title: A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks

city: Cambridge

first: Oren

last: Parnas

address: 415 main street

department: Biology

institute: BROAD INSTITUTE

summary: We introduced genome-wide pooled CRISPR-Cas9 libraries into primary mouse dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (TNF) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the TLR4 pathway. We found many of the known regulators of TLR4 signaling, as well as dozens of previously unknown candidates that we validated.

overall_design: We used stain base phenotype (staining for TNF) in order to search for negative and positive regulators of LPS response in differentiated BMDCs

last: Parans

first: Marko

last: Jovanovic

first: Thomas

last: Eisenhaure

first: Rebecca

middle: H

last: Herbst

first: Atray

last: Dixit

first: Chun

last: Ye

first: Feng

last: Zhang

first: Nir

last: Hacohen

first: Aviv

last: Regev


title: FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders

status: Public on Jul 16 2015

city: New Haven

first: Flora

last: Vaccarino

state: CT

address: 230 South Frontage Road

department: Child Study Center

country: USA

institute: Yale University

type: Expression profiling by high throughput sequencing

summary: Autism spectrum disorder (ASD) is a disorder of brain development believed, in most cases, to be of genetic origin. We use induced pluripotent stem cells (iPSCs)-derived 3-dimensional neural cultures (organoids) in patients with ASD and macrocephaly to investigate neurodevelopmental alterations that cause this form of ASD. By using transcriptome analyses, we identified modules of co-expressed genes significantly upregulated in ASD patients compared to non-ASD first-degree family members.

overall_design: Total RNA was prepared from terminal differentiation day 0, 11 and 31 of iPSCs-derived neural cultures from ASD patients and non-ASD first-degree family members. A total of 4 patients and 8 controls (unaffected family members) were analyzed in replicates (two to three iPSC clones per person).

first: Jessica

last: Mariani

first: Gianfilippo

last: Coppola

first: Ping

first: Alexej

last: Abyzov

first: Lauren

last: Provini

first: Livia

last: Tomasini

first: Mariangela

last: Amenduni

first: Anna

last: Szekely

first: Dean

last: Palejev

first: Michael

last: Wilson

first: Mark

last: Gerstein

first: Elena

middle: L

last: Grigorenko

first: Katarzyna

last: Chawarska

first: Kevin

middle: A

last: Pelphrey

first: James

middle: R

last: Howe

middle: M


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [mRNA-Seq]

city: Boston

first: Shannan

last: Ho Sui

state: Massachusetts

address: 401 Park Drive, Level 3 East

department: Biostatistics

institute: Harvard T.H. Chan School of Public Health

summary: Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies.

overall_design: mRNA sequencing of primary and secondary fibroblasts with reference BJ (supplementary file fibroblasts), reprogramming intermendiates from untreated hiF-T reprogramming (supplementary file reprogramming), or selective time points upon LSD1 inhibitor treatment (supplementary file LSD1i). RNA samples used for mRNA sequencing are the same used for smallRNA sequencing.

first: Davide

last: Cacchiarelli

first: Cole

last: Trapnell

middle: J

last: Ziller


title: Eed inactivation in Cdkn2a-null MLL-AF9 transformed cells

status: Public on Jul 15 2015

city: Aurora

first: Tobias

last: Neff

state: CO

department: Pediatric Oncology

institute: University of Colorado Denver AMC

summary: We sequenced mRNA from MLL-AF9 transformed Cdkn2ako murine LSK-cells with and without a functional Eed locus

overall_design: Comparison of mRNA levels between Eed_ff/Cdkn2ako and Eed_ko/Cdkn2ako cells in vitro

first: Etienne

last: Danis


title: The DAXX/ATRX Complex Protects Tandem Repetitive Elements in the Absence of DNA Methylation by Promoting H3K9 Trimethylation

city: Houston

first: Quanyuan

last: He

state: TX

address: One Baylor Plaza, Room S307

institute: Baylor College of Medicine

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: The DAXX/ATRX Complex Protects Tandem Repetitive Elements in the Absence of DNA Methylation by Promoting H3K9 Trimethylation [ChIP-Seq]

summary: In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription, translocation, and homologous recombination. However, DNA hypomethylation occurs during specific developmental stages (e.g. preimplantation embryos) and in certain cell types (e.g., primordial germ cells).  The absence of dysregulated repetitive elements in these cells suggests the existence of alternative mechanisms that prevent genome instability triggered by DNA hypomethylation. In this report, we seek to elucidate the factors that play a critical role in ensuring genome stability by focusing on DAXX and ATRX, two proteins that have been linked to transcriptional control and epigenetic regulation.  We carried out ChIP-seq and RNA-seq analyses to compare the genome-wide binding and transcriptome profiles of DAXX and ATRX in mouse ES (mES) cells triple knocked out for the three mammalian DNA methyltransferases (DNMTs) (TKO cells) to those in wildtype mES cells.  Our data indicate that DAXX and ATRX are distinct in their chromatin-binding profiles and highly co-enriched at tandem repetitive elements.  Global DNA hypomethylation, as was the case in TKO cells, further promoted the recruitment of the DAXX/ATRX complex to tandem repeat sequences including IAP (intracisternal A‐particle) retrotransposons and telomeres. Inhibition of DAXX or ATRX in cells with hypomethylated genomes (e.g., TKO cells, mES cells cultured in ground-state conditions, and preimplantation embryos) increased aberrant transcriptional de-repression of repeat elements and dysfunction at telomeres. Furthermore, we provide evidence that DAXX/ATRX-dependent silencing may occur through DAXX’s interaction with SUV39H1 and increased H3K9me3 on repetitive sequences. Our study suggests that DAXX and ATRX are important for safeguarding the genome, particularly in silencing repetitive elements in the absence of DNA methylation.

overall_design: We tested the hypothesis that the DAXX/ATRX complex participates in protecting repetitive elements in the absence of DNA methylation. To this end, we investigated genome-wide chromatin targeting of DAXX and ATRX in wildtype mES cells, and in mES cells that exhibit extensive loss of DNA methylation due to homozygous knockout of all three DNA.

first: Hyeung

last: Kim

first: Dan

last: Liu

first: Zhou

last: Songyang


title: The DAXX/ATRX Complex Protects Tandem Repetitive Elements in the Absence of DNA Methylation by Promoting H3K9 Trimethylation [RNA-Seq]

overall_design: We performed RNA-Seq experiments in four cell lines (J1, J1_DAXX_KO, TKO, and TKO_DAXX_KO) grown with or without media for ground state (vitamin C plus 2i). We then performed RNA-seq analyses for all these cell lines to compare the expression of DAXX/ATRX target genes.


