title: The transcription factor c-Maf is a positive regulator of IL-10 with context-specific roles in CD4+ T cell effector function and in vivo consequences [ATAC-seq]

first: Leona

last: Gabrysova

address: 1 Midland Road

institute: The Crick Institute

summary: CD4+ T cells differentiate into distinct subsets which produce unique patterns of cytokines to eradicate pathogens, alongside IL-10 which controls immune pathology. How Il10 expression is regulated alongside the subset-specific cytokines is unclear. Using transcriptional and epigenomic profiling of CD4+ T cells from TH1 (malaria), TH2 (allergy) and TH17 (autoimmunity) disease models, we show here that c-Maf, identified as the top candidate regulator of IL-10, directly induces IL-10 across all CD4+ T cell subsets, controlling pathology in TH1 and TH2-type responses. We also report direct negative effects of c-Maf on Il2 explaining the increased Foxp3+Tregs, and direct positive effects on Rorc, as mechanisms for the decreased pathology seen in the TH17 disease model in the absence of c-Maf. Thus, c-Maf has broad yet context specific roles in directly and indirectly regulating gene expression, allowing each type of T effector immune response to occur effectively yet in a controlled fashion.

overall_design: ATAC-seq analysis, in biological triplicates, of ex vivo CD4+ T cells from malaria (TH1), house dust mite (HDM) allergy (TH2) and experimental autoimmune encephalomyelitis (EAE) (TH17) models in c-Maf flfl and c-Maf flfl CD4Cre mice

last: Gabryšová

first: Marisol

last: Alvarez-Martinez

first: Raphaëlle

last: Luisier

first: Luke

last: Cox

first: Jan

last: Sodenkamp

first: Caroline

last: Hosking

first: Charlotte

last: Whicher

first: Yashaswini

last: Kannan

first: Krzysztof

last: Potempa

first: Xuemei

last: Wu

first: Leena

last: Bhaw

first: Hagen

last: Wende

first: Michael

last: Sieweke

first: Greg

last: Elgar

first: Mark

last: Wilson

first: James

last: Briscoe

first: Vicki

last: Metzis

first: Jean

last: Langhorne

first: Nicholas

last: Luscombe

first: Anne

last: O’Garra


title: The transcription factor c-Maf is a positive regulator of IL-10 with context-specific roles in CD4+ T cell effector function and in vivo consequences [RNA-seq in vivo]

status: Public on Feb 08 2018

city: London

first: Leona

last: Gabrysova

country: United Kingdom

type: Expression profiling by high throughput sequencing

overall_design: RNA-seq analysis, in biological triplicates, of ex vivo CD4+ T cells from malaria (TH1), house dust mite (HDM) allergy (TH2) and experimental autoimmune encephalomyelitis (EAE) (TH17) models in c-Maf flfl and c-Maf flfl CD4Cre mice

last: Gabryšová

first: Marisol

last: Alvarez-Martinez

first: Raphaëlle

last: Luisier

first: Luke

middle: S

last: Cox

middle: H

middle: M


title: Transcriptome analysis of SKI knock-out in HL60 cells

status: Public on Feb 07 2018

city: Marburg

first: Uta

middle: Maria

last: Bauer

state: Hessen

address: Hans-Meerwein-Str. 2

department: Institute of Molecular Biology and Tumor Research

country: Germany

institute: Philipps University Marburg

summary: Whole transcriptome for SKI knock-out and control HL60 cells was sequenced. SKI control and knockout samples were compared to find differentially expressed genes. Differentially expressed genes were further analysed to find the significance of SKI in HL60 cells.

overall_design: Examining of SKI dependent transcriptome in HL60 cells using RNAseq.

first: Christine

last: Feld

first: Peeyush

last: Sahu

first: Thorsten

last: Stiewe

first: Andreas

last: Neubauer


title: The transcription factor c-Maf is a positive regulator of IL-10 with context-specific roles in CD4+ T cell effector function and in vivo consequences [RNA-seq in vitro]

overall_design: RNA-Seq analysis, in 2-3 biological replicates, of in vitro differentiated CD4+ Th cells and ex vivo Treg cells

first: Jan

last: Sodenkamp

first: Caroline

last: Hosking

first: Charlotte

last: Whicher

first: Yashaswini

last: Kannan

first: Krzysztof

last: Potempa

first: Xuemei

last: Wu

first: Leena

last: Bhaw

first: Hagen

last: Wende

first: Michael

last: Sieweke

last: Elgar


title: Combined cistrome and transcriptome analysis of SKI in AML cells identifies SKI as a co-repressor for RUNX1

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Impact of high fat diet on Mus musculus domesticus secreted milk small RNA transcriptome

city: West Lafayette

first: Theresa

last: Casey

state: Indiana

address: 175 S. University Street

department: Animal Sciences

country: USA

institute: Purdue University

type: Non-coding RNA profiling by high throughput sequencing

summary: The impact of high fat diet on secreted milk small RNA transcriptome was studied by isolating total RNA from milk fat fraction collected on lactation day 10 from control diet fed (C; n=5; 10% fat; 7% sucrose; Research Diets #D12450J, Brunswick, NJ) and high fat diet fed (HF; n=4; Research Diets #D12492, 60% of total kcal energy is fat and match 7% of total kcal is sucrose; Brunswick, NJ) mice.

overall_design: Milk small RNA transcriptome profiles from fat fraction collected on lactation day 10 from control diet fed (n=5) and high fat diet fed (n=4) mice.


title: The transcription factor c-Maf is a positive regulator of IL-10 with context-specific roles in CD4+ T cell effector function and in vivo consequences

first: Leona

last: Gabrysova


title: Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes [RNA-seq2]

city: chengdu

first: Yong

last: Dong

state: sichuan

address: Xindu road 783, Xindu district

department: Immunology

country: China

institute: Chengdu medical college

summary: Here we  were performed RNAseq to analyze the transcriptome changing of Hoxb5 in Pro/pre-B by over expression.

overall_design: B220+ enriched cells from bone marrow were used to transduce Hoxb5 virus or GFP control virus. And 1000 transduced Pro/pre-B cells were sorted for RANseq analysis. The cDNA of sorted 1000-cell aliquots were generated and amplified as described previously (PMID；20203668). Then quality controls of the amplified cDNA were examined by qPCR analysis of housekeeping genes (B2m, Actb, Gapdh, Ecf1a1). RNAseq libraries of 1000 cells were generated by Nextera XT DNA Sample Preparation Kit (FC-131-1096) with 1 ng of generated dsDNA following the manufacturer’s protocol. The samples passed quality control were used for sequencing library preparation by illumine Nextera XT DNA Sample Preparation Kit (FC-131-1096). All libraries were sequenced by illumina sequencers NextSeq 500 (illumina).

first: Mengyun

last: Zhang

first: Jinyong

last: Wang


title: Cistrome analysis of SKI and RUNX1 in HL60

first: Uta

last: Bauer

summary: SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here we use ChIP-seq and RNA-seq to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which nearly 50% harbour SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI deletion causes a release of the myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets. RUNX1 ChIP-seq demonstrates that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 co-occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time on a genome-wide scale the predominant co-repressive function of SKI in AML cells and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression.

overall_design: Examining of SKI genome-wide binding and its comparison with other transcription factors and histone modifications.

first: Christine

last: Feld

first: Peeyush

last: Sahu

last: Stiewe


title: Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes [ChIP-Seq]

summary: Chipseq were performed to analyze the targets of Hoxb5 in pro-/pre-B by over expression.

overall_design: Chipseq: One Hoxb5 over expression sample and its sonication input (no replicates).


title: Verticillium dahliae-Arabidopsis interaction causes changes in gene expression profiles and jasmonate levels on different time scales

city: Jena

first: Sandra

last: Scholz

address: Dornburger Straße 159

department: Plantphysiology

institute: Friedrich-Schiller-University Jena

summary: Verticillium dahliae is a soil-borne vascular pathogen that causes severe wilt symptoms in a wide range of plants. Co-culture of the fungus with Arabidopsis roots for 24 hours induces many changes in the gene expression profiles of both partners, even before defense-related phytohormone levels are induced in the plant. Both partners reprogram sugar and amino acid metabolism, activate genes for signal perception and transduction, and induce defense and stress responsive genes. Furthermore, analysis of Arabidopsis expression profiles suggests a redirection from growth to defense. The plant and fungal genes that rapidly respond to the presence of the partner might be crucial for early recognition steps and the future development of the interaction. Thus they are potential targets for the control of V. dahliae-induced wilt diseases.

sample_organism:
Arabidopsis thaliana
Verticillium dahliae

overall_design: The aim of this study was to identify important targets regulated in both partners in the initial phase of interaction (24h). After 24 h of co-cultivation the fungus colonizes the plant root and first penetration of plant tissue by the fungus is visible while the vascular tissue of the plant is not yet colonized (pre-vascular phase).

first: Wolfgang

last: Schmidt-Heck

first: Reinhard

last: Guthke

first: Ralf

last: Oelmüller


title: mRNA Profiling of murine 4T1-T breast cancer cells with Asns knocked down after isolation from primary tumors and lung metastases with 6TG

city: Cold Spring Harbor

first: Simon

middle: Robert Vincent

last: Knott

state: NY

address: 1 Bungtown Rd

department: Biology

institute: Cold Spring Harbor Laboratory

summary: Murine 4T1-T breast cancer cells were infected with retroviral vectors harboring shRNAs for murine or a Renilla, as negative control. Cells were orthotopically injected into NSG mice. After 19 days mice were euthanized and tumors and lungs were removed and digested into single cells. Cells were then placed in culture dishes with 6TG. Surviving cells were then harvested and processed with the NuGen Ovation RNAseq system V2. Libraries were sequenced on an Illumina platform and mapped using Bowtie2 to the mm10 genome prior to transcript quantification using HT-Count.

