequencing, using the Ion Torrent Proton System.

first: Grant

last: Ramm


title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

city: NEW YORK

first: NADEJDA

last: TSANKOVA

state: NEW YORK

address: 1425 MADISON AVENUE 9TH FLOOR

department: PATHOLOGY

institute: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI

summary: The intrinsic drivers of migration in glioblastoma (GBM) are poorly understood. To better capture the native molecular imprint of GBM and its developmental context, we isolate human stem cell populations from GBM (GSC) and germinal matrix tissues and map their chromatin accessibility via ATAC-seq. We uncover two distinct regulatory GSC signatures, a developmentally-shared/proliferative and a tumor-specific/migratory one in which TEAD1/4 motifs are uniquely overrepresented. Using ChIP-PCR we validate TEAD1 trans occupancy at accessibility sites within EGFR, AQP4, and CDH4. To further characterize TEAD’s functional role in GBM, we knockout TEAD1 or TEAD4 in patient-derived GBM lines using CRISPR-Cas9. TEAD1 ablation robustly diminishes migration, both in vitro and in vivo, and alters migratory and EMT transcriptome signatures with consistent downregulation of its target AQP4. TEAD1 overexpression restores AQP4 expression, and both TEAD1 and AQP4 overexpression rescue migratory deficits in TEAD1-knockout cells, implicating a direct regulatory role for TEAD1-AQP4 in GBM migration.

overall_design:
Open chromatin areas were analyzed by ATAC-seq in stem cells from fetal germinal matrix (GM) and from human glioblastoma (GBM) in biological replicates (n ≥ 3).
Changes in transcriptome after ablation of TEAD1 and TEAD4 was analyzed by RNA-seq in TEAD1 and TEAD4 CRISPR-KO different GBM patient-derived cell lines (n = 4), as well as in TEAD1 KO gliomaspheres and compared to their corresponding SHAM lines.

first: Jessica

last: Tome-Garcia

first: Parsa

last: Erfani

first: German

last: Nudelman

first: Alexander

last: Tsankov

first: Igor

last: Katsyv

first: Rut

last: Tejero-Villalba

first: Bin

last: Zhang

first: Martin

last: Walsh

first: Roland

middle: H

last: Friedel

first: Elena

last: Zaslavsky

first: Nadejda

last: Tsankova


title: Inhibition of casein kinase 2 disrupts differentiation of myeloid cells in cancer and enhances the efficacy of immunotherapy in mice

city: Philadelphia

first: Priyankara

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: The role of myeloid cells as regulators of tumor progression that significantly impact the efficacy of cancer immunotherapies makes them an attractive target for inhibition. Here we explore the effect of a novel, potent, and selective inhibitor of serine/threonine protein kinase CK2 on modulating myeloid cells in the tumor microenvironment. Although inhibition of CK2 caused only a modest effect on dendritic cells in tumor-bearing mice, it substantially reduced the amount of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and tumor-associated macrophages (TAM). This effect was not caused by the induction of apoptosis, but rather by a block of differentiation. Our results implicated downregulation of CCAAT-enhancer binding protein-α (C/EBPα) in this effect. Although CK2 inhibition did not directly affect tumor cells, it dramatically enhanced the antitumor activity of immune checkpoint receptor blockade using anti-CTLA-4 antibody. These results suggest a potential role of CK2 inhibitors in combination therapies against cancer.

overall_design: Untreated and CK2 inhibitor treated hematopoietic progenitor cells cells assayed by RNA-seq

first: Dmitry

last: Gabrilovich

first: Ayumi

last: Hashimoto


title: Regulator of Ty1 transposition protein 105 (Rtt105) is an RPA chaperone and is required for genome stability maintenance

city: Beijing

first: Qing

last: Li

address: No.5 Yiheyuan Road Haidian District

department: School of Life Science

institute: Peking University

summary: Single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) is essential for protecting ssDNA at replication forks. However, how RPA is guided to DNA replication forks remains unclear. Here, we show that Regulator of Ty1 transposition protein 105 (Rtt105) is an RPA-binding protein and is required for the binding of RPA at replication forks. Cells lacking Rtt105 exhibit extensive genome instability and severe defects in DNA replication. Both the nuclear import of RPA and the extent of genome-wide binding of RPA at replication forks were greatly compromised in rtt105 mutant cells. Mechanistically, Rtt105 is required for the interaction of RPA with Kap95, an importin protein known to be essential for RPA’s nuclear import. Remarkably, Rtt105 strongly enhances RPA binding with ssDNA substrates in vitro. We therefore propose a model in which Rtt105 functions as a RPA chaperone that both escorts RPA during nuclear important and delivers RPA to replication forks.

overall_design: To examine strand bias of Rfa1 in WT and rtt105 deletion mutant cells, we used Enrichment and Sequencing of Protein Associated Nascent DNA (eSPAN) (Yu et al., 2014) for cells at G1 phase and S phase.

first: Shuqi

first: Jiawei

last: Xu

first: Zhiyun

first: Chuanhe

last: Yu

first: Pu

last: Zheng

first: Haiyun

last: Gan

first: Andrew

last: Chapes

first: Di

first: Kuiming

last: Chan

first: Jianxun

last: Feng


title: Next Generation Sequencing Facilitates Quantitative Analysis Of Wild Type And Epithelium-specific Cox10-/- Renal Transcriptomes

city: Cambridge

first: Jea Hyun

last: Baek

address: 225 Binney Street

institute: Biogen

summary:
Description: We find multiple evidence that mitochondria are directly and indirectly involved in the pathologic activation of cellular innate immunity. Previously, studies have shown that pathologic type I interferon signatures promote autoimmune diseases and noted mitochondrial and mtDNA dysregulation in chronic inflammatory diseases. Based upon previous data from our lab and others, we anticipated a direct link between the mitochondrial dysfunction and the elevated production of type I interferon or the increase of other innate immune signatures in chronic and general inflammatory diseases. In this project, we wished to test our overall hypothesis that inhibition of mtDNA-mediated immune activation pathway is a promising pharmacological target for chronic inflammatory diseases. To pursue this goal, we developed a murine conditional knockout model, where kidney epithelial cells show dysfunctioning mitochondrial phenotype, and verified that this mouse model has lethal kidney failure with features of chronic kidney disease. By using systems biology approaches, we aimed to examine which genes are regulated in the kidney with dysfunctioning mitochondrial phenotype and identify the main pathways leading to the chronic autoimmune disease.
Results: We found that interferon-stimulated genes are upregulated in Cox10 mouse kidney. Likely, mitochondrial dysregulation and mtDNA release induces expression of genes known as interferon-stimulated genes (IGS) (West et al., Nature 2015).