title: Study of gene expression changes in TH-MYCN murine neuroblastomas in response to 24hr of panobinostat treatment

city: East Melbourne

first: Paul

middle: James

last: Wood

state: Victoria

address: 2 St. Andrews Place

department: Translational Research Laboratory

country: Australia

institute: Peter MacCallum Cancer Centre

summary: Neuroblastoma is the most common extra-cranial malignancy in childhood and accounts for approximately 15% of childhood cancer deaths. Amplification of N-Myc in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N-Myc and regulate numerous cellular processes, including differentiation in neuroblastoma. We therefore investigated the antitumor activity of the histone deacetylase inhibitor, panobinostat in the setting of N-Myc amplified neuroblastoma using the TH-MYCN murine model. Continuous treatment of tumor bearing TH-MYCN transgenic mice with panobinostat for 9 weeks led to a significant improvement in survival as compared to mice treated with vehicle, or continuous treatment with panobinostat for a three week period. Panobinostat induced rapid tumor regression with no regrowth observed during the treatment period. Tumor response was associated with an initial apoptosis phenotype mediated via up-regulation of BMF and BIM.  When treated was stopped at three weeks 100% of mice relapsed with aggressive neuroblastoma.  Differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N-Myc expression, was observed in mice treated for nine weeks, resulting in a sustained remission on 90% of mice treated. RNA-sequencing analysis of tumors from treated animals confirmed significant up-regulation of gene pathways associated with apoptosis and differentiation as early as 24 hours after treatment. Together our data demonstrate the potential of panobinostat as a therapeutic strategy for high-risk neuroblastoma patients.

overall_design: Eight homozygous TH-MYCN mice bearing neuroblastomas were treated with either 5mg/kg panobinostat (4 animals) or vehicle (4 animals) for 24hr (two doses) and tumours harvested 4hr post the second dose.

first: Kelly

last: Waldeck

first: Carleen

last: Cullinane

first: Kerry

last: Ardley

first: Jake

last: Shortt

first: Ben

last: Martin

first: Richard

middle: W

last: Tothill

first: Jason

last: Li

first: Ricky

last: Johnstone

first: Grant

last: McArthur

first: Rodney

last: Hicks


title: PRMT5 is essential for sustaining normal adlut hematopoiesis

city: Miami

first: Fan

state: Florida

address: 1501 NW 10th Ave

department: Biochemistry and Molecular Biology

institute: University of Miami

summary: Examining the effects of PRMT5 loss on the gene expression profile of hematopoietic stem and progenitor cells

overall_design: We established the PRMT5 conditional KO mice, and bred the mice with Mx1 cre transgenic mice to delete PRMT5 in hematopoietic cells. We isolated hematopoetic stem and progenitor cells 3 days after PRMT5 deletion, and examined the gene expression files using RNA-sequencing.

first: Stephen

middle: D

last: Nimer


title: Cas9 gRNA engineering for selectable genome editing, activation and repression

first: George

last: Chruch

state: MA

address: 77 Ave Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: RNA-Seq after Cas9-gRNA transfection with different length gRNAs

overall_design: we performed PolyA Selection and RNA-Seq on cells transfected with dCas9-VPR and a gRNA of each length (20nt, 16nt, or 14nt) targeting ACTC1, MIAT, or HBG1/2

first: Alejandro

last: Chavez

first: Dmitry

last: Ter-Ovanesyan


title: Interleukin-1 signaling is involved in local self-renewal and maintenance of microglia

city: Mainz

first: Ari

last: Waisman

address: Obere Zahlbacher Straße 67

country: Germany

institute: Institute for Molecular Medicine

summary:
During early embryogenesis microglia arise from yolk sac progenitors populating the developing CNS, where they are maintained as tissue-resident macrophages throughout the organism’s lifespan.
Here, we describe an experimental system that allows the specific conditional ablation of microglia in vivo.
Strikingly, we found that the microglia compartment was reconstituted within one week following depletion. Microglia repopulation relied entirely on a CNS-resident, internal pool and was independent from bone marrow-derived precursors.
Newly formed microglia were found in highly proliferative, organized micro-clusters that dissolve once steady state was achieved. Gene expression profiling revealed prominent expression of Interleukin-1 (IL-1) receptor in proliferating microglia.
During the repopulation phase, IL-1 signaling was neutralized by treatment with IL-1 receptor antagonist that impaired microglia proliferation. Hence, microglia harbor a highly efficient potential to restore themselves without contribution of peripheral myeloid cells.
IL-1 signaling significantly participates in this restorative proliferation process and is involved in stabilizing microglia maintenance.

overall_design: bone marrow macrophages, wild type microglia, and repopulating microglia

first: Julia

last: Bruttger

first: Federico

last: Marini


title: Nurr1 and Retinoid X Receptor ligands stimulate Ret signaling in dopamine neurons and can alleviate α-synuclein disrupted gene expression

city: Stockholm

first: Nikolaos

last: Volakakis

address: Nobels vag 3

country: Sweden

institute: LICR

summary: We ovexpressed human alpha synuclein alone or together with Nurr1 in mouse primary midbrain cultures and identified the full spectrum of genes whose expression is affected by alpha synuclein, including genes whose expression is normalized after Nurr1 overexpression. Moreover we treated mouse primary midbrain cultures with Bexarotene or short hairpin RNA fro Nurr1, sorted out the dopamine neurons and assessed the effects of Bexarotene and of the Nurr1 downregulation on gene expression.

overall_design: Comparison of 3 Synuclein samples to 5 controls (RFP), Comparison of 3 Synuclein + Nurr1 samples to 5 controls (RFP), Comparison of 3 Bexarotene samples to 3 controls (DMSO), comparison of 1 short hairpin against Nurr1 to 1 control (scrambled).