middle: V


title: RNA sequencing of whole, ventral and dorsal hippocampus after different stress conditions

city: Zurich

first: Johannes

last: Bohacek

address: Winterthurerstrasse 190

country: Switzerland

institute: University of Zurich/ ETH Zurich

summary:
Purpose: The goal of this study was to characterize molecular stress responses on transcriptional level in dorsal and ventral hippocampus separately.
Methods: mRNA profiles of whole, dorsal and ventral hippocampus of mice 45min after first exposure to different acute stressors. The stressors were novelty (6min novel environment), restraint (30min immobilization), or cold swim (6min in 18 degree Celcius water). 5 mice were used per condition, using Illumina HiSeq4000. The sequence reads that passed quality filters were mapped with STAR, counted with RSEM and differential gene expression was calculated using the bioconducter package edgeR.
Results: With our workflow, we identified 13902 genes per sample. Approximately 20% of the genes were differentially expressed between dorsal and ventral hippocampus at baseline, with a log2 fold change >±0.3 and p value <0.005. Approximately 100 genes are differentially expressed upon stress treatment in the whole hippocampus. If ventral and dorsal hippocampus were analyzed separately, numbers increased to 150 and 250 respectively. Stress-responsive genes vary between dorsal and ventral hippocampus, and also vary between different stressors. Further analysis identified a functional epigenetic gene cluster specific for the stress response in the ventral hippocampus.
Conclusions: Our study represents the first RNA-seq analysis of dorsal and ventral hippocampus after different acute stress exposures.

overall_design: mRNA profiles of whole, dorsal and ventral hippocampus of mice 45min after different stress treatments, 5 mice per condition, using Illumina HiSeq4000.

first: Amalia

last: Floriou-Servou

first: Lukas

last: von Ziegler

first: Luzia

last: Stalder

first: Oliver

last: Sturman

first: Mattia

last: Privitera


title: mRNA profiling of 4T1-T cells with altered asparagine bioavailability


title: Lung Cancer Cell-Mediated Skeletal Muscle Mitochondrial Dysfunction is Mitigated by 1a,25-Dihydroxyvitamin D3

city: Rochester

first: Xuewei

address: 200 1st street

institute: Mayo Clinic

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Analysis of mRNA Expression in Lewis Lung Carcinoma (LLC1) Cells and MLE 12 cells

summary: Whole transcriptome sequencing was used to analyze mRNAs expressed by LLC1 and MLE 12 cells.

overall_design: LLC1 or MLE 12  cells were grown in appropriate media to approximately 80% confluence.  Cells were isolated and total and mRNA were isolated in order to prepare cDNA  libraries.

first: Rajiv

last: Kumar


title: mRNA Profiling of Murine 4T1 Breast Cancer Cells with Asns knocked down

summary: Murine 4T1-T Breast cancer cells were infected with retroviral vectors harboring shRNAs for murine or a Renilla, as negative control. After selection cells were harvested and processed with the NuGen Ovation RNAseq system V2. Libraries were sequenced on an Illumina platform and mapped using Bowtie2 to the mm10 genome prior to transcript quantification using HT-Count.


title: RNA-seq and ChIP-seq analysis of BMI1 or RING1B-silenced prostate cancer cells C4-2

city: Houston

first: Dongyu

last: Zhao

state: TX

address: 6670 Bertner Ave

institute: Houston Methodist Research Institute

summary: Purpose: Study of the role of BMI1 dependent and indpendent of PRC1 in castration-resistant prostate cancer(CRPC)   Method: The expression of BMI1 or RING1B was silenced by 2 independent siRNA strands targeted at BMI1 or RING1B in C4-2 cells, scramble RNA as control. mRNA profiles and genome-wide chromatin-state maps were generated by deep sequencing. AR, BMI1 and IgG ChIP was conducted in C4-2 cells.  Results: AR-induced genes were significantly down regulated by BMI1 knockdown but not RING1B. higher expression levels of BMI1 activated genes (those downregulated by BMI1 knockdown) were significantly associated with poorer disease-free  and poorer overall survival.

overall_design: Expression profiling by RNA-seq, profiling of BMI1 and RNF2 (wildtype and  knockdown) from C4-2 cell lines.  DNA was purified from chromatin immunoprecipitates for AR, BMI1 or IgG using the phenol/chloroform extraction. IgG was used as a negative control for ChIP. Then, DNA was amplified by quantitative PCR and normalized to input.

first: S

last: Zhu

first: D

first: K

last: Chen

first: Q

last: Cao


title: The COP9 Signalosome regulates seed germination by facilitating protein degradation of RGL2 and ABI5

city: Shenzhen

first: Bosheng

last: Li

state: Guangdong

address: SUStech Huiyuan #1 406

department: MCDB

institute: SUSTC (current, may different with previous institution )

summary: The control of seed germination and seed dormancy are critical for the successful propagation of plant species, and are important agricultural traits. Seed germination is tightly controlled by the balance of gibberellin (GA) and abscisic acid (ABA), and is influenced by environmental factors. The COP9 Signalosome (CSN) is a conserved multi-subunit protein complex that is best known as a regulator of the Cullin-RING family of ubiquitin E3 ligases (CRLs). Multiple viable mutants of the CSN showed poor germination, except for csn5b-1. Detailed analyses showed that csn1-10 has a stronger seed dormancy, while csn5a-1 mutants exhibit retarded seed germination in addition to hyperdormancy. Both csn5a-1 and csn1-10 plants show defects in the timely removal of the germination inhibitors: RGL2, a repressor of GA signaling, and ABI5, an effector of ABA responses. We provide genetic evidence to demonstrate that the germination phenotype of csn1-10 is caused by over-accumulation of RGL2, a substrate of the SCF (CRL1) ubiquitin E3 ligase, while the csn5a-1 phenotype is caused by over-accumulation of RGL2 as well as ABI5. The genetic data are consistent with the hypothesis that CSN5A regulates ABI5 by a mechanism that may not involve CSN1. Transcriptome analyses suggest that CSN1 has a more prominent role than CSN5A during seed maturation, but CSN5A plays a more important role than CSN1 during seed germination, further supporting the functional distinction of these two CSN genes. Our study delineates the molecular targets of the CSN complex in seed germination, and reveals that CSN5 has additional functions in regulating ABI5, thus the ABA signaling pathway.

overall_design:
Col-0, csn1-10, and csn5a-1 seeds were collected from plants grown side-by-side in the growth room. Seeds for Col-0 and csn1-10 were 1.5wks in storage, and csn5a-1 seeds were 4wk in storage. Approximately 100 microliter of settled seeds were used for RNA extraction for each sample. The 2-day imbibed seeds, prepared with the same volume of dry seeds as above, were sterilized, washed, and incubate on cell culture wells with 1ml of 0.5X liquid MS medium at 22oC under constant light for 2 days. Each sample points had three biological repeats. Seeds were centrifuged to remove the liquid, and were frozen in liquid nitrogen. The frozen seeds were ground to a fine powder using mortar and pestle in the presence of liquid nitrogen and small quantity of sterile quartz powder. RNA extraction was performed according to a published procedure [73].

High-throughput RNA-seq was carried out at Yale Center for Genome Analysis. The Single-End RNA-sequencing was carried out with Illumina Hi-seq 2000 platform (Genome Center, Yale West Campus). Specifically, libraries were analyzed with a Bioanalyzer 2100 instrument (Agilent, Santa Clara, CA), quantified by Qubit fluorometer (Life Technologies, Carlsbad, CA). The Arabidopsis thaliana genome obtained from TAIR10 (https://www.arabidopsis.org) was used as the genome reference. After adaptor trimming and contaminate sequence removing by fastqc (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastx-toolkits (hannonlab.cshl.edu/fastx_toolkit/), Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) was used for genome mapping and followed by the tophat (https://ccb.jhu.edu/software/tophat/index.shtml) transcript assembling. Gene differentially expression profiling was accomplished by cufflink and cuffdiff software package (cole-trapnell-lab.github.io/cufflinks/cuffdiff/) with default parameters and cutoffs, fold change cutoff was set to 2.

first: Dan

last: Jin

first: Ming

last: Wu

first: Birte

last: Bucker

first: Philipp

last: Keil

first: Shaoman

last: Zhang

first: Jigang

first: Dingming

last: Kang

first: Jie

last: Liu

last: Dong

first: Xing

middle: W

last: Deng

first: Vivian

last: Irish

first: Ning

last: Wei


title: PRC2 mediates paternal epigenetic inheritance: comparison of wild-type embryos

status: Public on Feb 09 2018

city: Clayton

first: Sam

last: Forster

state: Victoria

address: 27 - 31 Wright Street

department: Monash Institute

country: Australia

institute: Monash University

summary: Determining the mechanisms that establish and regulate the transmission of non-genetic (i.e. epigenetic) information from parent to offspring is critical for understanding disease heredity. Using an innovative model to assess transmission of epigenetic effects, we provide evidence that Polycomb Repressive Complex 2 (PRC2) mediates inheritance through the male germline. This study reveals a novel role for histone modifying complexes in epigenetic inheritance, with important implications for understanding disease inheritance.

overall_design: Comparison of gene expression in E8.5 embryos with the same wild-type genotype, produced from heterozygous (n=4) and wild-type (n=4) Eedl7Rn5-1989SB fathers reveal differences in gene expression possibly due to altered epigenetic patterning in the sperm.

first: Jessica

last: Stringer

first: Samuel

middle: C

first: Patrick

last: Western


title: Transcription factor Hoxb5 reprograms B cells into functional T lymphocytes [RNA-Seq]

first: Yong

summary: Natural hematopoietic stem cells are currently the only cell resource to generate entire T lymphopoiesis in vivo. Altenatively, functional and non-tumorigenic T lymphocytes converted from the other cell types remain unsuccessful. Evidence is still lacking about the concept that 'lineage-switching factors' (LSFs) exist in HSC, whose re-activation in progeny cells can accomplish lineage conversions. Here we show that upon ectopic expression of Hoxb5(a HSC-specific transcription factor), the Pro-/Pre-B cells were induced into functional early T cell progenitors(iETPs) in vivo. The reprogramming process initiated in the bone marrow and accomplished in the thymus at ETP stage. Strikingly, the T lymphocytes derived from iETPs resemble their natural counterparts in transcriptome pattern, hierarchical differentiation, tissue distribution and immune functions. Our observations pave the way for achieving lineage regeneration by direct reprogramming autologous somatic cells in vivo. Further, our success in haematopoietic system may build a paradigm for LSF-mediated regeneration of other tissues.

overall_design: Mus musculus mRNA profiles of 24 cell types, including HSC, MPP, Mature lineage cells (T cells_B cells_Myeloid cells), Pro-_Pre-_B cells, BM-ETPs, BM-iETPs, Thy-ETPs, Thy-iETPs, DN1, DN2, DN3, DN4, DP, CD4-SP,CD8-SP, iDN1, iDN2, iDN3, iDN4, iDP, iCD4-SP and iCD8-SP. 1000 of each cell type was sorted for RANseq analysis refered to published protocol (PMID:20203668).