overall_design: Renal mRNA profiles of 3-month old wild type (WT) and epithelial-specific Cox10-/- mice were generated by deep sequencing, in quadruplicate, on the Illumina HiSeq 2500 platform.

first: Jea-Hyun

first: Patrick

last: Cullen


title: RNAseq data of two week-old soybean leaves response to cold stress

city: Tottori

first: Yuji

last: Yamasaki

address: 1390 Hamasaka

institute: Tottori University

summary: Transcriptome analysis of cold-treated leaves (unifoliates) of soybean seedlings were performed. RNAseq analysis was performed using two lanes on a Illumina HiSeq2000 and sequenced on a 100bp, paired-end run.

overall_design: Two-weeks old soybean (c.v. ‘Williams 82’) seedlings were cold-treated (4 °C) starting at 4 h after the lights turned on (Zeitgeber Time, ZT4 h, 18 hours light/6 hours dark) and maintaining 4 °C  continuously with the light cycle till harvest time (0, 1, and 24 hours). All treatment samples were performed in triplicate (with n≥6 plants per replication).

first: Stephen

middle: K

last: Randall


title: An atlas of genetic variation for linking pathogen-induced cellular traits to human disease

status: Public on Jul 26 2018

city: Durham

first: Liuyang

last: Wang

state: NC

address: 0049 CARL Building

institute: Duke University

summary: Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with disease. To facilitate moving from associations to disease mechanisms, we leveraged the role of pathogens in shaping human evolution with the Hi-HOST Phenome Project (H2P2): a catalog of cellular GWAS comprised of 79 phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance (p<5x10-8) for phenotypes ranging from pathogen replication to cytokine production. Combining H2P2 with clinical association data on 83,717 patients from the eMERGE Network and experimental validation revealed evidence for mechanisms and connections with diseases. We identified a SNP near CXCL10 as a cis-cytokine-QTL and risk factor for inflammatory bowel disease. A SNP in ZBTB20 demonstrated pleiotropy, likely through suppression of multiple target genes, and was associated with viral hepatitis. Data are in an H2P2 web portal to facilitate interpreting human genome variation through the lens of cell biology.

overall_design: Three human Huh7s' mRNA profiles from non-target, ZBTB20 siRNA Chlamydia infection were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000/2500 platform.

first: Kelly

last: Pittman

first: Jeffrey

last: Barker

first: Raul

middle: E

last: Salinas

first: Ian

last: Stanaway

first: Graham

middle: D

last: Williams

first: Robert

last: Carroll

first: Tom

last: Balmat

first: Andy

last: Ingham

first: Anusha

last: Gopalakrishnan

first: Kyle

last: Gibbs

first: Alejandro

last: Antonia

first: Joseph

last: Heitman

first: Soo

last: Lee

first: Gail

middle: P

last: Jarvick

first: Joshua

last: Denny

first: Stacy

last: Horner

first: Mark

last: Delong

first: Raphael

last: Valdivia

first: David

last: Crosslin

first: Dennis

last: Ko


title: Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones

city: Birmingham

first: Peter

last: Keane

address: Vincent Drive

department: Institute for Cancer and Genomic Sciences

institute: University of Birmingham

summary: Intra-tumour heterogeneity caused by clonal evolution is a major problem in cancer treatment stratification. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane (PM) proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth and engraftment behavior, as determined by RNA sequencing, DNaseI hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.

overall_design: DNaseI-Hypersensitivity profiles for 8 AML subclones isolated from 4 patients

first: Bauke

last: De Boer

first: Janine

last: Prick

first: Maurien

last: Pruis

first: Maria

last: Imperato

first: Jennifer

last: Jaques

first: Annet

middle: Z

last: Brouwers-Vos

first: Shanna

middle: M

last: Hogeling

first: Carolien

last: Woolthuis

first: Marije

last: Nijk

first: Arjan

last: Diepstra

first: Sebastian

last: Wandinger

first: Matthias

last: Versele

first: Ricardo

last: Attar

middle: N

last: Cockerill

first: Gerwin

last: Huls

first: Edo

last: Vellenga

first: Andre

last: Mulder

first: Constanze

last: Bonifer

first: Jan

middle: J

last: Schuringa


title: Schizosaccharomyces pombe Pol II Transcription Elongation Factor ELL Functions as Part of a Rudimentary Super Elongation Complex

city: Kansas City

first: Dana

last: Gibbon

state: MO

institute: Stowers Institute for Medical Research

summary: ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause.  In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including S. pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes.  In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC.  This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.

overall_design: All experminents were performed in duplicates. For Ell1, Eaf1, and Ebp1 ChIP experiments, ell1Δ, eaf1Δ and ebp1Δ strains were used as controls.

first: Sneha

last: Gopalan

first: Ronald

middle: C

last: Conaway

first: Joan


title: Transcriptional effects of the BAF inhibitor BRD-K98645985 on mouse embryonic stem cells

city: Bethesda

first: Benjamin

last: Carter

state: MD

address: 10 Center Drive

department: NHLBI

institute: National Institutes of Health

summary: We used RNA-seq to characterize the effects of the inhibitor on gene expression in mouse embryonic stem cells (ESCs).

overall_design: RNA was purified from ESCs treated with BAF inhibitor or a DMSO mock treatment. cDNA libraries were sequenced using Illumina HiSeq technology. Differentially expressed genes were identified using DESeq2.