title: RNA-seq in bas1 and ino4 mutants

city: New York

first: Xuan

last: Zhu

state: NY

address: 430 East 67th Street

institute: Memorial Sloan Kettering Cancer Center

summary: Meiotic recombination is initiated by developmentally programmed DNA double-strand breaks (DSBs). In S. cerevisiae, the vast majority of DSBs occur in the nucleosome-depleted regions at gene promoters, where transcription factors (TFs) B296bind. It has been proposed that TF binding can stimulate DSB formation nearby by modulating local chromatin structure. However, a prior study in TF bas1 mutant suggested that the role of TF binding in determining break formation is complex. Here, we examined fine-scale DSB distributions in TF mutant (bas1Δ and ino4Δ) strains. In bas1Δ mutants, 239 out of the 2468 hotspots showed reduced DSB activity, whereas 87 hotspots showed increased DSB activity. Similarly, in ino4Δ mutant, 415 out of the 2468 hotspots showed reduced DSB activity, whereas 322 hotspots showed increased DSB activity. We also mapped Bas1 and Ino4 binding sites in meiosis and found that only a small portion of the affected hotspots contained TF binding sites. This indicates that TF can influence DSB distribution both directly and indirectly. Surprisingly, these DSB changes in TF mutants did not correlate with change in chromatin structure and histone H3K4me3 modification, suggesting that the role of TF on DSB distribution cannot be simply explained by affecting local chromatin status.

overall_design: Six samples total: wild type, bas1 mutants, ino4 mutants; replicates for each genotype

first: Scott

last: Keeney


title: mRNA and small RNA associated with Crohn's disease behavior [ncRNA-Seq]

city: Chapel Hill

first: Bailey

last: Peck

state: NC

address: 120 Mason Farm Rd, 5100H Genetic Medicine Bldg

institute: University of North Carolina at Chapel Hill

type: Non-coding RNA profiling by high throughput sequencing

summary: There is a need for reliable prognostic markers that can guide therapeutic intervention in Crohn’s disease (CD). We examined whether different behavioral phenotypes in CD can be classified based on colonic miRNA or mRNA expression and if miRNAs have prognostic utility for CD. We perform high-throughput sequencing of small RNA and mRNA isolated from colon tissue from CD patients and non-IBD (NIBD) controls. To identify miRNA and genes associated with specific behavioral phenotypes of CD, patients were stratified according to disease behavior (non-stricturing, non-penetrating; stricturing; penetrating) and compared miRNA profiles in each class with those of the NIBD group. Using a novel statistical simulation approach applied to colonic RNA-seq data for CD patients and NIBD controls, we identify at drivers of gene expression profiles associated with CD.

overall_design: Macroscopically non-inflamed colon tissue from characterized Crohn's disease patients and normal controls were obtained. Small RNA-seq and RNA-seq were performed on these samples. Additionally, we investigated the effect of inflammation on miRNA expression by performing small RNA-seq on matched colon samples obtained from macroscopically inflamed regions from a subset (six) of these patients with CD.

first: Shezhad

middle: Z

last: Sheikh

first: Praveen

last: Sethupathy

first: Terrence

middle: S

last: Furey

middle: C

first: Mathew

last: Weiser


title: Genome-wide reprogramming of transcript architecture by temperature specifies the developmental states of the human pathogen Histoplasma

city: San Francisco

first: Mark

last: Voorhies

state: CA

address: 513 Parnassus, S472

department: Microbiology and Immunology

institute: University of California


title: mRNA and small RNA associated with Crohn's disease behavior [RNA-Seq]

overall_design: Macroscopically non-inflamed colon tissue from well-characterized Crohn's disease patients and normal controls were obtained. Small RNA-seq and RNA-seq were performed on these samples. Additionally, we investigated the effect of inflammation on miRNA expression by performing small RNA-seq on matched colon samples obtained from macroscopically inflamed regions from a subset (six) of these patients with Crohn's Disease.


title: mRNA and small RNA associated with Crohn's disease behavior


title: High resolution mapping of enhancer-promoter interactions [ChIA-PET]

first: David

last: Gifford

address: 32 Vassar St.

department: Computer Science and Artificial Intelligence Laboratory

institute: Massachusetts Institute of Technology

summary: RNA Polymerase II ChIA-PET data has revealed enhancers that are active in a profiled cell type and the genes that the enhancers regulate through chromatin interactions. The most commonly used computational method for analyzing ChIA-PET data, the ChIA-PET Tool, discovers interaction anchors at a spatial resolution that is insufficient to accurately identify individual enhancers. We introduce $Germ$, a computational method that estimates the likelihood that any two narrowly defined genomic locations are jointly occupied by RNA Polymerase II. $Germ$ takes a blind deconvolution approach to simultaneously estimate the likelihood of RNA Polymerase II occupation as well as a model of the arrangement of read alignments relative to locations occupied by RNA Polymerase II. Both types of information are utilized to estimate the likelihood that RNA Polymerase II jointly occupies any two genomic locations. We apply $Germ$ to RNA Polymerase II ChIA-PET data from embryonic stem cells to identify the genomic locations that are jointly occupied along with transcription start sites. We show that these genomic locations align more closely with features of active enhancers measured by ChIP-Seq than the locations identified using the ChIA-PET Tool. We also apply $Germ$ to RNA Polymerase II ChIA-PET data from motor neuron progenitors. Based on the $Germ$ results, we observe that a combination of cell type specific and cell type independent regulatory interactions are utilized by cells to regulate gene expression.

overall_design: RNA Polymerase II ChIA-PET data from murine motor neuron progenitors were generated by deep sequencing.

first: Christopher

last: Reeder

first: Michael

last: Closser

first: Huay

last: Poh

first: Guoliang

last: Li

first: Kuljeet

last: Sandhu

first: Xiaoan

last: Ruan

first: Yijun

first: Hynek

last: Wichterle


title: Polycomb PRC2 antagonizes de novo DNA methylation at the maternal Gtl2-Rian-Mirg locus [Next-Gen Sequence Data]

city: Corvallis

middle: Anthony

last: Hendrix

state: OR

address: 2011 Ag & Life Sciences Bldg

department: Biochemistry and Biophysics/EECS

institute: Oregon State University

summary: Wild-type mouse embryonic stem cells are compared with mutants for components of PRC2 including  Ezh2-/-, Eed-/-, and Jarid2-/- cells. Chromatin modifications, Gene expression, Pol-II, small-RNA sequencing, and DNA methylation are compared for both cell types.

overall_design: To study genomic and epigentic control of PRC2 in mESCs, we designed gene expression analysis (RNA-Seq and small RNA-Seq), combining with ChIP-Seq analysis of several factors and histone marks from wild-type and distinct PRC2 mutants including wild-type, Ezh2-/-, Eed-/-, and Jarid2-/-.