last: Yang

first: Jinyong

last: Wang


title: mRNA profiling of Asns-silenced murine 4T1-T breast cancer cells grown in the presence and absence of Asparagine

first: Simon

last: Knott

summary: Murine 4T1-T breast cancer cells were infected with an shRNA targeting Asparagine Synthetase (Asns) and grown in DMEM +/- asparagine. Cells were harvested and processed with the NuGen Ovation RNAseq system V2. Libraries were sequenced on an Illumina platform and mapped using Bowtie2 to the mm10 genome prior to transcript quantification using HT-Count.

overall_design: Asns-silenced 4T1-T breast cancer cells grown in the presence and absence of Asparagine


title: The developmental and metabolic roles of the diabetes candidate gene TCF7L2 in adipose tissue

city: Kuopio

first: Sami

last: Heikkinen

address: Yliopistonranta 1E

department: Institute of Biomedicine

country: Finland

institute: University of Eastern Finland

summary: The gene encoding for transcription factor 7-like 2 (TCF7L2) is the strongest type 2 diabetes (T2DM) candidate gene discovered to date. While its association with T2DM has been replicated in most populations worldwide, the molecular and physiological mechanisms that underlie this association remain largely unknown. As a key transcriptional effector of the Wnt/β-catenin signaling pathway, we hypothesized that TCF7L2 plays an important role in the development and function of adipocytes. Using a combination of in vitro and in vivo approaches, we show that TCF7L2 is critical for proper adipogenesis and that inactivating TCF7L2 mediated transcription by removing its DNA binding domain in mature adipocytes leads to whole-body glucose intolerance and hepatic insulin resistance in mice. This effect is secondary to an increase in subcutaneous adipose tissue mass caused by adipocyte hypertrophy that worsens with age and with high-fat feeding. Finally, in humans with adipocyte insulin resistance we demonstrate that TCF7L2 expression is downregulated, highlighting the translational importance of our findings. In summary our data indicate that TCF7L2 is an important mediator of adipocyte biology and has key roles in adipose tissue development and function.

overall_design: For the identification of genes regulated during adipogenesis by Tcf7l2, Tcf7l2 was silenced in murine 3T3-L1 preadipocyte cells using lentivirally delivered stable silencing of Tcf7l2 with a scrambled shRNA as control. Sampling times were at 0, 2, 4, 6, and 8 days of differentiation. RNA-seq timecourse was performed in duplicate.

first: Luke

last: Norton


title: PARP14 controls the nuclear accumulation of a subset of type I Interferon-inducible proteins [RNA-seq1]

city: Milano

first: Viviana

last: Piccolo

state: Milano

address: Via Adamello 16

department: Department of Experimental Oncology

country: Italy

institute: European Institute of Oncology (IEO)

summary: The enzymes of the poly-ADP-ribose polymerase (PARP) super-family control many relevant cellular processes, but a precise understanding of their activities in different physiological or disease contexts is largely incomplete. We found that transcription of several PARP genes was dynamically regulated upon macrophage activation by several inflammatory stimuli. Specifically, PARP14 was strongly induced by endotoxin stimulation and translocated to the nucleus in stimulated cells. Quantitative mass spectrometry analysis showed that PARP14 bound to a group of interferon-stimulated gene (ISG)-encoded proteins, most with an unknown function, and it was required for their nuclear accumulation. Moreover, PARP14 depletion attenuated transcription of primary antiviral response genes regulated by the transcription factor IRF3, including Ifnb1, thus reducing IFNβ production and activation of ISGs involved in the secondary antiviral response. Overall, these data hint at a role of PARP14 in the control of antimicrobial responses and specifically in nuclear activities of a subgroup of ISG-encoded proteins.

overall_design: mRNA  sequencing of differentially expressed genes in PARP14 WT and KO RAW 264.7 cells, upon: no treatment, LPS,  Jak inhibitor or LPS plus Jak inhibitor treatment.

first: Greta

last: Caprara

first: Elena

last: Prosperini

first: Gianluca

last: Sigismondo

first: Alessia

last: Melacarne

first: Alessandro

last: Cuomo

first: Mark

last: Boothby

first: Maria

last: Rescigno

first: Tiziana

last: Bonaldi

first: Gioacchino

last: Natoli


title: Analysis by Whole Transcriptome Sequencing of the Effects of  LLC1 conditioned medium, LLC1 conditioned plus Calcitriol,  non-conditioned medium,  and non-conditioned medium plus Calcitriol on mRNA Expression in Primary Human Skeletal Muscle Cells

first: Xuewei

summary: Primary human skeletal muscle cells (Lonza) were treated with  LLC1 conditioned medium, LLC1 conditioned medium plus Calcitriol, LLC1 non-conditioned medium or LLC1 non-conditioned medium plus Calcitriol for a period of 24 hours prior to isolation of RNA.

overall_design: To assess the effects of  molecules produced by LLC1 cells on primary human skeletal muscle cells, LLC1 cells were grown and conditioned medium from LLC1 cells with or without added calcitriol was added to primary human skeletal muscle cells for 24 hours prior to the isolation of RNA.

first: Rajiv

last: Kumar


title: PARP14 controls the nuclear accumulation of a subset of type I Interferon-inducible proteins [RNA-seq2]

overall_design: mRNA  sequencing of differentially expressed genes in PARP14 WT RAW 264.7 cells, with or without LPS treatment


title: The human circulating miRNome reflects multiple diseases associated with short-term exposure to traffic-related air pollution

status: Public on Feb 06 2018

city: Maastricht

first: Julian

last: Krauskopf

address: Universiteitssingel 50

country: Netherlands

institute: Maastricht University

summary: The study comprises of 24 subjects that have been exposed to low (H) and high dose (O) ambient traffic pollution. Each subject has been exposed to 2 different exposure site. Blood samples were taken at both sites of which circulating miRNAs in plasma have been prepared

overall_design: 48 Samples total

first: Jos


title: Diurnal transcriptome atlas of a primate across all major neural and peripheral tissues

city: La Jolla

first: Max

last: Chang

state: CA

address: 9500 Gilman Drive

institute: University of California, San Diego

summary: Spatio-temporal gene expression patterns underlie time-of-the day specific functional specialization of different organs. Although circadian gene expression atlases from model organisms including the nocturnal mouse are available, they are limited to a few peripheral organs. Here we report the circadian transcriptome of 64 different tissues, including 22 different brain regions, sampled every 2 h over 24 h, from the diurnal primate Papio Anubis. We found that the primate genome is vastly rhythmic with up to 67% of genes showing daily rhythms in expression. In addition to the diversity of transcripts in each tissue, the repertoire and phases of expression of cycling transcripts impart another layer of functional specialization to each tissue. Transcripts that are ubiquitously expressed and participate in essential cellular functions are more likely to exhibit rhythmic expression in a tissue-specific manner. At the whole organism level, the peak phases of rhythmic transcripts are largely clustered around dawn and dusk with a "quiescent period" during the first half of the night. Our findings also unveil a different temporal organization of central and peripheral tissues between diurnal and nocturnal animals. This study will contribute significantly to understanding circadian regulation of organs function and contribution of circadian rhythm disruption to diseases.

overall_design: transcriptome of 64 different tissues from the diurnal primate Papio Anubis sampled every 2 h over 24 h

first: Ludovic

last: Mure

first: Satchidananda

last: Panda


title: CpG on carbon nanotubes inhibits migration of brain tumor cells while activating immune cells

city: Duarte

first: Jacob

last: Berlin

state: California

address: 1500 E Duarte Rd

institute: City of Hope

summary: Even when treated with aggressive current therapies, patients with glioblastoma usually survive less than two years and exhibit a high rate of recurrence. CpG is an oligonucleotide that activates the innate immune system via TLR9 activation. Injection of CpG into glioblastoma tumors showed promise as an immunotherapy in mouse models but proved disappointing in human trials. One aspect of glioma that is not addressed by CpG therapy alone is the highly invasive nature of glioma cells, which is associated with resistance to radiation and chemotherapy. Here, we demonstrate that single-walled carbon nanotubes non-covalently functionalized with CpG (SWNT/CpG) not only retain the immunostimulatory property of the CpG, but interestingly also inhibit the migration of glioma cells without affecting cell viability or proliferation. SWNT/CpG activated macrophages by induction of the TLR9/NF-κB pathway, but actually decreased NF-κB activation in glioma cells. By using RNA-seq to compare the effect of SWNT/CpG treatment on glioma cells and macrophages, we confirmed that SWNT/CpG treatment has cell type-specific effects on gene expression. Through RNA-seq analysis, we identified a number of differentially affected pathways which may be involved in glioma cell migration inhibition and observed reduced gene expression in DNA repair pathways for SWNT/CpG-treated glioma cells. The migration inhibition of glioma cells was correlated with reduced intracellular levels of reactive oxygen species (ROS), suggesting that an antioxidant-based mechanism mediates the observed effects. To our knowledge, SWNT/CpG is the first therapy that inhibits the migration of cancer cells while stimulating the immune system.

overall_design: K-Luc glioma or RAW-Blue macrophage cells were plated in a scratch assay (ibidi 2-well insert) and treated with either  1) water (untreated control), 2) CpG-only control, 3) SWNT/LP-only control (SWNTs coated with a control material), 4) CpG + SWNT/LP co-treatment control, or 5) SWNT/CpG treatment (SWNTs coated with CpG DNA). At the conclusion of the migration assay, each treatment group was compared to the untreated control for that cell line to determine if there was any observable migration inhibition. After this observation was noted, total RNA was purified for each replicate.