first: Emily

last: Dykhuizen

first: Basudev

last: Chowdhury


title: Analysis of gene expression heteroegeneity in adult undifferentiated spermatogonia [single-cell RNA-Seq]

city: CLAYTON

first: Robin

middle: Mark

last: Hobbs

address: 27-31 Wright Street

department: Centre for Reproductive Health

institute: Hudson Institute of Medical Research

summary: The undifferentiated spermatogonial population of mouse testis is functionally heterogeneous and contains stem cells and committed progenitor cells. However, gene expression patterns marking these distinct cell fractions are poorly defined. Therefore, our aim was to profile gene expression of undifferentiated cells by RNA-Seq at the single cell level. Undifferentiated cells were therefore isolated from adult mouse testes based on expression of a Plzf/Zbtb16 gene reporter, which marks both stem and progenitor spermatogonia. Isolated cells were then processed using the 10X Chromium system and analysed by RNA-Seq. Our goal was to characterize distinct subsets of undifferentiated spermatogonia based on gene expression patterns.

overall_design: Undifferentiated spermatogonia were independently isolated from 2 adult Plzf-mCherry/CreER reporter adult mice and samples processed independently.

first: Hue

last: La

first: Juho-Antti

last: Mäkelä

first: Ai-Leen

last: Chan

first: Fernando

last: Rossello

first: Christian

last: Nefzger

first: Julien

last: Legrand

first: Mia

last: De Seram

first: Jose

last: Polo


title: Precise and Predictable CRISPR Chromosomal Rearrangement Editing Reveals Principles of Cas9-mediated Nucleotide Insertion

city: Shanghai

first: Jia

last: Shou

state: Shanghai

address: 800 Dongchuan Rd

department: Institute of Systems Biomedicine

institute: Shanghai Jiao Tong University

summary: Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate structural genome variations and developmental gene regulation, but little is known about the underlying mechanism. Here we report that disrupting CtIP or FANCD2, which is thought to function in NHEJ, enhances precise DNA-fragment deletion. In addition, by analyzing the inserted nucleotides at the junctions of DNA-fragment deletions, inversions, duplications, and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of -3 position of the PAM site in vivo and in vitro, generating overhanged DSB ends. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations. These findings have important implications regarding 3D chromatin folding and enhancer insulation during gene regulation.

overall_design: HEK293T expression profiles of CtIP knockout

first: Jinhuan

last: Li

first: Yingbin

last: Liu

first: Qiang

last: Wu


title: DNA methylation analysis for target regions in swim-up sperm and HRCS.

city: Fukuoka

first: Hiromitsu

last: Araki

address: 744 Motooka Nishi-Ku

institute: Kyushu University

type: Methylation profiling by high throughput sequencing

summary: Majority of histones are replaced by protamine during spermatogenesis, but small amount of histones are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important how histones are distributed on sperm genome. We have found that the swim-up sperm used in many studies contains about 10% of immature sperm which do not yet complete the histone-to-protamine replacement. We have developed the novel method to purify the histone replacement-completed sperm (HRCS), and to completely solubilize nucleosomes from cross-linked HRCS without MNase digestion. Here, we analyzed H3 binding profiles in total sperm and HRCS by Chromatin IP – sequencing (ChIP-seq) analysis, and DNA methylation profiles in swim-up sperm and HRCS by target bisulfite sequencing (TGSB).

overall_design: Examination of DNA methylation status in 2 fractions of sperm samples.

first: Keisuke

last: Yoshida

first: Fumihito

last: Miura

first: Takashi

last: Ito


city: La Jolla

first: Mariana

last: Babor

address: 9420 Athena Cir

institute: La Jolla Institute for Allergy and Immunology

summary:
In the mid-1990s vaccine-related side effects prompted the replacement of the whole Pertussis (wP) by a new acellular Pertussis vaccine (aP). Unexpectedly, whooping cough cases have recently increased, particularly in teenagers. Here we compared individuals born before 1995 and vaccinated in infancy with wP, with individuals born in 1996 or later and vaccinated with aP in infancy. Remarkably, we detected differences in the two populations in response to a contemporary aP booster, despite the fact that the original aP or wP priming occurred more than 18 years previously. The nature of the differences in T cells between aP and wP original priming was further investigated by evaluation of memory subset composition in respect to function, activation and exhaustion expression protein markers and RNA transcriptomic profiles of the PT-specific T cells from aP vs. wP donors.
Transcriptomic profile of circulating CD4+ T cells from TCM and TEM  memory compartments from donors vaccinated at birth either with whole or acellular Pertussis vaccine. All donors were boosted with the aP vaccine  1-3 months before the PBMCs were collected.

overall_design: RNA-sequencing of sorted CD3+CD4+ cells from central (TCM) or effector (TEM) memory  compartments from cryopreserved PBMC of 8 subjects vaccinated at birth with whole Pertussis vaccine and  8 subjects vaccinated at birth with acellular Pertussis vaccine. All donors were boosted with the aP vaccine  1-3 months before the PBMCs were collected. Stimulation with either Pertussis (PT) or Tetanus (TT) petides with a 24h AIM assay were used to capture and sort the antigen-specific cells (average of 10,000 cells per sample).

first: Ricardo

last: Da Silva Antunes

first: Gregory

last: Seumois

first: Zheng

last: Fu

first: Jason

last: Greenbaum

first: Bjoern

last: Peters

first: Pandurangan

last: Vijayanand

first: Alessandro

last: Sette


title: RNA-seq for MP, AML and MEFs from mice that carry a "knock-in" allele of the mutant splicing factor, U2af1(S34F), with or without homozygous deletion of the transcription factor, Runx1

city: New York

first: Harold

last: Varmus

state: NY

address: 1300 York Ave

department: Department of Medicine

institute: Weill Cornell Medicine

summary: We have generated mice that carry Cre-dependent "knock-in" alleles (MGS34F and IES34F) of U2af1(S34F), a mutation commonly encountered in human myelodysplastic syndromes and acute myeloid leukemia (AML). MEFs were generated from the progenies of these mice with those carrying a UBC-CreERT2 transgene, and were treated with 4-Hydroxytamoxifen (4OHT) to induce Cre-mediated recombination and subsequent U2af1(S34F) expression. Mice carrying the MGS34F allele were further crossed with those carrying a floxed Runx1 allele and Mx1-Cre. U2af1(S34F) expression and Runx1 deletion caused changes in gene expression and RNA splicing. Mice with a conditional "knock-in" U2af1(S34F) allele and conditional "knockout" alleles of Runx1 were mutagenized with low-dose N-Ethyl-N-Nitrosourea (ENU). Within 1.5 year, one of fourteen ENU-treated mice with U2af1(S34F) and Runx1 deletion developed AML, as did recipients of allografted splenic cells from two other such mice, but AML did not arise from cells with other genotypes or mice without ENU treatment.