first: Partha

middle: P

last: Das


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

first: Shannan

last: Ho Sui

type: Methylation profiling by high throughput sequencing

sample_organism:
Homo sapiens
Mus musculus
synthetic construct


title: Chip-seq BRD4NUT-BioTAP mapping in 293Trex and 797 cells

status: Public on Jul 14 2015

first: Peter

last: Kharchenko

address: 10 Shattuck St.

department: DBMI

summary: BRD4-NUT megadomains is an order of magnitude larger than super-enhancer regions and displays a more continuously enriched profile rather than appearing as a cluster of individual peaks.

first: Xin

last: Wang


title: Changes of axonal transcriptomes in response to local Abeta

first: Ulrich

last: Hengst

institute: Columbia University

summary: We report that local application of Abeta(1-42) for 24h triggers changes in the localized transcriptome, while the transcriptome in cell bodies remains largely unchanged.

overall_design: Axonal and somatic mRNA profiles of rat embryonic hippocampal neurons grown in microfluidic chambers were generated by RNA-Seq in duplicates using an Illumina MiSeq. Axons only were treated for 24 h with soluble oligomeric Abeta(1-42) or vehicle control.

first: Jimena

last: Baleriola

last: Nagy


title: Diversity in the polyadenylation and decay of human cellular microRNAs during Vaccinia virus infection

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

country: United Kingdom

institute: Centre for Immunity, Infection and Evolution

summary: Vaccinia virus (VACV) is a large cytoplasmic dsDNA virus that causes dramatic alterations to many cellular pathways including microRNA biogenesis. Here we use small RNA sequencing to comprehensively quantify the impact of VACV infection on the expression of endogenous small non-coding RNAs (sncRNAs) at both early (6 h) and late (24 h) times post infection.

overall_design: Non-coding RNAs (sncRNAs) were assessed at 6 hours and 24 hours, in naive and VACV-infected HeLa cells, in the presence or absence of the inhibitor AraC.  There were three replicates for each of the eight groups.

first: Amy

last: Buck

first: Beard

last: Philippa


title: Polycomb PRC2 antagonizes de novo DNA methylation at the maternal Gtl2-Rian-Mirg locus

first: David


title: Chip-seq mapping of active chromatin marks in BRD4-NUT and different NMC cells


title: Tribbles-1 regulates hepatic lipogenesis through post-transcriptional regulation of C/EBPα

city: Philadelphia

first: Robert

last: Bauer

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania


title: The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains


title: Tribbles-1 regulates hepatic lipogenesis through post-transcriptional regulation of C/EBPα [ChIP-Seq]

summary: We report ChIP-Seq data for C/EBPa in livers of mice with liver-specific KO (LSKO) of Trib1 as compared to WT controls, or in livers of mice overexpressing C/EBPa via adeno-associated virus (AAV) as compared to controls.

overall_design: 8-10 week old Trib1 flox/flox mice treated with AAV_Null (WT) or AAV_Cre (LSKO); 8-10 week old C57B/6 WT mice treated with AAV_Null or AAV_Cebpa.

first: Daniel

last: Cohen

last: Steger

last: Rader


title: BisPCR2 method for targeted bisulfite sequencing

first: Diana

last: Bernstein

address: 3400 Civic Center Blvd Bldg 421

department: Department of Genetics, IDOM

summary: We present a novel method, termed BisPCR2, for targeted bisulfite sequencing and apply it in the setting of validating type 2 diabetes CpG susceptibility loci

overall_design: Targeted bisulfite sequencing of five non-diabetic and five type 2 diabetic human pancreatic islets

first: Klaus

last: Kaestner


title: HIV-1 RNAs are not part of the Argonaute 2 associated RNA interference pathway in macrophages

first: Pejman

last: Mohammadi

address: 101 Av. of the Americas

institute: New York Genome Center

summary: MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte-derived macrophages (MDM). The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs. PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified, suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

sample_organism:
Homo sapiens
Human immunodeficiency virus 1

overall_design: PAR-CLIP data (2 donors) and HITS-CLIP data (3 donor mix) of HIV-1 infected and noninfected MDM. In addition, small RNA sequencing data (4 donors) of HIV-1 infected and noninfected MDM.

first: Valentina

last: Vongrad

first: Jochen

last: Imig


title: Transcription Profiling of Malaria-Naïve and Semi-Immune Colombian Volunteers in a Plasmodium vivax Sporozoite Challenge

city: Atlanta

first: Monica

middle: L.

last: Rojas-Peña

state: GA

address: 310 Ferst Dr.

department: School of Biology

institute: Georgia Institute of Technology

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

overall_design: Refer to individual Series


title: Transcription Profiling of Malaria-Naïve and Semi-Immune Colombian Volunteers in a Plasmodium vivax Sporozoite Challenge [RNA-Seq]

summary: We report two clusters in the overall profiles of expression among the samples. At the parasitemia onset, there is a strong interferon response reflected in up-regulation of co-regulated transcripts, while unexpectedly we also see down-regulation of transcripts related to TLR signaling and innate immunity. RNASeq also suggested differential expression of reticulocytes and a subset of T cell function. No obvious difference in the transcriptomes of naïve and semi-immune volunteers was seen, however several hundred genes were up-regulated in naïve individuals.

overall_design: RNA-seq analysis was performed for 12 individuals (6 each from Buenaventura and Cali) for two of the time points, namely the diagnosis day and baseline (pre-challenge day).

first: Andres

last: Vallejo

first: Sócrates

last: Herrera

first: Greg

last: Gibson

first: Myriam

last: Arévalo-Herrera


title: Calorie restriction suppresses age-dependent hippocampal transcriptional signatures.