first: Ethan

last: White

first: Behnam

last: Badie


title: ZNF131 suppresses centrosome fragmentation in Glioblastoma stem-like cells through regulation of HAUS5

city: Seattle

first: Sonali

last: Arora

state: WA

address: 1100 Fairview Ave N,

institute: FHCRC

summary: To identify new Glioblastoma multiforme (GBM) therapeutic strategies, we previously performed genome-wide RNAi lethality screens in patient-derived GBM stem-like cells (GSCs) and neural progenitor cells (NPCs) to identify genes required for the self-renewal of GSC isolates, but which are dispensable for NPCs.  Here we report the retest of the GSC-lethal gene ZNF131, which encodes a novel vertebrate-specific BTB domain zinc finger transcription factor that is broadly required for GSC viability.  Examination of gene expression changes after ZNF131 knockdown (kd) revealed that ZNF131 activity notably promotes expression of Joubert Syndrome ciliopathy genes, including KIF7, NPHP1, and TMEM237, as well as HAUS5, a component of Augmin/HAUS complex that facilitates microtubule (MT) nucleation along the mitotic spindle.  Of these genes only kd of HAUS5 displayed GSC-specific viability loss, and, critically, HAUS5 ectopic expression was sufficient to suppress viability defects of ZNF131 kd cells.  Moreover, ZNF131 and HAUS5 kd phenocopied each other in GSCs, each causing: mitotic arrest, centrosome fragmentation, loss of Augmin/HAUS complex on the mitotic spindle, and loss of GSC self-renewal and tumor formation capacity.  In control NPCs, we observed centrosome fragmentation and lethality only when HAUS5 kd was combined with kd of HAUS2 or HAUS4, two other Augmin complex members, demonstrating that the complex is essential in NPCs, but that GSCs have heightened requirement. Our results suggest that GSCs differentially rely on ZNF131-dependent expression of HAUS5 as well as the Augmin/HAUS complex activity to maintain the integrity of centrosome function and viability.  We speculate that this requirement likely arises from aneuploidy frequently observed in late stage GBM tumors, rather than supernumerary centrosomes.

first: Yu

last: Ding

middle: A

last: Herman

first: Chad

last: Toledo

first: Jacquelyn

last: Lang

first: Philip

last: Corrin

first: Emily

middle: J

last: Girard

first: Ryan

last: Basom

first: Jeffery

last: Delrow

first: James

last: Olson

first: Patrick

last: Paddison


title: Modeling signaling-dependent pluripotent cell states with boolean logic can predict cell fate transitions [II]

city: Toronto

first: Peter

last: Zandstra

state: Ontario

address: 160 College Street, University of Toronto

department: Institute of Biomaterials and Biomedical Engineering

country: Canada

institute: University of Toronto

summary: Pluripotent stem cells (PSCs) exist in multiple stable states, each with specific cellular properties and molecular signatures. The process by which pluripotency is either maintained or destabilized to initiate specific developmental programs is poorly understood. We have developed a model to predict stabilized PSC gene regulatory network (GRN) states in response to combinations of input signals.  While previous attempts to model PSC fate have been limited to static cell compositions, our approach enables simulations of dynamic heterogeneity by combining an Asynchronous Boolean Simulation (ABS) strategy with simulated single cell fate transitions using a Strongly Connected Components (SCCs). This computational framework was applied to a reverse-engineered and curated core GRN for mouse embryonic stem cells (mESCs) to simulate responses to LIF, Wnt/β-catenin, FGF/ERK, BMP4, and Activin A/Nodal pathway activation. For these input signals, our simulations exhibit strong predictive power for gene expression patterns, cell population composition, and nodes controlling cell fate transitions. The model predictions extend into early PSC differentiation, demonstrating, for example, that a Cdx2-high/Oct4-low state can be efficiently generated from mESCs residing in a naïve and signal-receptive state sustained by combinations of signaling activators and inhibitors.

overall_design:
Examination of perturbed PSCs versus control PSCs and mesoderm progenitors
Mouse pluripotent stem cells were grown on tissue culture plates for two days in serum-containing, feeder free medium supplemented with the following cytokines/small molecules:
2i = CHIR99021 (Reagents Direct 27-H76 – 3µM) & PD0325901 (Reagents Direct 39-C68 – 1µM)
Jaki = JAK inhibitor (EMD Millipore 420097 – 2.0µM)
BMP = BMP4 (R&D Systems 314-BP-010 – 10ng/ml)
Alk5i = ALK5 inhibitor II (Cedarlane ALX-270-445 - 10µM)

first: Ayako

last: Yachie-Kinoshita

first: Kento

last: Onishi

first: Joel

middle: E

last: Ostblom

first: Eszter

last: Posfai

first: Janet

last: Rossant


title: Single-cell RNA-seq of NR5A1-eGFP positive cells of the developing mouse testis from E10.5 to E16.5

city: Ramat Gan

first: Isabelle

last: Stévant

address: Institute of Nanotechnology and Advanced Materials, Building 206

country: Israel

institute: University of Bar Ilan

summary:
Purpose: We performed a time-course single-cell RNA-seq of the somatic cells of the XY mouse gonads to study the cell population heterogeneity and the genetic program during their differentiation.
Methods: We collected gonads from NR5A1-eGFP transgenic embryos at five embryonic stages: E10.5, E11.5, E12.5, E13.5, E16.5. Cells were captured with the C1 autoprep system and cDNA sequenced with Illumina HiSeq 2000.
Results: One cell population was detected at E10.5 and gives rise to both Sertoli and fetal Leydig cells. A precursor cell population remains undifferentiated at E16.5 and are likely to be adult Leydig cell precursors.
Conclusion: Our study is, to date, the most granular transcriptomic study of the developing mouse testis and provide a more complete model of somatic cell differentiation during male sex determination.

overall_design: 435 cells were collected in total. 64 cells at E10.5, 98 cells at E11.5, 76 cells at E12.5, 120 cells at E13.5, 77 cells at E16.5. We performed two independent captures for each embryonic stage to reach a reasonable number of cells except for E10.5 where we captured enough cells in one experiment.

first: Yasmine

last: Neirjinck

first: Christelle

last: Borel

first: Jessica

last: Escoffier

first: Lee

middle: B

last: Smith

first: Stylianos

last: Antonarakis

first: Emmanouil

middle: T

last: Dermitzakis

first: Serge

last: Nef


title: Modeling signaling-dependent pluripotent cell states with boolean logic can predict cell fate transitions [I]

overall_design:
Examination of perturbed PSCs versus control PSCs and TSCs
Mouse pluripotent stem cells were grown on tissue culture plates for two days in serum-containing, feeder free medium supplemented with the following cytokines/small molecules:
2i = CHIR99021 (Reagents Direct 27-H76 – 3µM) & PD0325901 (Reagents Direct 39-C68 – 1µM)
Jaki = JAK inhibitor (EMD Millipore 420097 – 2.0µM)
BMP = BMP4 (R&D Systems 314-BP-010 – 10ng/ml)
Alk5i = ALK5 inhibitor II (Cedarlane ALX-270-445 - 10µM)


title: CHRAC/ACF Contribute to the Repressive Ground State of Chromatin [RNA-seq]

status: Public on Feb 05 2018

city: Munich

first: Tamas

last: Schauer

address: Feodor-Lynen-Straße 21

department: Institute of Epigenetics and Stem Cells

institute: Helmholtz Zentrum München

summary: The chromatin remodeling complexes CHRAC and ACF combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression is unclear. Here we explored the influence of Drosophila CHRAC/ACF on transcription by complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of a Acf knock-out allele showed that only lowly expressed genes are de-repressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.

overall_design: Comparison of Acf mutant embryos to wild-type (Oregon-R) in two embryonic stages

first: Alessandro

last: Scacchetti


title: Genome-wide maps of H3k56ac in HSCs

status: Public on Feb 04 2018

city: hangzhou

first: Hu

last: Wang

address: wenyixi road 1378

institute: hangzhou normal university

summary: H3K56ac is a target of SIRT6. So we measured the H3K56ac modifications in SIRT6 KO and control HSCs.

overall_design: Examination of H3K56ac modifications in SIRT6 KO and control HSCs.

first: Zhenyu

last: Ju


title: miR25/93 mediates the hypoxia-induced immunosuppression by repression of cGAS

first: Max

last: Chang

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

overall_design: Refer to individual Series


title: Transcriptomic Analysis of Adult Zebrafish Inner Ear Hair Cells

city: Omaha

first: David

last: He

state: Nebraska

address: 2500 California Plaza

department: Department of Biomedical Sciences

institute: Creighton University

summary: To understand the basic biological property of hair cells (HCs) from lower vertebrates, we examined transcriptomes of adult zebrafish HCs.  GFP-labeled HCs were isolated from the utricle, saccule, and lagena, the three inner-ear sensory epithelia of a pou4f3 promoter-driven GAP-GFP line of transgenic zebrafish.  2,000 HCs and 2,000 non-sensory cells from the inner ear were individually collected by suction pipet technique.  RNA sequencing was performed and the resulting sequences were mapped, analyzed, and compared. Comparisons allow us to identify enriched genes in HCs, which may underlie HC specialization.

overall_design: Examination of transcriptomes of adult zebrafish inner ear hair cells and surrounding cells individually collected and sorted using pou4f3 promoter-driven GFP marking hair cells.

first: Cody

middle: L

last: Barta

first: Huizhan

last: Liu

first: Lei

last: Chen

first: Kimberlee

middle: P

last: Giffen

first: Yi

last: Li

first: Kenneth

last: Kramer

first: Kirk

last: Beisel

middle: Z


title: RNA sequencing for identifying downstream targets of miR25/93

summary: we identify several downstream targets that are under control of miR25/93 cluster

overall_design: examination of global changes in mRNAs in two different lines

first: Min-Zu

last: Wu

first: Juan

last: Izpisua Belmonte


title: small RNA sequencing for identifying hypoxic responsive miRNAs

summary: we identify certain hypoxia responsive miRNAs that associate with hypoxia-induced tumor malignancy

overall_design: examination of global changes in small RNAs in four different lines


title: Gene expression changes upon activation of RIG-I by a minimal potent RIG-I ligand

status: Public on Feb 03 2018

city: New Haven

first: Anna

middle: Marie

last: Pyle

state: CT

address: 219 Prospect St.

institute: Yale University

summary: We have developed highly potent synthetic activators of the vertebrate immune system that specifically target the RIG-I receptor.  When introduced into mice, a family of short, triphosphorylated Stem Loop RNAs (SLRs) induces a potent interferon response and the activation of specific genes essential for antiviral defense.  Using RNAseq, we provide the first in-vivo genome-wide view of the expression networks that are initiated upon RIG-I activation.  We observe that SLRs specifically induce type I interferons, subsets of interferon-stimulated genes (ISGs), and cellular remodeling factors.  By contrast, poly(I:C), which binds and activates multiple RNA sensors, induces type III interferons and several unique ISGs. The short length (10-14 base pairs) and robust function of SLRs in mice demonstrate that RIG-I forms active signaling complexes without oligomerizing on RNA. These findings demonstrate that SLRs are potent therapeutic and investigative tools for targeted modulation of the innate immune system.