overall_design: mRNA sequencing for MEFs, MPs, and AML samples of the indicated genotypes in mice

first: Dennis

middle: L

last: Fei

first: Tao

last: Zhen

first: Tuo

last: Zhang

first: Robert

last: Bradley

first: Paul


title: Activation of Th1 Immunity Within the Tumor Microenvironment is Associated with Clinical Response to Lenalidomide in Chronic Lymphocytic Leukemia

status: Public on Jul 25 2018

first: Delong

last: Liu

institute: NHLBI/NIH

summary: Immune stimulation contributes to lenalidomide’s anti-tumor activity. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature, autoreactive B cells in secondary lymphoid tissues, blood and bone marrow and progressive immune dysfunction. Previous studies in CLL indicated that lenalidomide can repair defective T-cell function in vitro. Whether T-cell activation is required for clinical response to lenalidomide remains unclear. Here we report changes in the immune microenvironment in patients with CLL treated with single-agent lenalidomide and associate the immunologic effects of lenalidomide with anti-tumor response. Within days of starting lenalidomide, CD3+ cells increased in the tumor microenvironment and showed Th1-type polarization. Gene expression profiling of pre-treatment and on-treatment lymph node biopsies revealed upregulation of IFN and many of its target genes in response to lenalidomide. The IFNγ-mediated Th1 response was limited to patients achieving a clinical response defined by a reduction in lymphadenopathy. Deep sequencing of T-cell receptor genes revealed decreasing diversity of the T-cell repertoire and an expansion of select clonotypes in responders. To validate our observations, we stimulated T cells and CLL cells with lenalidomide in culture and detected lenalidomide-dependent increases in T-cell proliferation. Taken together, our data demonstrate that lenalidomide induced Th1 immunity in the lymph node that is associated with clinical response.

overall_design: Patients with relapsed CLL were enrolled on a phase 2 study of lenalidomide at 10 mg or 20 mg daily for 3 weeks followed by 3 weeks off for up to 8 cycles.  Blood and lyphm node samples were collected from the patients prior treatment and treated on day 8 for studying change in expression of genes in blood and lymph nodes.

first: Georg

last: Aue

first: Clare

last: Sun

first: Adrian

last: Wiestner


title: RNA-seq of PC9 cells tolerant to gefitinib

city: Richmond

first: Timothy

last: Lochmann

state: VA

address: 521 N 11th St

department: OCMB

institute: Virginia Commonwealth University

overall_design:
The NSCLC cell line PC9 was made tolerant to gefitinib over 6-days. Replicates were performed at a minimum of duplicates.
EGFR inhibitors (EGFRi) are effective against EGFR mutant lung cancers. The efficacy of these drugs however is mitigated by the outgrowth of resistant cells, most often driven by a secondary acquired mutation in EGFR, T790M. We recently demonstrated that T790M can arise de novo during treatment (Hata et al., Nature Medicine 2016); it follows that one potential therapeutic strategy to thwart resistance would be identifying and eliminating these cells (referred to as drug tolerant cells (DTCs)) prior to acquiring secondary mutations like T790M. We have developed DTCs to EGFRi in EGFR mutant lung cancer cell lines. Subsequent analyses of DTCs included RNA-seq, high-content microscopy, and protein translational assays. Based on these results, we tested the ability of MCL-1 BH3 mimetics to combine with EGFR inhibitors to eliminate DTCs and shrink EGFR mutant lung cancer tumors in vivo.

first: Lochmann

last: TL

first: Faber

last: AC


title: Transcriptional changes in mouse models in SCA3

city: ANN ARBOR

first: Yao

last: Xiao

state: MICHIGAN

address: 100 Washtenaw Ave

institute: UNIVERSITY OF MICHIGAN

summary: The study examined early transcriptional changes in the brain of different mouse models of spinocerebellar ataxia type 3, a dominantly-inherited neurodegenerative disease caused by a CAG repeat expansion in the ATXN3 gene. The goal was to identify early transcriptional signatures that are strongly associated with the accumulation and aggregation of the disease protein, ataxin-3, in the brain. The study also investigated the extent to which the observed transcriptional changes might be contributors to disease pathogenesis.

overall_design: The overall study includes multiple different RNA-seq runs utilizing wild-type, two different knock-in mouse models of SCA3 (a traditional and variant), two different transgenic models of SCA3 (Q84 and Q15), and an ataxin-3 knock-out mouse. In total, 19 wild-type mice, 13 homozygous variant knock-in mice, 6 heterozygous variant knock-in mice, 4 traditional homozygous knock-in mice, 4 traditional heterozygous knock-in mice, 4 Q84 transgenic mice, 4 Q15 transgenic mice, and 3 ataxin-3 knock-out mice. The majority of the study examined the pons of the mice, and with one smaller run examining the deep cerebellar nuclei of wild-type and variant homozygous knock-in mice (n=3 each).

first: Yuanfang

last: Guan

first: Bharat

last: Panwar

first: Biswarathan

last: Ramani


title: Inflammation induced by influenza virus impairs innate control of human pneumococcal carriage

city: Liverpool

first: Simon

last: Jochems

address: Pembroke Place

institute: Liverpool School of Tropical Medicine

summary: Secondary bacterial pneumonia following influenza infection is a significant cause of mortality worldwide. Upper respiratory tract pneumococcal carriage is important as both determinants of disease and population transmission. The immunological mechanisms that contain pneumococcal carriage are well-studied in mice but remain unclear in humans. Loss of this control of carriage following influenza infection is associated with secondary bacterial pneumonia during seasonal and pandemic outbreaks. We used a human type 6B pneumococcal challenge model to show that carriage acquisition induces early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function associated with clearance of pneumococcal carriage. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate function and altered genome-wide nasal gene responses to pneumococcal carriage. Levels of the cytokine IP-10 promoted by viral infection at the time of pneumococcal encounter was positively associated with bacterial density. These findings provide novel insights in nasal immunity to pneumococcus and viral-bacterial interactions during co-infection.