city: Orangeburg

first: Stephen

last: Ginsberg

address: 140 Old Orangeburg Road

department: Center for Dementia Research

institute: Nathan Kline Institute

summary: Calorie restriction (CR) enhances longevity and mitigates aging phenotypes in numerous species. Physiological responses to CR are cell-type specific and variable throughout the lifespan; however, the mosaic of molecular changes responsible CR benefits remain unclear, particularly in brain regions susceptible to deterioration throughout aging. Thus, we examined the influence of long-term CR on the CA1 hippocampal region, a key learning and memory brain area that is vulnerable to age-related pathologies, such as Alzheimer’s disease (AD). Through mRNA sequencing and NanoString nCounter analysis, we demonstrate that one year of CR feeding suppresses an age-dependent signature of 882 genes functionally associated with synaptic transmission-related pathways, including calcium signaling, long-term potentiation (LTP), and Creb signaling in wild-type mice. By comparing the influence of CR on hippocampal CA1 region transcriptional profiles at younger- (5 months) and older-adult (15 months) timepoints, we identify conserved upregulation of proteome quality control and calcium buffering genes, including heat shock 70 kDa proteins 1b and 5 (Hspa1b and Hspa5), protein disulfide isomerase family A members 4 and 6 (Pdia4 and Pdia6), and calreticulin (Calr). Expression levels of putative neuroprotective factors, klotho (Kl) and transthyretin (Ttr), are also elevated by CR throughout adulthood, although the global CR-specific expression profiles at young and older timepoints are highly divergent. At a previously unachieved resolution, our results demonstrate conserved activation of neuroprotective gene signatures and broad CR-suppression of age-dependent hippocampal CA1 region expression changes, indicating that CR functionally maintains a more youthful transcriptional state within hippocampal CA1 throughout aging.

overall_design: Hippocampal CA1 region mRNA profiles of younger- (5 months) and older-adult (15 months) mice on calorie-restricted (CR) and normal (AD) diets were generated by deep sequencing using Illumina HiSeq 2500.

first: Marissa

last: Schafer

first: Igor

last: Dolgalev

first: Melissa

last: Alldred

first: Adriana

last: Heguy


title: Genome wide DNA methylation analysis of leukemia and reprogrammed leukemia cells

city: Saarbrücken

first: Fabian

last: Müller

address: Campus A 2.4

institute: Saarland University

summary: We apply cellular reprogramming to an induced pluripotent cell state to human cell lines and primary samples exhibiting Chronic Myoloid Leukemia (CML). Addtionally, we generated an inducible CML BCR-ABL transgenic mouse model. Biological replicates (duplicates) were available for all samples. DNA methylation profiles for all cells were generated using Reduced Representation Bisulfite Sequencing (RRBS).

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: DNA methylation profiling of human leukemia cell lines, primary cells, derived iPS cells as well as transgenic mouse models for CML in duplicates using RRBS.

first: Giovanni

last: Amabile

first: Annalisa

last: Di Ruscio

first: Alexander

last: Ebralidze

first: Sarawut

last: Wongphayak

first: Christoph

last: Bock

last: Tenen


title: A DNA Hypomethylation Signature Predicts Novel Anti-Tumor Activity of LSD1 Inhibitors in SCLC (ChIP-Seq)

city: Collegeville

first: David

last: Soong

address: 1250 S Collegeville Rd

institute: GlaxoSmithKline

summary: Epigenetic dysregulation has emerged as an important mechanism in cancer.  Alterations in epigenetic machinery have become a major focus for new targeted therapies.  The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552.  This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inhibitor of LSD1.  A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition.  The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes suggesting this may be used as a predictive biomarker of activity.  The targeted mechanism coupled with a novel predictive biomarker make LSD1 inhibition an exciting potential therapy for SCLC, a highly prevalent, rarely cured, tumor type representing approximately 15% of all lung cancers.

overall_design: To investigate the mechanism of LSD1 efficacy in SCLC cell lines we used chromatin immunoprecipitation (ChIP) sequencing studies to examine the genomic distribution of LSD1 as well as H3K4me2 and H3K4me1 in NCI-H526 SCLC cells in the absence and presence of LSD1 inhibition.

first: Helai

last: Mohammad

first: Kimberly

last: Smitheman

first: Chandrashekhar

last: Kamat

first: Kelly

last: Federowicz

first: Glenn

last: Van Aller

first: Jess

last: Schneck

first: Jeff

last: Carson

first: Yan

last: Liu

first: Michael

last: Butticello

first: William

last: Bonnette

first: Shelby

last: Gorman

last: Degenhardt

first: Yuchen

last: Bai

last: McCabe

last: Pappalardi

first: Jiri

last: Kasparec

first: Xinrong

last: Tian

first: Ken

last: McNulty

first: Megan

last: Rouse

first: Patrick

last: McDevitt

first: Thau

last: Ho

first: Michelle

last: Crouthamel

first: Nestor

last: Concha

first: Charles

last: McHugh

last: Miller

first: Dashyant

last: Dhanak

first: Peter

last: Tummino

first: Christopher

last: Carpenter

first: Neil

last: Johnson

first: Christine

last: Hann

first: Ryan

middle: G

last: Kruger


title: Ezh2 maintains proliferation, transcriptional integrity, and the timing of late differentiation during mouse retina development

city: Salt Lake City

first: Monica

last: Vetter

state: UT

address: 20 South, 2030 East Rm. 320 BPRB

department: Neurobiology & Anatomy

institute: University of Utah

summary: We investigated the gene expression profile changes after Ezh2 conditional knockout in the mouse retina at E16.5. Loss of Ezh2 leads to up-regulation of PRC2 targeted genes including cell cycle regulators and multiple genes which are not normally expressed in the retina, including many Hox genes. Loss of Ezh2 resulted in a dramatic decline in progenitor proliferation by postnatal day 3, such that there is an early end to neurogenesis, and disruption of laminar organization. Although there are only minor effects on embryonic retinal development, there is accelerated differentiation of several late born cell types postnatally, including photoreceptors and Mueller glia, which become reactive by postnatal day 14.

overall_design: Peripheral retina was dissected at E16.5 from Pax6alpha-Cre:Ezh2fl/+ and Pax6alpha-Cre:Ezh2fl/null mouse embryos. Total RNA was purified and RNA deep sequencing was done using 4 controls and 4 conditional knockout samples.

first: Jianmin

last: Zhang


title: A DNA Hypomethylation Signature Predicts Novel Anti-Tumor Activity of LSD1 Inhibitors in SCLC

first: David

last: Soong


title: Recovery and analysis of nascent RNA

city: Boston

last: Kharchenko

institute: Harvard Medical School

summary: Nascent transcription profiles are shown for scaled megadomains and 100kb flanking regions before BRD4-NUT induction (0h) and at different time points (2h, 3h, 7h) following induction in 293T cells. Increase of the transcription from 0h to 7h after induction. Average level of transcriptional activity is reduced within the megadomains and their flanking regions following JQ1 treatment of TC-797 cells. Profile of nascent RNA-seq is shown for cells without JQ1 treatment, and for cells 1hr, 2.5hr and 4hr following JQ1 treatment.