overall_design: We have triplicate samples of spleens from mice treated with SLR10, SLR14, poly(I:C) and pppNS, we have duplicate samples of mice treated with jetPEI vehicle control, and we have triplicate samples of untreated control mice

first: Thayne

last: Dickey

first: Emanuela

last: Molinari

first: Melissa

last: Linehan

first: Megan

last: Fitzgerald

first: Olga

last: Potapova

first: Akiko

last: Iwasaki


title: Characterizing steroid hormone receptor chromatin binding landscapes in male and female breast cancer

status: Public on Feb 02 2018

city: Amsterdam

first: Tesa

last: Severson

address: 121 Plesmanlaan

department: Molecular Carcinogensis

institute: Netherlands Cancer Institute

summary: Male breast cancer (MBC) is rare and poorly characterized. Like the female counterpart, most MBCs are hormonally driven, but resistance to hormonal treatment is common. We use transcriptomics to reveal gender-selective and genomic location-specific hormone receptor actions, which associated with survival in MBC patients.

overall_design: Gene expression analysis of male breast tumors (RNA-seq on 46 samples).

first: Yongsoo

last: Kim

first: Stacey

last: Joosten

first: Karianne

last: Schuurman

first: Petra

last: van der Groep

first: Paul

last: van Diest

first: Wilbert

last: Zwart


title: H3K27 acetylation and gene expression during late spermatogenesis

city: Cincinnati

first: So

last: Maezawa

address: 3333 Burnet Ave.

department: Reproductive Sciences

institute: Cincinnati Children's Hospital Medical Center

summary: ChIP-seq for H3K27 acetylation and RNA-seq were performed during spermatogenesis. We analyzed two representative stages of spermatogenesis: purified pachytene spermatocytes (PS) undergoing meiosis; and postmeiotic round spermatids (RS) from adult testes.

overall_design: ChIP-seq analyses using spermatocytes and spermatides from wild-type mice and RNA-seq analysis using spermatocytes and spermatides from Rnf8- and Scml2-dKO mice

first: Artem

last: Barski

first: Satoshi

last: Namekawa


title: Genetic dissection of chromatin regulators reveals the INO80 chromatin remodeling complex as a key metabolic regulator utilizing TORC1 and histone acetylation pathways

city: Stanford

first: Sean

last: Beckwith

address: 371 Serra Mall

institute: Stanford University

summary: We performed RNA-seq in wild-type yeast, a strain lacking INO80, and a strain lacking ARP5, central components of the INO80 chromatin remodelling complex, treated with rapamycin

overall_design: Yeast mRNA profiles were generated for WT, arp5, ino80 mutant strains treated with rapamycin

first: Graeme

last: Gowans

first: Ashby

last: Morrison


title: Transcriptome profiling of Drosophila Wild Type and Sc overexpressed intestinal progenitor cells

city: Beijing

first: huanwei

last: huang

address: 7 Science Park Rd., ZGC life science park

institute: NIBS

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study  are to compare transcriptome profiling (RNA-seq) of intestinal progenitor cells in Drosophila wild type and Sc overexpressed midgut.
Methods: mRNA profiles of intestinal progenitor cells isolated from 2-day-old wild-type (Ctrl) and Sc overexpressed(ScOE) Drosophila midgut were generated by deep sequencing using Illumina HiSeq 4000.
Results:

first: Jun

last: Chen

first: Rongwen

last: Xi


title: CHRAC/ACF Contribute to the Repressive Ground State of Chromatin [Mnase-seq]

first: Tamas

last: Schauer

summary: The chromatin remodeling complexes CHRAC and ACF combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression is unclear. Here we explored the influence of Drosophila CHRAC/ACF on transcription by complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of a Acf knock-out allele showed that only lowly expressed genes are de-repressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together our results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.

overall_design: MNase-Seq in Drosophila melanogaster embryos carrying a UAS-LacZ-mini-white transgene and expressing either Gal4DBD, Gal4-DBD-Acf1 or Acf1-Gal4DBD

first: Alessandro

last: Scacchetti

first: Laura

last: Brueckner

first: Dhawal

last: Jain

first: Xu

last: Zhang

first: Frank

last: Schnorrer

first: Bas

last: van Steensel

first: Tobias

last: Straub

first: Peter

last: Becker


title: CHRAC/ACF Contribute to the Repressive Ground State of Chromatin


title: Polysomal profiling in control and Ddx27-/- C2C12 myoblasts

city: Boston

first: Vandana

last: Gupta

state: Massachusetts

address: 77 Avenue Louis pasteur, 168A

department: Genetics

institute: Brigham and Women's Hospital

summary: Purpose: DDX27 regulates skeletal muscle developmental by regulating ribosomal biogenesis. Therefore, to identify translational changes in DDx27 deficiency, polysomal profiling was performed.

overall_design: Polysomal profiling was performed in control and Ddx27 knockout myoblasts in triplicates and RNA was isolated. Total RNA from cells was also also isolated and used to compare enrichment in polysomes.


title: Mouse natural killer cells response to DKK2 treatment

first: Weijia

last: Wang

address: 10 Amistad St

summary: Analysis of mouse primary natural killer (NK) cells and NK cells treated with DKK2 for 24 hours.Sequencing of the mRNAs from DKK2-treated primary NK cells in comparison of those from mock-treated cells suggest an alteration in STAT signaling.

overall_design: Mouse primary NK cells were isolated from the spleens and cultured in the presence of 50 ng/ml recombinant murine IL-15 for 24 hours. And then NK cells were treated with mock or 200ng/ml DKK2 for another 24 hours before mRNA was isolated and purified by using RNeasy Plus Mini Kit (Qiagen). A total of two groups of Control NK cells and two groups of DKK2-treated NK cells were individually micromanipulated.

first: Qian

last: Xiao

first: Jibo

last: Wu

first: Wei-Jia

first: Yingxia

last: Zheng

first: Xiaoqing

last: Yu

first: Lin

last: Li

first: Wenwen

last: Tang

first: Dianqing


title: Arabidopsis Messenger RNA N6-methyladenosine Demethylation Regulates Plant Development

first: Guifang

last: Jia

address: college of chemistry and molecular engineering, Peking University

institute: Peking University

summary: N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and α-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants.

overall_design: m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in three biological replicates

first: Hong-Chao

last: Duan


title: Small RNA profiling of WT E14, Ago2_KO and Ago2_KO-AGO1 mouse embryonic stem cells.

city: Zurich

first: Constance

last: Ciaudo

email: cciaudo@ethz.ch, ciaudobeyer@gmail.com

address: Otto-Stern-Weg 7

department: D-BIOL

institute: ETH

summary: Purpose:  The goals of this study was to obtain the profile total miRNAs in WT E14, Ago2_KO mESCs to compare with Ago2_KO-AGO1 overexpressing AGO1.

overall_design: ALL small RNA profiles from total RNA were generated by deep sequencing, in duplicate, using Illumina HiSeq2000.


title: Transcriptome of Ago2_KO mouse embryonic stem cells (mESCs) before and after XEN differentiation

summary: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study was to obtain the trasncriptome of Ago2_KO mESCs to compare it with the transcriptome of WT mESCs before and after XEN conversion (deposit separately).

overall_design: mRNA profiles of Ago2_KO mESCs,  wildtype XEN and Ago2_KO XEN were generated by deep sequencing, using Illumina HiSeq2000. Wild type mESCs were deposited in GSE78971.


title: Modeling signaling-dependent pluripotent cell states with boolean logic can predict cell fate transitions

last: Zandstra


title: Small RNA profiling of WT E14, Ago2_KO and Ago1_KO mouse embryonic stem cells before and after XEN conversion and small RNA profiling after Immunoprecipitation of AGO1&2 proteins in WT E14, Ago1_KO and Ago2_KO mESCs.

summary: Purpose:  The goals of this study was to obtain the profile total miRNAs in WT E14, Ago2_KO and Ago1_KO mESCs to compare it with Immunoprecipitated microRNAs in AGO1&2 proteins.

overall_design:
ALL small RNA profiles from total RNA or after immunoprecipitation were generated by deep sequencing, in duplicate, using Illumina HiSeq2000.
Small RNA profiling in Dicer_KO mESCs as been perform as a control library.


title: Roles of Argonaute protein in mouse embryonic stem cells


title: Gene expression changes in Hutchinson Gilford Progeria Syndrome (HGPS) are counteracted by calcitriol

city: St Louis

first: Susana

last: Gonzalo

state: MO

address: 1100 S Grand Blvd

department: Biochemistry and Molecular Biology

institute: Saint Louis University

summary: RNA expression was measured using RNA-seq

overall_design: RNA levels of normal human primary fibroblasts and HGPS patient-derived fibroblasts subjected to short (4 days) and long (3 months) treatment with calcitriol (1,25D). RNA levels were compared among all different samples.

first: Dale

last: Dorsett

first: Ray

last: Kreienkamp


title: Transcriptome dynamics at Arabidopsis graft junctions reveal an intertissue recognition mechanism that activates vascular regeneration

city: Würzburg

first: Alexander

last: Gabel

address: Josef-Schneider-Str. 2 / D15

institute: Helmholtz Institute for RNA-based Infection Research

summary: The ability for cut tissues to join together and form a chimeric organism is a remarkable property of many plants, however, grafting is poorly characterized at the molecular level. To better understand this process we monitored genome-wide temporal and spatial gene expression changes in grafted Arabidopsis thaliana hypocotyls. Tissues above and below the graft rapidly developed an asymmetry such that many genes were more highly expressed on one side than the other. This asymmetry correlated with sugar responsive genes and we observed an accumulation of starch above the graft that decreased along with asymmetry once the sugar-transporting vascular tissues reconnected. Despite the initial starvation response below the graft, many genes associated with vascular formation were rapidly activated in grafted tissues but not in cut and separated tissues indicating that a recognition mechanism activated that was independent of functional vascular connections. Auxin which is transported cell-to-cell, had a rapidly elevated response that was symmetric, suggesting that auxin was perceived by the root within hours of tissue attachment to activate the vascular regeneration process. A subset of genes were expressed only in grafted tissues, indicating that wound healing proceeded via different mechanisms depending on the presence or absence of adjoining tissues. Such a recognition process could have broader relevance for tissue regeneration, inter-tissue communication and tissue fusion events.