overall_design: 96 nasal samples from healthy volunteers experimentally challenged with pneumococcus, 3  days after receiving live attenuated influenza vaccine or tetravalent inactivated influenza vaccine underwent RNA-Sequencing. Nasal cells were collected at baseline (D-4) before vaccination, and at 5 days after vaccination (or 2 days after pneumococcal inoculation, D+2) and at 12 days after vaccination (or 9 days after pneumoocccal inoculation, D9)

first: Fernando

last: Marcon


city: Rochester

first: Xuewei

last: Wang

address: 200 1st street

institute: Mayo Clinic

summary: Alzheimer’s disease (AD) is an age-associated neurodegenerative disease characterized by amyloidosis, tauopathy, and activation of microglia, the brain resident innate immune cells. We show that a RiboTag translational profiling approach can bypass biases due to cellular enrichment/cell sorting. In our recent study entitled “Microglial translational profiling reveals a convergent APOE pathway from aging, amyloid, and tau”, we utilized data acquired using this approach in models of amyloidosis, tauopathy, and aging, to reveal a common set of alterations and identified a central APOE-driven network that converged on CCL3 and CCL4 across all conditions. Notably, examination of the aged female dataset demonstrated a significant exacerbation of many of these shared transcripts in this APOE network, revealing a potential mechanism for increased AD susceptibility in females. This study has broad implications for microglial transcriptomic approaches and provides new insights into microglial pathways associated with different pathological aspects of aging and AD.

overall_design: Microglial transcriptomes from the forebrain were generated using RNAseq using an Illumina HiSeq 4000 were done for N=3-5 animals per condition in the context of aging, amyloidosis, tauopathy, and systemic inflammation (i.e. LPS or Poly(I:C) stimulation) in RiboTag mice. In addition, a cellular isolation procedure using enzymatic digestion and cell sorting was also used to compare this protocol versus RiboTag isolation during basal conditions and LPS induced inflammation.

first: John

last: Fryer

first: Silvia

last: Kang


title: Osteoblastic loss of Ezh2

first: Asha

last: Nair

address: 200 1ST ST SW

summary: Osteblastic loss of Ezh2 was achieved by breeding Ezh2flox/flox (Su et al., PMID: 12496962) with Osx-Cre (Rodda et al., PMID:16854976).

overall_design: Calvaria were dissected out from 2 to 3 day old wild type (WT, Ezh2_WT_Osx) and Ezh2 conditional knockout (Ezh2 cKO, Ezh2_cKO-Osx), washed in PBS, digested for 20 minutes in collagenase digestion medium (αMEM, 2mg/ml Collagenase type II, and 0.005% trypsin) at 37oC, washed in PBS, and snap frozen with liquid nitrogen and stored at -80oC. RNA was then isolated utilizing Ultra Turrax T25 (IKA) and Direct-zol™ RNA kit (Zymo Research).

first: Amel

last: Dudakovic

first: Andre

last: van Wijnen


title: PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing

first: Jinguo

last: Chen

department: NIAID

institute: NIH

summary:
Background:  Interest in single-cell whole transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works, investigators have used live cells which represent several inconveniences and limitations. Some recent cell fixation methods did not work with most primary cells including immune cells.
Methods:    The methanol-fixation and new processing method was introduced to preserve PBMCs for single-cell RNA sequencing (scRNA-Seq) analysis on 10X Chromium platform.
Results:     When methanol fixation protocol was broken up into three steps, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to three-month storage steps. Resuspension but not rehydration in 3X saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3X SSC were successfully implemented into 10X Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol was validated in CD8+ T cell and several other cell types.
Conclusions:  We expect that the methanol-based cell fixation procedure presented here will substantially enable complex experimental design with primary cells at single cell resolution.

overall_design: single cell RNA sequencing of PBMC, CD8+ T cell or KLM1 cell line after fixation with methonal and resuspension in SSC buffer

first: Foo

last: Cheung

last: Tsang


title: Diet-induced circadian enhancer remodeling synchronizes opposing hepatic lipid metabolic processes

city: Houston

first: Dongyin

state: TX

address: 1 Baylor Plaza, ABBR

institute: Baylor College of Medicine

summary: Overnutrition disrupts circadian rhythms leading to dysregulated metabolism by mechanisms that are not well understood. Here we show that diet-induced obesity (DIO) causes massive remodeling of circadian enhancer activity and gene transcription in mouse liver. Remarkably, DIO triggers synchronous, high amplitude circadian rhythms of both fatty acid (FA) synthesis and oxidation. This gain of circadian rhythmicity in lipid metabolic pathways that oppose each other emphasizes the importance of balance and flux in normal hepatic lipid metabolism. DIO-promoted rhythmicity of Sterol Regulatory Element-Binding Protein (SREBP) activation, which was required not only for the induction of FA synthesis but also, surprisingly, for FA oxidation (FAO).  DIO also brought about a high amplitude circadian rhythm of peroxisome proliferated receptor a (PPARa), which was required for FAO. Provision of a pharmacological ligand for PPARa abrogated the requirement of SREBP for FA oxidation (but not FA synthesis), suggesting that SREBP indirectly controls FA oxidation via production of an endogenous PPARa ligand. Moreover, the high amplitude circadian rhythm of PPARa imparts time-of-day-dependent responsiveness to lipid-lowering drugs. Thus, acquisition of rhythmicity for the non-core clock components PPARa and SREBP1 remodels metabolic gene transcription in response to a challenging nutritive environment and enables a chronopharmacological approach to metabolic disorders.

overall_design: Nascent RNA transcripts in liver of DIO mice (Research Diet D12492) were profiled at 8 time points of the 24 hour light-dark cycle. H3K27ac ChIP-seq in liver of DIO mice were performed at ZT10 and ZT12.