overall_design: Recovery and analysis of nascent RNA

first: Xin

last: Wang


title: Spatially distinct neutrophil responses within the inflammatory lesions of pneumonic plague

status: Public on Jul 13 2015

first: William

middle: Evan

last: Goldman

address: 130 Mason Farm Rd.

department: Division of Infectious Diseases

summary: During pneumonic plague, the bacterium Yersinia pestis elicits the development of inflammatory lung lesions that continue to expand throughout infection. This lesion development and persistence is poorly understood. Here, we examine spatially distinct regions of lung lesions using laser capture microdissection and RNAseq analysis to identify transcriptional differences between lesion microenvironments. We show that cellular pathways involved in leukocyte migration and apoptosis are down regulated in the center of lung lesions compared to the periphery. Probing for the bacterial factor(s) important for the alteration in neutrophil survival, we show both in vitro and in vivo that Y. pestis increases neutrophil survival in a manner that is dependent on the type-III secretion system effector YopM. This research explores the complexity of spatially distinct host - microbe interactions and emphasizes the importance of cell relevance in assays in order to fully understand Y. pestis virulence.

overall_design: We examine spatially distinct regions of lung lesions using laser capture microdissection and RNAseq analysis to identify transcriptional differences between lesion microenvironments.  Sample types: uninfected BM-PMN, infected BM-PMN, lesion periphery, lesion center.

first: Nikolas

last: Stasulli

first: Joel

last: Parker

middle: E


title: Single cell transcriptomic analysis of thymic epithelial cells

first: CBDM

last: Lab

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

summary: Single cell transcriptomic analysis of wildtype and AireKO thymic epithelial cells

overall_design: Single cells were sorted by FACS for single cell RNAseq library preparation

first: Christophe

last: Benoist


title: MOF maintains Transcriptional Programs required to protect cells against Stress

city: Freiburg im Breisgau

first: Thomas

last: Manke

address: Stuebeweg 51

department: Bioinformatics and Deep-Sequencing Unit

institute: Max-Planck-Institute for Immunobiology and Epigenetics


title: MYC is a major determinant of mitotic cell fate

city: La Jolla

first: Don

middle: W.

last: Cleveland

address: 9500 Gilman Drive

institute: Ludwig Institute for Cancer Research

summary:
Purpose: To determine global gene expression changes following siRNA knockdown of Myc, Kcnk1, and Snta1 compared to non-targeting siRNAs or mock-transfected cells
Methods: Total RNA was processed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit according to manufacturer’s protocol. Generated cDNA libraries were sequenced using an Illumina HiSeq 2000 sequencer with four biological replicates sequenced per condition using single read, 50 cycle runs. Quality of sequencing reads were assessed using FastQC (Babraham Bioinformatics) and then aligned to a reference genome (hg19, UCSC Genome Browser) using TopHat. Sequencing yielded, on average, 23.7 million unique reads per sample with a 60.7 - 65.7% mapping rate. Cufflinks was used to generate transcript abundance for each annotated protein-coding gene as Fragments Per Kilobase of transcript per Million mapped reads (FPKM), and statistical analysis and comparison of FPKM values was calculated using R (Bioconductor).
Results: Fold changes comparing mock and a non-targeting siRNA were highly congruent. Myc RNAi induced numerous changes, with 955 downregulated genes and 1214 upregulated genes. The effect on Myc itself was relatively modest, possibly reflecting its ability to negatively auto-regulate its own expression. Gene ontology analysis highlighted ribosome biogenesis, metabolism, gene expression, cell cycle, and apoptosis pathways, consistent with known Myc functions. The Kcnk1 siRNA affected 424 genes, with KCNK1 itself one of the most repressed. While gene ontology analysis also highlighted metabolism and biosynthesis pathways, the p-values and fold enrichment scores were substantially lower, indicating that DiM can be suppressed without major effects on metabolism and biosynthesis pathways. The Snta1 siRNA deregulated 575 genes, with SNTA1 itself the most repressed gene. Cell cycle and mitosis-related gene ontology terms feature heavily, consistent with this siRNA accelerating mitotic exit. Interestingly, FoxM1, which drives G2/M gene expression was reduced 1.75-fold, indicating that this siRNA may disrupt mitotic controls by deregulating FoxM1.
Conclusions: Global gene expression profiling identifies Egr1 as regulator of mitotic cell fate.

overall_design: Total RNA was extracted from at least four replicates of siRNA-transfected cells and libraries for sequencing was prepared for each replicate.

last: Ly

first: Ying

last: Sun

first: Stephen

last: Taylor

first: Caroline

last: Topham


title: Unique gene program of rat mesenteric arteries as revealed by Deep RNA Sequencing

city: Baltimore

first: Amol

middle: Carl

last: Shetty

state: Maryland

address: 670 W. Baltimore Street, Floor 3

department: Institute for Genome Sciences

institute: University of Maryland Baltimore

summary:
This study used Deep RNA sequencing to define gene expression in the small resistance arteries and compare to a reference conduit artery, the aorta
Methods: Deep RNA Sequencing (~90 million reads) was performed on 2 samples of adult SD rat mesenteric arteries and compared to that of 2 samples of aorta.
Results: ~900 genes were identified that were expressed at least 2-fold difference between meseteric artery and aorta

overall_design: mRNA profiles of 2 samples of rat mesenteric artery and 2 samples of rat aorta were sequenced using Illumina HiSeq

first: John

last: Reho

first: Rachael

last: Dippold

first: Anup

last: Mahurkar

first: Steven

middle: A

last: Fisher


title: Deep sequencing analysis of microRNA expression in human melanocyte and melanoma cell lines

city: Beijing

first: yadong

last: yang

address: No.1-104 Beichen West Road, Chaoyang

country: China

institute: Beijing Institute of Genomics

summary: We investigated the miRNAome in human melanocyte and melanoma cell lines using high-throughput RNA sequencing. We identified a group of dysregulated miRNAs by comparing the miRNA expression profiles among melanoma cell lines. Target genes of these miRNAs participate in functions associated with the cell cycle and apoptosis. Gene networks were built to investigate the interactions of genes during melanoma progression. We identified that the key genes that regulate melanoma cell proliferation were regulated by miRNAs. Our findings provide further knowledge regarding the mechanisms of melanoma development.