overall_design: We analyzed the poly-adenylated transcriptomes of Arabidopsis thaliana hypocotyle tissue during grafting. Our dataset contains 82 strand-specific samples, whereas each condition is represented by two biological replicates.

first: Charles

last: Melnyk

first: Thomas

last: Hardcastle

first: Sarah

last: Robinson

first: Shunsuke

last: Miyashima

first: Ivo

last: Grosse

first: Elliot

last: Meyerowitz


title: Precursors of human CD4+ cytotoxic T lymphocytes identified by single-cell transcriptome analysis [10X genomics]

city: La Jolla

first: Pandurangan

last: Vijayanand

address: 9420 Athena Circle

department: Division of Vaccine Discovery

institute: La Jolla Institute for Allergy and Immunology

summary: CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, heterogeneity and clonal diversity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA-seq in over 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile and clonality in humans. The single-cell differential gene expression analysis, revealed a spectrum of known transcripts, including several linked to cytotoxic and co-stimulatory function, and transcripts of unknown cytotoxicity-related function that are expressed at higher levels in the TEMRA subset, which is highly enriched for CD4-CTLs, compared to cells in the central and effector memory subsets (TCM, TEM). Simultaneous T cells antigen receptor (TCR) analysis in single-cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared to TCM and TEM cells and that the majority of CD4-TEMRA were dengue virus (DENV)-specific in subjects with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across subjects, with four distinct clusters identified by the single-cell analysis. Most importantly, we identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and thus could provide insights into the mechanisms that may be utilized to generate durable and effective CD4-CTL immunity.

overall_design: Single cell RNA-seq analysis of  purified populations of human CD4 memory cell subsets by both bulk TCR-sequencing.

first: Veena

middle: S

last: Patil

first: Ariel

last: Madrigal

first: Benjamin

last: Schmiedel

first: James

last: Clarke

first: Patrick

last: O'Rourke

first: Eva

last: Harris

first: Aruna

middle: D

last: de Silva

first: Bjoern

last: Peters

first: Grégory

last: Seumois

first: Daniela

last: Weiskopf

first: Alessandro

last: Sette


title: Comparing murine lung resident alveolar Siglec-F(high) macrophages to CD11b(high) macrophages following bleomycin injury

city: Denver

first: Alexandra

last: McCubbrey

state: CO

address: 1400 Jackson St

institute: National Jewish Health

summary: Macrophages (MΦ) have been shown to contribute to fibrogenesis, however the underlying mechanisms and specific MΦ subsets involved remain unclear.  Lung MΦ can be divided into two subsets: Siglec-Fhi resident alveolar MΦ and CD11bhi MΦ that primarily arise from immigrating monocytes. RNA-seq analysis was performed to compare these MΦ subsets during fibrosis.  CD11bhi MΦ, not Siglec-Fhi MΦ, expressed high levels of pro-fibrotic chemokines and growth factors.

overall_design: C56BL/6 WT mice were treated intratracheally with bleomycin.  8 days later, CD64+Mertk+ MΦ were sorted into Siglec-F(high) and CD11b(high) subsets.  SiglecF(high) MΦ from naïve mice were also sorted.  RNA was isolated and RNA-seq was performed to compare  MΦ subsets.

first: Thomas

last: Danhorn

first: Sonia

middle: M

last: Leach

first: William

last: Janssen


title: A transcriptomic and functional analysis of the interaction between Caenorhabditis elegans and Stenotrophomonas maltophilia

status: Public on Feb 01 2018

city: Manhattan

first: Michael

last: Herman

state: KS

address: 266 Chalmers Hall

department: Division of Biology

institute: Kansas State University, Ecological Genomics Institute

summary: The goal of this study was to elucidate genes that are employed by the bacterivorous nematode Caenorhabditis elegans to respond to the emerging nosocomial bacterial pathogen Stenotrophomonas maltophilia.

overall_design: 12 microarrays (4 per bacterial treatment) were used to identify genes that were differentially expressed when exposed to virulent (JCMS) and avirulent (K279a) strains of S. maltophilia and an avirulent (OP50) strain of E. coli.

first: Corin

last: White


title: Sequence variation within similar cis-elements promotes context-specific functions of two Drosophila GA-binding transcription factors (ChIP-seq data set)

last: Tolstorukov

state: MA

address: 185 Cambridge Street

department: Molecular Biology

institute: Massachusetts General Hospital

summary: A key model for understanding how large transcription complexes are targeted is the Drosophila dosage compensation system in which the Male-Specific Lethal (MSL) transcription complex specifically identifies and regulates the male X-chromosome. MSL complex is targeted to GA-containing sequences, but the most well-studied GA-binding transcription factor, GAGA Associated Factor (GAF), does not physically associate with MSL complex. Instead the Chromatin Linked Adapter for MSL Proteins (CLAMP) zinc-finger protein specifically targets MSL complex to GA-rich sequences on the X-chromosome. Here, we compare the binding relationships of CLAMP, GAF, and the MSL3 dosage compensation complex protein using ChIP-seq.

overall_design: To map the occupancy of CLAMP and GAF relative to each other and to MSL complex at high resolution, we performed three to four replicates of ChIP-seq on all three factors in male Drosophila S2 cells under RNAi knockdown conditions of GFP (control), GAF (trl), and CLAMP.

first: Emily

middle: G

last: Kaye

first: Matthew

last: Booker


title: Inter-replicon gene flow contributes to transcriptional integration in the Sinorhizobium meliloti multipartite genome

city: Hamilton

first: Turlough

last: Finan

address: 1280 Main St. W.

department: Department of Biology

institute: McMaster University

summary: Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT). Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT.  The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid (termed pSymB) consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid (termed pSymA) consisting primarily of recently acquired genes.  Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid.  Removal of the megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ~ 8% of chromosomal genes and ~ 4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome.  No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched.  Down-regulated genes were enriched in motility and sensory transduction pathways.  Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions.  In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome.

overall_design: There was a total of 10 samples for the RNA-sequencing analysis: five strains performed in biological duplicates.  Total RNA was isolated from S. meliloti RmP3496 (∆pSymAB), RmP3497 (∆pSymB), RmP3498 (∆pSymA), RmP3499 (wild-type), and RmP3500 (∆pSymB with the ETR region of S. fredii integrated into the chromosome), grown in minimal medium with 10 mM sucrose as the carbon source and ammonia as the nitrogen source.  Sequencing was performed using Illumina HiSeq 1500 with 75 nt single reads.

first: George

last: diCenzo

first: Deelaka

last: Weelapilli

first: Brian

last: Golding


title: Transcriptional Pause Sites Delineate Stable Nucleosome-Associated Premature Polyadenylation Suppressed by U1 snRNP [2P-seq, Pabpn1 CKO]

city: Nagoya

first: Hiroshi

middle: I.

last: Suzuki

address: 65 Tsurumai-cho, Showa-ku

department: DIvision of Molecular Oncology, Graduate School of Medicine

country: Japan

institute: Nagoya University

summary: Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.

overall_design: 3'-End sequencing (2P-seq) in Pabpn1 conditional KO mESCs

first: Anthony

last: Chiu

middle: I

first: Phillip

last: Sharp


title: A TAD boundary is preserved upon deletion of the CTCF-rich Firre locus

status: Public on Feb 12 2018

city: Durham

first: Rasim

last: Barutcu

state: NC

address: 6 Davis Drive

institute: ScitoVation

summary:
ABSTRACT
The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). However, the general mechanisms of folding the genome into TADs are not fully understood. Here, we tested the effects of deleting a CTCF-rich locus on TAD boundary formation. Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~15 CTCF binding sites, and located at the long non-coding RNA (lncRNA) locus Firre. Specifically, this TAD boundary is invariant across evolution, tissues, and temporal dynamics of X-chromosome inactivation. We demonstrate that neither the deletion of this locus, nor the ectopic insertion of Firre cDNA or its ectopic expression are sufficient to alter TADs in a sex-, or allele-specific manner. In contrast, Firre’s deletion disrupts the chromatin super-loop formation of the inactive X-chromosome. Collectively, our findings suggest that apart from CTCF binding, additional mechanisms may play roles in establishing TAD boundary formation.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series.

first: A

middle: R

first: Philipp

last: Maass

first: Jordan

last: Lewandowski

first: Catherine

last: Weiner

first: John

last: Rinn


title: Genome-wide relationship between R-loop formation and antisense transcription in Escherichia coli

city: Hyderabad

first: J

last: Gowrishankar

state: Telangana

address: Survey Nos. 728-730,Uppal

country: India

institute: Centre for DNA Fingerprinting and Diagnostics

summary: Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA-DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW. We also provide evidence that in Rho-deficient cells, R-loop formation blocks subsequent rounds of antisense transcription at more than 500 chromosomal loci. Hence these antisense transcripts, which can extend beyond 10 kb in their length, are only detected when Rho function is absent or compromised and the UvsW helicase is concurrently expressed. Thus the potential for antisense transcription in bacteria is much greater than hitherto recognized; and the cells are able to retain viablity even when nearly one-quarter of their total non-rRNA abundance is accounted for by antisense transcripts, provided that R-loop formation from them is curtailed.

overall_design: Strand specific RNA seq experiments were performed on ribosome-depleted RNA from 5 E.coli cultures, each in triplicate, total= 15 samples

first: Nalini

last: Raghunathan

first: Jayaraman


title: RNA-sequencing based analysis of Listeria monocytogenes 10403S and H7858 and corresponding ΔsigB mutants under exposure to pH 5.5 with or without bile

city: Ithaca

first: Veronica

last: Guariglia

state: New York

address: 320 Stocking Hall

department: Food Science

institute: Cornell University

summary: In this study, RNA-seq was used to compare the transcriptomes of Listeria monocytogenes 10403S and H7858 and corresponding ΔsigB mutants under exposure to pH 5.5 with or without bile.  RNA-seq was performed on all strains and conditions in four independent biological replicates. Indexed and purified cDNA libraries (12 libraries per strains) were loaded together onto an independent flow cell without any other samples; sequencing was carried out by running Hiseq 2500 (single-end, 100-bp per read). Reads alignment was carried out using the Burrows-Wheeler Aligner (BWA). Differential expression of genes in different strains and conditions was statistically assessed using the BaySeq method.