first: Ying

last: Xiong

first: Patricia

last: Borck

first: Cholsoon

last: Jang

first: Paschalis-Thomas

last: Doulias

first: Romeo

last: Papazyan

first: Bin

last: Fang

first: Chunjie

last: Jiang

first: Yuxiang

last: Zhang

first: Erika

middle: R

last: Briggs

first: Wenxiang

last: Hu

first: David

last: Steger

first: Harry

last: Ischiropoulos

first: Joshua

last: Rabinowitz

first: Mitchell

middle: A

last: Lazar


title: Acquisition of a side population fraction through downregulation of MSL3, ZNF691, VPS45, ITGB3BP, TLE2, and ZNF498 augments malignant phenotype in ovarian cancer.

status: Public on Jul 24 2018

city: Kyoto

first: Koji

last: Yamanoi

address: 54 Shogoin Kawahara, Sakyo

institute: Kyoto University Graduate School of Medicine

summary: Side population (SP) cells harbor malignant phenotypes, such as sphere forming capacity, single cell clonogenicity and in vivo tumorigenicity. These malignant phenotypes are related to a poor prognosis for women with ovarian cancer. The aim of this study was to identify key factor(s) that increase the proportion of ovarian cancer SP cells through a functional genomics screen. A library of 81 000 shRNAs targeting 15 000 genes was transfected into CH1 and SKOV3 cells, followed by SP analysis. We found that suppression of MSL3, ZNF691, VPS45, ITGB3BP, TLE2, and ZNF498 markedly increased the proportion of SP cells. Newly generated SP cells exhibit significantly greater capacity for sphere formation, single cell clonogenicity, and in vivo tumorigenicity. On the contrary, overexpression of MSL3, VPS45, ITGB3BP, TLE2, and ZNF498 significantly decreased the proportion of SP cells, sphere formation capacity and single cell clonogenicity. In ovarian cancer cases, low expression of MSL3, ZNF691 and VPS45 was related to poor prognosis. Suppression of these six genes tended to increase some stem cell-related pathways, and significantly enhanced activity of the hedgehog pathway. Cyclopamine, a hedgehog pathway inhibitor, significantly decreased the number of newly generated SP cells and their sphere forming ability. Our results provide new information regarding molecular mechanisms favoring SP cells and suggest that Hedgehog signaling may provide a viable target for improving ovarian cancer survival.

overall_design: CH1 human ovarian cancer cells were transfected with TRC Lentivial shRNA of MSL3, ZNF691, VPS45 and non-silencing control. SKOV3 human ovarian cancer cells were transfected with TRX Lentivial shRNA of ITGB3BP, TLE2, ZNF498 and non-silencing control.

first: Noriomi

last: Matsumura


title: RNA-sequencing of liver samples derived from offspring sired by males chronically exposed to alcohol prior to conception

city: College Station

first: MICHAEL

middle: C.

last: GOLDING

state: Texas

address: 4466 TAMU

department: Physiology

institute: Texas A&M UNiversity

summary: Using an established mouse model of chronic exposure, previous work by our group had linked preconception paternal alcohol use to sex-specific patterns of fetal growth restriction and placental dysfunction. The goal of the present study was to investigate the long-term impact chronic preconception paternal alcohol exposure has on offspring growth and metabolic programming. In the male offspring, we observed increases in both the expression of genes within the pro-fibrotic TGF-ß signaling pathway as well as total levels of cellular hydroxyproline within the liver. Further, we identified suppressed cytokine profiles, which could be linked to the up-regulation of genes in the LiverX/RetinoidX/FarnesoidX Receptor pathways.

overall_design: Postnatal day 90 male mice were provided access to either a solution of 10% (w⁄v) alcohol and 0.066% (w⁄v) Sweet ‘N Low (experimental) or 0.066% (w⁄v) Sweet ‘N Low alone (control) for four hours a day. Once consistent patterns of drinking were established, males were maintained on this protocol for a period of 70 days, which corresponds to the length of approximately two complete spermatogenic cycles. Once the 70-day milestone was surpassed, males were mated to unexposed dams and the offspring evaluated for growth.

first: Michael

last: Golding

first: Richard

last: Chang


title: Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice

city: Philadelphia

first: Colin

last: Conine

state: Pennsylvania

address: 421 Curie Blvd BRB 1213

department: Genetics and Neonatology

institute: UPenn Perelman SOM and CHOP

summary: The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects, but also suppressed the postimplantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm, and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.

overall_design: Zygotes were generated by ICSI from sperm isolated from the testis, caput epididymis, or cauda epididiymis. Following fertilization by ICSI zygotes were developed to different stages of preimplantation development and were harvested for single-embryo RNA-Seq (Smart-Seq 2 protocol). For RNA injection experiments 3 hours after fertilization by ICSI RNA was injected using an Eppendorf Femtojet injection setup.

first: Oliver

last: Rando


title: RNA Sequencing analysis of different genetically characterized lung cancer cell lines

city: Barcelona

first: Anna

last: Esteve-Codina

address: Carrer Baldiri Reixac 4-8

country: Spain

institute: CNAG-CRG

summary: We performed RNA sequencing to assess changes in gene expression in lung cancer cell lines with MET genetic alterations with or without co-occurrence of JAK2 inactivating mutations. Different treatments have been administrated to activate or inhibit selected pathways in order to define MET signature and IFNg (or JAK/STAT) signature.

overall_design: Differential expression analysis of RNA sequencing of 4 different lung cancer cell lines with MET genetic alterations  treated with different treatements to activate or inhibit selected pathways

first: Montserrat

last: Sanchez-Cespedes

first: Maria

last: Saigi-Morgui


title: Gene expression analysis and p53 ChIP-seq in PHA-stimulated human T-lymphocytes

city: RTP

first: NIEHS

last: Microarray Core

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

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

overall_design: Refer to individual Series


title: TGF-β1 negatively regulates cycling short- and long-term hematopoietic stem cells

status: Public on Jul 23 2018

city: Beijing

first: Ji

last: Dong

address: Yiheyuan 5#

institute: Peking University

summary: Transforming growth factor-β (TGF-β) is considered to play a role in the maintenance of quiescent hematopoietic stem cells (HSCs) in vivo. We asked a question of whether TGF-β could be used to control the cell cycle status of HSCs in vitro. To examine the effect of TGF-β on the HSC function, we used in vitro culture system in which HSCs divide with retention of short- and long-term reconstitution ability. Single-cell analyses showed that regardless of its concentrations, TGF-β slowed down cell cycle progression of HSCs, but consequently suppressed the self-renewal potential, particularly in cycling HSCs. This study highlighted a role of TGF-β in the negative regulation of HSC number and function.