overall_design: miRNA profiles of melanocyte (HEMn-LP), low metastatic melanoma (A375) and high metastatic melanoma (A2058) cell line were generated using Illumina GA

first: Xiangdong

last: Fang


title: Identification and characterization of microRNA expression in Ginkgo biloba L. leaves

city: Yangzhou

first: Biao

last: Jin

state: Jiangsu

address: Wenhui east road no. 48

department: College of Horticulture and Plant Protection

institute: Yangzhou University

summary: High-throughput small RNA sequencing were performed to identify a large number of miRNAs and their targets in mature female and male G. biloba leaves for the first time. We ascertained that the regulatory networks of the miRNAs are involved in many different primary biological processes based on potential target designations. Our study is the first to provide useful information for uncovering the regulatory networks of miRNAs in basal gymnosperm G. biloba leaves.

overall_design: small RNA sequencing in female and male leaves of G. biloba

first: Jian

last: Zhao


title: Impact of Tcf1 and Lef1 deficiency on follicular helper T (Tfh) cells

city: Washington

first: Weiqun

last: Peng

state: DC

address: 1918 F Street, NW , DC

institute: The George Washington University

summary: Comparison of transcriptome between control and Tcf1/Lef1-deficient germinal center Tfh (GC-Tfh) cells

overall_design: Control mice or those are deficient for Tcf1 and Lef1 transcription factors were infected with vaccinia virus. On day 8 after infection, the splenocytes were isolated and surface stained to identify CD44-high, CD62L-low, GFP+, CD4+ T cells. From these cells, PD-1+, CXCR5+ GC-Tfh cells were sorted for RNAseq analysis

first: Hai-Hui

last: Xue


title: RNA-seq analysis of Myt3 suppression and over-expression in mouse pancreatic islets

status: Public on Jul 10 2015

city: Vancouver

first: Brad

last: Hoffman

state: BC

country: Canada

institute: University of British Columbia

summary:
Purpose: Myt3 is a putative pro-survival factor in pancreatic islets and is expressed from E18.5 onwards. The genes that Myt3 regulates and its role in islet-cell development, function and survival are not fully known. The purpose of this study was to determine the changes in gene expression following the suppression or over-expression of Myt3 to identify possible pathways that may be regulated by Myt3.
Methods: Pancreatic islets were isolated from both male and female C57/B6 mice by collagenase digestion and were picked by hand. Hand-picked islets were plated onto 804G, a complete extracellular-matrix (ECM) produced by a rat bladder carcinoma cell line, treated tissue culture dishes and transduced with 1x106 pfu virus overnight. The next  day media was changed to fresh RPMI and islets were cultured for 7 days. At the end of the culture period islets were dispersed and FACs sorted for GFP+ cells. Sorted cells from at least 5 experiments were pooled and RNA was isolated using Trizol reagent. 800ng of RNA was used in the Illumina TruSeq kit for library preparation.
Results: Comparison of gene expression levels between control-transduced islets and either shMyt3- or Myt3-transduced islets resulted in the identification of 51 and 89 significantly altered genes respectively.
Conclusions: Our study represents the first comprehensive analysis of genes that are potentially regulated by Myt3 and highlight potential pathways that are likely important for mediating Myt3's effects of islet development, function and survival.

overall_design: Isolated islets were transduced with adenoviruses, cultured for 7 days on ECM and FACS sorted. Cells from up to 5 experiments were pooled and sequenced.

first: Bryan

last: Tennant


title: Gene Regulatory Circuitry of JNK Signaling Identifies Novel Regulators of Epithelial to Mesenchymal Transition

city: Cambridge

first: Sudhir

last: Thakurela

address: 7 Divinity Avenue

institute: HSCRB

summary:
Purpose: The purpose of this study is to investigate role of JNK signaling during EMT.
Method: Transcriptome of TGF-b treated NmuMG cells along with DMSO and JNKi treated NMuMG was generated using next generation high throughput sequencing in triplicates and duplicates respectively. Reads were mapped using Tophat and transcript abundance and differential expression was calculated using HTSeq-Count and DESeq programs.
Results: Using time course RNA-Seq data, we uncover a large number of coding and  noncoding RNAs that are modulated during stepwise progression of TGF-β-induced EMT. Concomitant with their activation behavior, Smad and JNK pathway are required for onset and  progression of EMT respectively, a finding that was also confirmed in patients. Transcriptome analysis further revealed a progressive dependency of EMT on JNK signaling.
Conclusion: We identified several novel transcription factors that require JNK signaling for their  enhanced expression upon EMT and show that depletion of these factors during EMT hampers acquisition of transcriptional and phenotypic changes hallmark of this process. These factors are similarly induced during neurogenesis, a process also involving  JNK activation and EMT.

first: Sanjeeb

last: Sahu

first: Angela

last: Garding

first: Vijay

middle: K

last: Tiwari


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and ZmMADS47 RNAi mutant Transcriptomes

city: Shanghai

first: zhenyi

last: qiao

address: 333 Nanchen Road, Life Science Building, Room 322

department: School of Life Science

institute: Shanghai University

summary: Analysis of  gene expression level. The hypothesis tested in the present study was that ZmMADS47 RNAi mutant influence the expression of nutritional metabolism genes. Results provide important information of the gene expression level regulation of zein family.

overall_design: Maize kernel mRNA profiles of 15DAP wild type (WT) and ZmMADS47 RNAi mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2000 (Zea mays).

first: Weiwei

last: Qi

first: Zhenyi

last: Qiao


title: Differential expression profiles of type I JAK inhibitor persistent vs. naïve MPN cells

status: Public on Jul 11 2015

city: New York

first: Jeffrey

address: 1275 York Avenue

department: Genomics Core

institute: Memorial Sloan Kettering Cancer Center

summary: The type I JAK inhibitor ruxolitinib is approved for therapy of MPN patients but evokes resistance with longer exposure. Several novel type I JAK inhibitors were studied and we show that they uniformly induce resistance via a shared mechanism of JAK family heterodimer formation.Here we studied the expression profiles of SET2 cell lines persistent to several different type I JAK inhibitors in comparison to naive SET2 cells or in comparison to SET2 cells with acute exposure to ruxolitinib.