overall_design: Transcriptome profiles of L. monocytogenes 10403S and H7858 and corresponding ΔsigB mutants under exposure to pH 5.5 with or without bile were generated by deep sequencing, in quadruplicate, using Illumina Hiseq 2500.


title: Next Generation Sequencing Study of Circadian Changes in Transcriptome of Zebrafish Pineal Gland and Eye

city: New York

first: Marjan

last: Bozinoski

state: NY

address: 1305 York Avenue, Box 140

department: Physiology and Biophysics

institute: Weill Cornell Medicine

summary:
Purpose: We performed an NGS study on the circadian changes in zebrafish eye and pineal gland transcriptome in order to elucidate novel and conserved elements in the circadian clock.
Methods: Poly-A selected RNA from zebrafish eyes and pineaal glands of animals eutanized at 2 timepoints (Mid-Day, Midnight)  was deep sequenced, using Illumina HiSeq2000. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2.
Results: We discover a variety of genes that show circadian activivty in both eye and pineal gland of Zebrafish.
Conclusions: Our study represents part of a comparative analysis of retinal(eye) and pineal gland transcriptome of several species, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: Adult (0.5-1.5 years old) transgenic zebrafish, Tg(aanat2:EGFP), which express enhanced green fluorescent protein (EGFP) in the pineal gland under the control of the aanat2 regulatory regions, were used. Fish were raised under 12-hr light:12-hr dark (LD) cycles in a temperature controlled room. For tissue collection fish were anesthetized in 1.5 mM Tricane (Sigma), and sacrificed by decapitation. Dissections were conducted at mid-day (ZT6) and mid-night (ZT18). Fluorescent pineal glands, whole eyes and other tissues were removed under a dissecting microscope. A pool of 18 pineal glands 6 eyes and 4-8 of each peripheral tissue was collected at each time point. Total RNA for mRNA analysis was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions. The mixed tissue samples consisted of equal amounts of total RNA (1mg) from muscle, ovary, kidney, gill, liver, intestines, heart and brain.  Poly-A selected RNA from zebrafish eyes, mixed tissue and pineaal glands of animals eutanized at 2 timepoints (Mid-Day, Midnight)  was deep sequenced, using Illumina HiSeq2000. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2.

first: David

middle: C

last: Klein

first: Christopher

last: Mason

first: Yoav

last: Gothilf

first: Steven

last: Coon


title: Functional characterization and genomic studies of a novel murine submandibular gland epithelial cell line.

city: Buffalo

first: Rose-Anne

last: Romano

address: 3435 Main Street

department: Oral Biology

institute: State University of New York at Buffalo

summary: Gene expression analysis of spontenously immortalized mouse submandibular salivary gland epithelial cells grown in culture at different passages and in different media conditions

overall_design:
Submandibular salivary gland epithelial cells isolated from adult, (12 week old) C57BL/6 mice were spontaneously immortalized in culture, and RNA was isolated and analyzed for early passage, mid passage and late passage cells
Mouse oral keratinocyte cells isolated from adult, (12 week old) C57BL/6 mice were spontaneously immortalized in culture, and RNA was isolated and analyzed in triplicates

first: Sangwon

last: Min

first: Christine

last: Song

first: Akinsola

last: Oyelakin

first: Christian

last: Gluck

first: Kirsten

last: Smalley

first: Satrajit

last: Sinha


title: Phosphotyrosine-mediated regulation of enterohemorhagic Escherichia coli virulence

city: Baltimore

first: Tracy

last: Hazen

state: MD

address: 801 W. Baltimore St.

institute: Institute for Genome Sciences

summary: Enteric pathogens with low infectious doses rely on the ability to orchestrate expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link expression of type III secretion system (T3SS) factors to nutrient availability. While phosphorylation of histidine and aspartate on two-component system response regulators is recognized as an integral part of signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli revealed 342 proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. Here, we demonstrate that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression in glycolytic conditions typical for the colon lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating expression of ler, encoding the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA-binding, thereby altering expression of metabolism and virulence-associated genes including those of the LEE pathogenicity island encoding the T3SS. Hence, phosphotyrosine-mediated regulation provides a mechanism to regulate Cra activity. Our data further suggest that tyrosine phosphorylation influences DNA binding by PurR and LacI, thereby  phosphotyrosine-mediated control could provide a means to regulate DNA-binding of LacI/GalR family regulators in general. Our study provides an initial effort to unravel the role of phosphotyrosine-mediated global signaling in controlling the EHEC virulence potential

overall_design: The EHEC EDL933 stx- strain TUV93-0 carrying an in-frame deletion of cra was used in this study. Transcriptomes were determined of biological duplicates of TUV93-0 ∆cra::FRT producing plasmid-encoded wild type Cra, non-phosphorylatable Cra Y47F and phosphomimetic Cra Y47D, which represent a total of six samples.

first: Colin

last: Robertson

middle: H

first: James

last: Kaper

middle: A

last: Rasko

first: Anne-Marie

last: Hansen


title: MiR-129-5p downregulates HuD expression impairing neuritogenesis in Amyotrophic Lateral Sclerosis

city: Torino

first: Raffaele

last: Calogero

state: To

address: Via Nizza 52

department: Molecular Biotechnology Center

institute: University of Torino

summary: Amyotrophic lateral sclerosis (ALS) involves the degeneration of brain and spinal cord motor neurons. Mutations in Superoxide Dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43) and Fused-in-Sarcoma (FUS) account for 20-30 % of the familial ALS (fALS) cases. The RNA-binding proteins TDP-43 and FUS function in mRNA and miRNA biogenesis. MiRNAs are required for survival of neurons and deregulation of miRNA expression has been reported in several neurodegenerative disorders. Here, we report the dysregulation of DROSHA, DGCR8, and DICER in human neuroblastoma SH-SY5Y cells expressing the ALS-associated SOD1(G93A) mutant protein. MiRNA profiling in SH-SY5Y/SOD1(G93A) cells and transgenic SOD1(G93A) mice revealed upregulation of miR-129-5p at the early stage of disease. Moreover, miR-129-5p is also upregulated in lymphocytes of sporadic ALS patients. We demonstrate that miR-129-5p targets ELAVL4/HuD mRNA by binding to its 3’ UTR, which reduces HuD expression and impairs differentiation and neurite outgrowth. Conversely, treatment with an antagomir or complementation with HuD protein restores neuritogenesis. Collectively, our study identifies miR-129-5p and HuD as key regulators of neuronal differentiation and as potential therapeutic targets for ALS.

overall_design: Human neuroblastoma SHSY-5Y cells stably transfected with cDNAs coding for wild type SOD1, four biological replicates, or the mutant SOD1(G93A), four biological replicates, were sequenced

first: Alessia

last: Loffreda

first: Alessandro

last: Arosio

first: Marc-David

last: Ruepp

first: Stefano

last: Volinia

first: Caterina

last: Bendotti

first: Carlo

last: Ferrarese

last: Lunetta

first: Oliver

last: Mühlemann

first: Lucio

last: Tremolizzo

first: Silvia

last: Barabino


title: Cross-Site Comparison of Ribosomal Depletion Kits for Illumina RNAseq Library Construction

city: West Lafayette

first: Jyothi

last: Thimmapuram

state: IN

address: YONG 1022, 155 S. Grant Street

department: Bioinformatics Core

institute: Purdue University

summary: In a cross-site study we evaluated the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. We found that all of the kits were capable of performing significant ribosomal depletion, though there were differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes among kits suggested that transcript length may be a key factor in library production efficiency. These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.

overall_design:
The Universal Human Reference RNA (Agilent) was diluted to 500 ng/ul in 200ul of RNase-free water and 3.94ul of the Spike-in RNA Variant Control E2 Mix (Lexogen) were added. The sample was split into two aliquots, one of which was then heated at 94° C for 1 hour and 27 minutes. 1ul of ERCC RNA Spike-In Mix 1 was added to both the intact and degraded samples. The final intact and degraded RNA samples were then diluted to 25 ng/uL and were distributed to each of the ten genomics core facilities (members of ABRF) for ribo-depletion and library preparation following vendor protocol. Each site prepared between one and four library types. Indices were assigned by the group to prevent overlapping among libraries. Libraries were pooled at an equimolar concentration from each kit using site-specific quantification and pooling SOPs and return each pool along with individual un-pooled libraries to the designated sequencing site. The sequencing site quantified each pool, multiplexed and sequenced over three high output paired-end 75bp runs on the Illumina NextSeq 500.
contributor: The Association of Biomolecular Resource Facilities (ABRF) DNA Sequencing Research Group (DSRG) members


title: A human mitochondrial DNA genetic bottleneck prevents mutational meltdown by purifying the early maternal germ line

first: Sabine

last: Dietmann

address: 4565 McKinley Avenue

institute: Washington University School of Medicine

summary: Mitochondrial DNA (mtDNA) mutations cause inherited diseases and are implicated in the pathogenesis of common late-onset disorders, but it is not clear how they arise and propagate in the humans. Here we show that mtDNA mutations are present in primordial germ cells (PGCs) within healthy female human embryos. Close scrutiny revealed the signature of selection against non-synonymous variants in the protein-coding region, tRNA gene variants, and variants in specific regions of the non-coding D-loop. In isolated single PGCs we saw a profound reduction in the cellular mtDNA content, with discrete mitochondria containing ~5 mtDNA molecules during early germline development. Single cell deep mtDNA sequencing showed rare variants reaching higher heteroplasmy levels in later PGCs, consistent with the observed genetic bottleneck, and predicting >80% levels within isolated organelles. Genome-wide RNA-seq showed a progressive upregulation of genes involving mtDNA replication and transcription, linked to a transition from glycolytic to oxidative metabolism. The metabolic shift exposes deleterious mutations to selection at the organellar level during early germ cell development. In this way, the genetic bottleneck prevents the relentless accumulation of mtDNA mutations in the human population predicted by Muller’s ratchet. Mutations escaping this mechanism will, however, show massive shifts in heteroplasmy levels within one human generation, explaining the extreme phenotypic variation seen in human pedigrees with inherited mtDNA disorders.