overall_design:
Single-cell RNA-sequence analysis was performed on single cells isolated from the culture with SCF+TPO and SCF+TPO+TGF-β1 on day5. Seven or eight cells were isolated from individual wells with or without TGF-β1. A total of 135 single cells were randomly manipulated by mouth-pipette and transferred into cell lysis buffer.
In the attached file "Sample_Barcode_Information.xlsx", the information of all sequenced cellls is listed in sheet1 and barcode information is listed in sheet2. Each cell is named as IVHSC_X(treatment)_X(batch information)_scX(barcode information, see sheet2)

first: Xiaofang

last: Wang

first: Hideo

last: Ema


title: Human bone marrow resident natural killer cells have a unique transcriptional profile and resemble resident memory CD8+ T cells

city: Leiden

first: Janine

last: Melsen

address: Albinusdreef 2

department: Pediatrics

institute: LUMC

summary: Human lymphoid tissues harbor, in addition to CD56bright and CD56dim natural killer (NK) cells, a third NK cell population: CD69+CXCR6+ lymphoid tissue (lt)NK cells. The function and development of ltNK cells remain poorly understood. In this study we performed RNA sequencing on the CD56bright and CD56dim NK cells (from bone marrow and blood), and the ltNK cells (from bone marrow). In addition, the blood derived CD56dim, and bone marrow derived ltNK cells were further subdivided into a NKG2A+ and NKG2A- fraction. Paired blood and bone marrow samples of 4 healthy donors were included. When comparing the NKG2A fractions, only 3 genes (of 9382 genes included) had a significantly differential expression. Therefore, we pooled the expression data proportionally from the NKG2A+ and NKG2A- fractions in subsequent analyses. In ltNK cells, 1353 genes were differentially expressed compared to circulating NK cells. Several molecules involved in migration were downregulated in ltNK cells: S1PR1, SELPLG and CD62L. By flow cytometry we confirmed that the expression profile of adhesion molecules (CD49e-, CD29low, CD81high, CD62L-, CD11c-) and transcription factors (Eomeshigh, Tbetlow) of ltNK cells differed from their circulating counterparts. LtNK cells were characterized by enhanced expression of inhibitory receptors TIGIT and CD96 and low expression of DNAM1 and cytolytic molecules (GZMB, GZMH, GNLY). Their proliferative capacity was reduced compared to the circulating NK cells. By performing gene set enrichment analysis we identified DUSP6 and EGR2 as potential regulators of the ltNK cell transcriptome. Remarkably, comparison of the ltNK cell transcriptome to the published human spleen-resident memory CD8+ T (Trm) cell transcriptome revealed an overlapping gene signature. Moreover, the phenotypic profile of ltNK cells resembled that of CD8+ Trm cells in bone marrow. Together, we provide a comprehensive molecular framework of the conventional CD56bright and CD56dim NK cells as well as the tissue-resident ltNK cells and provide a core gene signature which might be involved in promoting tissue-residency.

overall_design: mRNA sequencing of NK cell populations isolated from blood: CD56bright, NKG2A+ CD56dim and NKG2A- CD56dim, and bone marrow: CD56bright, CD56dim, NKG2A+ ltNK, and NKG2A- ltNK. Each sample has 4 biological replicates.

first: Gertjan

last: Lugthart

first: Szymon

last: Kielbasa

first: Sander

last: van der Zeeuw

first: Henk

last: Buermans

first: Marco

middle: W

last: Schilham


summary: Gene expression analysis of the p53 response in PHA-stimulated human T-lymphocytes treated ex vivo with either Doxorubicin, Nutlin-3, or DMSO

overall_design: Gene expression analysis (using Affymetrix Human Genome U133 Plus 2.0 GeneChip® arrays) of the p53 response in PHA-stimulated human T-lymphocytes (from two healthy volunteers) treated ex vivo with either Doxorubicin or Nutlin-3, relative to the DMSO control.

first: Thuy-Ai

middle: T

last: Nguyen

first: Daniel

last: Menendez


title: Single Sample Sequencing (S3EQ) of epigenome and transcriptome in nucleus accumbens

status: Public on Jul 21 2018

first: Song-Jun

last: Xu

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: Single Sample Sequencing (S3EQ) is an innovative approach to analyze both epigenetic and transcriptomic regulation within a single neuronal sample.

overall_design: We analyzed chromatin immunoprecipitation (ChIP)- and RNA-sequencing data from the nucleus accumbens (NAc) of the same animal.

first: S

first: E

last: Heller


title: Mediator and Pol II form diffraction-sized condensates dependent on active transcription in living stem cells

status: Public on Jul 22 2018

city: Cambridge

first: Charles

last: Li

address: 455 Main Street

institute: Whitehead Institute for Biomedical Research

summary: Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites and recruit the Mediator complex which contacts the pre-initiating RNA Polymerase II (Pol II) complex assembled at the start site of the gene. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. Here we use quantitative live cell super-resolution and light sheet imaging to study the organization and dynamics of endogenous Mediator and Pol II directly in living mouse embryonic stem cells. In addition to forming transient clusters with average lifetimes of 11.1 (± 0.9) s, and 12.1 (± 1.4) s respectively, Mediator and Pol II also form large and stable clusters in stem cells (~15 stable clusters per cell). The large and stable Mediator and Pol II clusters gradually disappear within hours after induction of stem cell differentiation. Mediator and Pol II colocalize in the large clusters. Inhibition of Brd4 bromodomains necessary for enhancer association eliminates both Mediator and Pol II stable clusters, and inhibition of transcription elongation selectively eliminates stable Pol II but not stable Mediator clusters. Tracking of Mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property associated with phase separated droplets. We conclude that Mediator and Pol II associate in diffraction-sized condensates with a defined lifetime dependent on active transcription in living stem cells.

overall_design: H3K27ac, RPB1, and Dendra2 ChIP-seq in WT and Dendra2-RPB1, Halo-MED19 tagged (DRHM) R1 mouse ES cells in the ES state and the EpiLC state.

middle: H

first: Won-Ki

last: Cho


title: RNA-seq and flow-cytometry of conventional, scalp, and palmoplantar psoriasis reveal shared and distinct molecular pathways

status: Public on Jul 20 2018

city: Los Angeles

first: Richard

middle: Sungho

last: Ahn

state: CA

address: 611 Charles E. Young Drive East, UCLA, Boyer Hall, 510C

department: Microbiology, Immunology, and Molecular Genetics

institute: University of California Los Angeles

summary: It has long been recognized that anatomic location is an important feature for defining distinct subtypes of plaque psoriasis. However, little is known about the molecular differences between scalp, palmoplantar, and conventional plaque psoriasis. To investigate the molecular heterogeneity of these psoriasis subtypes, we performed RNA-seq and flow cytometry on skin samples from individuals with scalp, palmoplantar, and conventional plaque psoriasis, along with samples from healthy control patients. We performed differential expression analysis and network analysis using weighted gene coexpression network analysis (WGCNA). Our analysis revealed a core set of 763 differentially expressed genes common to all sub-types of psoriasis. In contrast, we identified 605, 632, and 262 genes uniquely differentially expressed in conventional, scalp, and palmoplantar psoriasis, respectively. WGCNA and pathway analysis revealed biological processes for the core genes as well as subtype-specific genes. Flow cytometry analysis revealed a shared increase in the percentage of CD4+ T regulatory cells in all psoriasis subtypes relative to controls, whereas distinct psoriasis subtypes displayed differences in IL-17A, IFN-gamma, and IL-22 production. This work reveals the molecular heterogeneity of plaque psoriasis and identifies subtype-specific signaling pathways that will aid in the development of therapy that is appropriate for each subtype of plaque psoriasis.

overall_design: Transcriptomic profiles were obtained from palmoplantar (n = 3), scalp (n = 8), and conventional psoriatic skin (n = 8) as well as healthy control skin (n = 9) biopsies on the Illumina HiSeq 2000/4000 platforms. Multi-parameter FACS was also performed on each biopsy sample to obtain T cell populations (CD4+ T effectors, CD8+ T cells, and CD4+Foxp3+ Tregs).

first: Di

last: Yan

first: Hsin-Wen

last: Chang

first: Kristina

last: Lee

first: Shrishti

last: Bhattarai

first: Zhi-Ming

last: Huang

first: Mio

last: Nakamura

first: Rasnik

last: Singh

first: Ladan

last: Afifi

first: Keyon

last: Taravati

first: Priscila

last: Munoz-Sandoval

first: Mariela

last: Pauli

first: Michael

last: Rosenblum

first: Wilson

last: Liao


title: Exploration of the genetic makeup and expression of the glycolytic and fermentative pathways within the Saccharomyces genus

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

institute: Delft University of Technology

summary: The ability of the yeast Saccharomyces cerevisiae to convert glucose, even in the presence of oxygen, via glycolysis and the fermentative pathway to ethanol has played an important role in its domestication. Despite the extensive knowledge on these pathways in S. cerevisiae, relatively little is known about these pathways in other industrially-relevant Saccharomyces yeast species. In this study we explore the diversity of the glycolytic and fermentative pathways within the Saccharomyces genus using S. cerevisiae, S. kudriavzevii and S. eubayanus as paradigms. Sequencing data revealed a highly conserved genetic makeup of the glycolytic and fermentative pathways in the three species in terms of number of paralogous genes. Although promoter regions were less conserved between the three species as compared to coding sequences, binding sites for Rap1, Gcr1 and Abf1, main transcriptional regulators of glycolytic and fermentative genes, were highly conserved. Transcriptome profiling of these three strains grown in aerobic batch cultivation in chemically defined medium with glucose as carbon source, revealed a remarkably similar expression of the glycolytic and fermentative genes across species, and the conserved classification of genes into major and minor paralogs. Furthermore, transplantation of the promoters of major paralogs of S. kudriavzevii and S. eubayanus into S. cerevisiae demonstrated not only the transferability of these promoters, but also the similarity of their strength and response to various environmental stimuli.  The relatively low homology of S. kudriavzevii and S. eubayanus promoters to their S. cerevisiae relatives makes them very attractive alternatives for strain construction in S. cerevisiae, thereby expanding S. cerevisiae molecular toolbox.

sample_organism:
Saccharomyces cerevisiae
Saccharomyces kudriavzevii
Saccharomyces eubayanus

overall_design: Transcriptome profiling of these three strains S. cerevisiae, S. krudiavzevii and S. cerevisiae grown in aerobic batch cultivation in chemically defined medium with glucose as carbon source, revealed a remarkably similar expression of the glycolytic and fermentative genes across species, and the conserved classification of genes into major and minor paralogs.

first: Marcel

last: van den Broek


title: Domain-focused CRISPR-screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells

city: University Park

first: Ross

last: Hardison

address: 303 Wartik Lab

institute: Pennsylvania State University

summary: Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of beta-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase-domain focused CRISPR/Cas9-based genetic screen with a newly optimized sgRNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.

overall_design: A CRISPR-screen reveals HRI kinase as a fetal hemoglobin repressor and further validated in HUDEP2 and CD34+ derived primary erythroid cultures.

first: Jeremy

last: Grevet

first: Xianjiang

last: Lan

first: Nicole

last: Hamagami

first: Christopher

last: Edwards

first: Laavanya

last: Sankaranarayanan

first: Xinjun

last: Ji

first: Saurabh

middle: K

last: Brardwaj

first: Carolyne

last: Face

first: David

middle: F

last: Posocco

first: Osheiza

last: Abdulmalik

first: Cheryl

last: Keller

first: Belinda

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