overall_design: RNA-Seq and NGS analysis to investigate transcriptomes and mtDNA sequences of fetal hPGCs

first: V

first: S

first: W

last: Tang

first: M

last: Surani

last: Chinnery


title: Natural Killer cells control tumor growth by sensing a growth factor

city: San Diego

first: John

last: Walker

state: CA

address: 10675 John Jay Hopkins

institute: Genomics Institute of the Novartis Research Foundation

summary: Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44 encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of IFN-γ and TNF-α that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell cycle genes correlated with NCR2 and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, whilst cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.

overall_design: RNAseq of NK cell and tumor cell samples in reponse to various stimuli

first: Alexander

last: Barrow

first: Melissa

last: Edeling

first: Vladimir

last: Trifonov

first: Jingqin

last: Luo

first: Piyush

last: Goyal

first: Benjamin

last: Bohl

first: Jennifer

last: Bando

first: Albert

last: Kim

first: Mary

last: Andahazy

first: Mattia

last: Bugatti

first: Laura

last: Melocchi

first: William

last: Vermi

first: Daved

last: Fremont

first: Sarah

last: Cox

first: Marina

last: Cella

first: Christian

last: Schmedt

first: Marco

last: Colonna


title: A Brain Penetrant Mutant IDH1 Inhibitor Provides In Vivo Survival Benefit

city: Kenilworth

first: Diane

last: Levitan

state: NJ

address: 2015 Galloping Hill Road

department: Translational Medicine

institute: Merck

summary: We profiled two IDH1 mutant glioma models, grown as intracranial xenografts, treated with an IDH1 inhbitor.  One model, BT424, was responsive to IDH1 inhibition by compound treatement and also displayed more transcriptional changes in response to treatment.  The other model, GB10, was not responsive to IDH1 inhibition and was transcriptionally quiet after treatment with compound.

overall_design: BT142 or GB10 xenograft bearing animals were treated with 30 mpk MRK-A or vehicle for 4 weeks


title: RNA sequencing (RNA-SEQ) of PSPC1 knockdown by shRNA in A549 cells

city: Taipei

first: HSI-Wen

last: Yeh

state: TAIWAN

address: Academia Sinica, No. 128

department: Institute of biomedical  science

country: Taiwan

institute: Academia Sinica

summary: We show that PSPC1 is required for cancer cell motilities and stemness properties in vitro and lung metastasis in vivo. We used high throughput sequencing to analyze the PSPC1 regulated gene expression. Loss of PSPC1 results in significant gene expression level changes in thousands of individual transcripts in key regulators of the EMT, CSCs and TGFb signaling.

overall_design: Methods: Gene expression profiles of A549 shPSPC1 knockdown cells were generated by Illumna RNA sequencing and compared to profiles derived from control cells (shLacZ knockdown).

first: Hsi

middle: W

first: Yuh

last: Jou


title: Genome-wide maps of Ad4BP/SF-1 binding in mouse Leydig cells

city: Fukuoka

first: Tetsuya

last: Sato

address: 3-1-1, Maidashi Higashi-ku

institute: Kyushu University

summary: To determine whole target genes of Ad4BP/SF-1, ChIP-seq was performed. Soluble chromatin was prepared from mouse Leydig cells treated or non-treated with cAMP. As a result, 5,721 and 4,025 ChIP-peaks were identified in Leydig and cAMP-treated Leydig cells, respectively.

overall_design: Determination of target sites of Ad4BP/SF-1 by ChIP-seq in mouse testicular Leydig cells

first: Takashi

last: Baba

first: Ken-ichirou

last: Morohashi


title: Genome-wide genetic variation identification and fruit-associated trait comparisons between kumquat (Fortunella japonica) and Clementine mandarin

city: WuHan

first: jin

middle: zhi

last: zhang

address: College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan 430070, China

institute: Huazhong Agricultural University

summary: We performed whole genome re-sequencing to reveal the comprehensive genetic variation of the fruit development between kumquat (Fortunella japonica) and Clementine mandarin. Total 5,865,235 single-nucleotide polymorphisms (SNPs) and 414,447 insertion/deletion (InDels) were identified in the two citrus species. Meanwhile, a total of 640,801 SNPs and 20,733 InDels were identified based on integrative analysis of genome and transcriptome of fruit. The variation feature, genomic distribution, functional effect and other characteristics of these genetic variation were explored. Total 1,090 differentially expressed genes (DEGs) were found during fruit development process of kumquat and Clementine mandarin by RNA-sequencing. Gene Ontology revealed that these genes were involved in various molecular functional and biological processes. Meanwhile, the genetic variation of 939 DEGs and 74 multiple fruit development pathway genes from previous reported were also identified. In addition, a global survey of genes splicing events identified 24,237 specific alternative splicing (AS) events in the two citrus species and showed that intron retention is the most prevalent pattern of alternative splicing.

sample_organism:
Citrus japonica
Citrus x clementina

platform_organism:
Citrus japonica
Citrus x clementina

overall_design: The materials from Clementine mandarin and Kumquat were collected in the experiment fields of the National Citrus Breeding Center at Huazhong Agricultural University (30°28′ N, 114°21′ E, 30). Healthy leaves without insect damage of Clementine mandarin and Kumquat were used as materials for re-sequencing. The five developmental stages of Clementine mandarin fruit were collected at 15, 30, 45, 100 and 180 days after flowering (DAF), respectively. Meanwhile, the five developmental stages of Kumquat fruit were also collected at 15, 30, 45, 80 and 120 DAF, respectively. And fruit growth including length, width, and fresh weight during fruit developmental stage can be seen in Figure 5A. To prepare a representative sample of total RNA from Clementine mandarin and kumquat tissues for RNA-sequencing (RNA-Seq), different developmental stages of fruit from roughly equal numbers of Clementine mandarin and kumquat were pooled for each sample. All plant tissues were sampled according to the demands of each experiment, immediately frozen in liquid nitrogen, and stored at -80℃ until used. All materials were collected from three individual plants for RNA and DNA extraction.

first: Chun-Gen

last: Hu

first: Jin-Zhi

last: Zhang


title: Next Generation Sequencing Study of Circadian Changes in Transcriptome of Human Pineal Gland

first: Marjan

last: Bozinoski

summary:
Purpose: We performed an NGS study on the circadian changes in human pineal gland transcriptome in order to elucidate novel and conserved elements in the circadian clock, as well as to conduct a comparative analysis of pineal transcriptomes of several animal species.
Methods: Total RNA from human pineal glands of individuals that died at 2 timepoints (Mid-Day, Midnight)  was deep sequenced, using Illumina HiSeq2500. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2.
Results: We discover a variety of genes that show circadian activivty in human pineal gland.
Conclusions: Our study represents part of a comparative analysis of  pineal gland transcriptome of several species, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: Pineal glands were obtained from NDRI (National Disease Research Interchange), (Pennsylvania Medical Center, Philadelphia, PA). Two glands were from individuals that died at mid-day (11:00-13:00) and four from deaths occurring at mid-night (23:00-01:00). Samples were stored at -80 o C until use. RNA was extracted from individual glands with Trizol (Invitrogen, Carlsbad,CA) and subsequently cleaned up using an RNeasy Micro Kit with on-column DNase treatment(Qiagen, Valencia, CA). The amount and quality of RNA were determined using a NanoDropspectrophotometer (NanoDrop, Wilmington, DE) and an Agilent 2100 Bioanalyzer (AgilentTechnologies, Santa Clara, CA), respectively. RIN values ranged from 7.3-8.7. RNA-Seq library preparation and sequencing: Ribosomal-depleted RNA was used to construct stranded libraries from 1 µg aliquots of total RNA using a TruSeq Stranded Total RNA Sample Prep Kit (Illumina cat. no. RS-122- 2301) according to the manufacturer's instructions. The library insert sizes were approximately 170 bp. Unique barcode adapters were appended to each library. Equal volumes of individual libraries were pooled and run on a MiSeq (Illumina, San Diego, CA). The libraries were then re-pooled based on the MiSeq demultiplexing results. The pooled libraries were sequenced on a HiSeq 2500 (Illumina, San Diego, CA) using version 4 chemistry. The  data was processed using RTA version 1.18.64 and CASAVA 1.8.2. This yielded an average of 81 million 126-bp read-pairs for each sample. Reads were aligned using STAR aligner and differential expression was asssessed using DESeq2. GRCh37/hg19 genome build with Gencode 19 annotation was used.

first: David

last: Klein

first: Christopher

middle: E

last: Mason

first: Steven

last: Coon


title: Single-cell RNA-seq of mouse dopaminergic neurons informs candidate gene selection for sporadic Parkinson's disease

status: Public on Jan 31 2018

first: Loyal

last: Goff

address: 733 N. Broadway Avenue

department: Institute of Genetic Medicine

institute: Johns Hopkins University

summary: Genetic variation modulating risk of sporadic Parkinson’s disease (PD) has been primarily explored through genome wide association studies (GWAS). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal timepoints. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a novel postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including known PD genes and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1 null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research.

overall_design: 473 single cell RNA-Seq samples from sorted mouse Th-eGFP+ dopaminergic neurons collected at two timepoints from three distinct brain regions.

first: Paul

last: Hook

first: Sarah

last: MyClymont

first: Gabrielle

last: Cannon

first: William

last: Law

first: A

middle: J

last: Morton

first: Andrew

last: McCallion


title: Transctriptional profiling of murine hematopoietic stem and progenitor cells upon β-glucan administration

city: Dresden

first: Triantafyllos

last: Chavakis

address: Fiedlerstraße 42

department: Klinische Pathobiochemie

institute: Medizinische Fakultät der TU Dresden

summary: Objective: Transcriptional profiling of murine HSPC in response to  β-glucan-induced innate immune training

overall_design: HSPC mRNA profiles of  wild type (WT) mice injected with PBS or  β-glucan. Wild type (WT) C57BL/6 mice were intraperitoneally injected with PBS or 1 mg   β-glucan in PBS. Mice were sacrificed on day 7 or day 28 and long-term heematopoietic stem cells (LT-HSC) and/or multipotent progenitors (MPP) were sorted. In another group, mice were injected with PBS or 1 mg   β-glucan in PBS and on day 7 they were additionally injected with 150 mg/kg 5-fluouracil. Mice were sacrificed on day 14 after 5-FU administration and LT-HSC were sorted.

first: Ioannis

last: Mitroulis

first: Mathias

last: Lesche