overall_design: Analysis of RNA isolated from several type I JAK inhibitor SET2 cell lines in comparison to naïve SET2 cells

first: Sara

last: Meyer

first: Franck

last: Rapaport

first: Ross

last: Levine


title: Active Promoters give rise to false positive 'Phantom Peaks' in ChIP-Seq experiments

city: Martinsried

first: Tobias

last: Straub

address: Großhadener Str. 9

department: Biomedical Center, Bioinformatics

institute: LMU Munich

summary: We applied ChIP-seq to map the chromosomal binding sites for two nucleosome remodeling complexes containing the ATPase ISWI, ACF and RSF, in Drosophila embryos. Employing a panel of polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. For further validation we repeated the mapping using chromatin of mutant embryos that do not express ACF1 or RSF1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and give rise to ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin.

overall_design: Examination of various ACF1 and RSF1 antibodies in Drosophila melanogaster embryos which are wildtype or mutant for the antibody targets.

first: Dhawal

last: Jain

first: Sandro

last: Baldi

first: Angelika

last: Zabel

first: Peter

middle: B

last: Becker


title: Checking the origin utilisation of Haloferax mediterranei H13 through high-throughput DNA sequencing

first: Haibo

last: Yang

address: No.1 Beichen West Road, Chaoyang District

institute: Institute of Microbiology, Chinese Academy of Sciences

summary: A potential origin that appears to stay dormant in its native host Haloferax volcanii lacking the main active origins becomes activated and competent for replication of the entire chromosome when integrated into the chromosome of the origin-deleted H. mediterranei.

overall_design: Measurement of replication dynamics (marker frequency analysis; MFA) for Haloferax mediterranei H13

first: Lei

last: Wang

first: Hua

last: Xiang


title: Chordoma ATAC-Seq

first: Charles

last: Lin

address: 415 Main St.

institute: Broad Institute

summary: Chromatin profiling of chordoma collected by the Broad chordoma target discovery project

overall_design: paired end ATAC-Seq profiling in the UCH2 and MUGCHOR chordoma cell lines

first: Stuart

last: Schreiber

first: Tanaz

last: Sharifnia

first: Joanne

last: Kotz


title: Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq

city: Sheffiled

first: Bin

last: Hu

address: West Bank

department: MBB

institute: University of Sheffield

summary: Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein’s distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C.glabrata cells (the calibration genome) with S.cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this “internal standard” calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using PCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.

sample_organism:
Saccharomyces cerevisiae
Nakaseomyces glabratus

overall_design: Develop a method for quantitative ChIP-seq

first: Naomi

last: Petela

first: Kim

last: Nasmyth


title: Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens

city: Los Angeles

first: Sanjai

last: Sharma

department: Hematology Oncology

institute: UCLA West LA VA Medical Center

summary:
Background: To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.
Methods:  Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.
Results: An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q<0.05, fold change >2) were identified. Expression of selected DEGs (n=32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90% of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1).
Conclusion: The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR  for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis.

first: Wei

last: Liao

first: Gwen

last: Jordaan

first: Phillipp

last: Nham

first: Ryan

middle: T

last: Phandaan

first: Matteo

last: Pelegrini


title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-TAB-seq

city: Athens

first: Robert

middle: J

last: Schmitz

address: B416 Davison Life Sciences

department: Genetics

institute: University of Georgia

summary: N4-methylcytosine is a major DNA modification integral to restriction-modification (R-M) systems in bacterial genomes. Here we describe 4mC-Tet-Assisted Bisulfite-sequencing (4mC-TAB-seq), a method that accurately and rapidly reveals the genome-wide locations of N4-methylcytosines at single-base resolution. By coupling Tet-mediated oxidation with a modified sodium bisulfite conversion reaction, unmodified cytosines and 5-methylcytosines are read out as thymines, whereas N4-methylcytosines are read out as cytosines revealing their positions throughout the genome.

sample_organism:
synthetic construct
Caldicellulosiruptor acetigenus


title: Heterogeneous effects of massive hypoxia pathway activation in kidney cancer

city: Oxford

first: Rafik

last: Salama

address: Roosevelt Drive

institute: University of Oxford

summary: General activation of hypoxia-inducible factor (HIF) pathways is classically associated with adverse prognosis in cancer and has been proposed to contribute to oncogenic drive. In clear cell renal carcinoma (CCRC) HIF pathways are upregulated by inactivation of the von-Hippel-Lindau tumour suppressor. However HIF-1a and HIF-2a have contrasting effects on experimental tumour progression. To better understand this paradox we examined pan-genomic patterns of HIF DNA binding and associated gene expression in response to manipulation of HIF-1a and HIF-2a and related the findings to CCRC prognosis. Our findings reveal distinct pan-genomic organization of HIF isoform-specific DNA binding at thousands of sites. Overall associations were observed between HIF-1a-specific binding, and genes associated with favourable prognosis and between HIF-2a-specific binding and adverse prognosis.  However within each isoform-specific set, individual gene associations were heterogeneous in sign and magnitude, suggesting that activation of each HIF-a isoform contributes a highly complex mix of pro- and anti-tumorigenic effects

overall_design: ChIP and RNASeq of HIF-1a and HIF-2a transfection in 786-O cell lines

first: Salama

last: Rafik

first: Mole

last: David


title: Pyrococcus furiosus crRNAs associated with Cmr, Csa and Cst CRISPR-Cas systems

city: Farmington

first: Brenton

middle: R

last: Graveley

state: CT

address: 400 Farmington Avenue

department: Genetics and Genome Sciences

institute: University of Connecticut Health Center

summary: CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism.

overall_design: 4 Samples: Protein-associated small RNAs

first: Sonali

last: Majumdar

first: Peng

last: Zhao

first: Neil

last: Pfister

first: Mark

last: Compton

first: Sara

last: Olson

first: Claiborne

last: Glover

first: Lance

last: Wells

first: Rebecca

middle: M

last: Terns

first: Michael


title: Analysis of MIZ1, MYC-ER and MYC-ERVD binding sites in MCF10A cells

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

summary: Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli and this protects long-lived organisms from cancer development. How cells discriminate physiological from supra-physiological levels of Myc is largely unknown. Here we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-sequencing experiments show that oncogenic levels of Myc, but not of MycV394D, a point mutant that does not bind Miz1, recruit Miz1 to core promoters and enable binding of Myc/Miz1 complexes to low-affinity target sites, correlating with repression of a specific set of target genes. Repressed genes encode proteins involved in cell adhesion, migration and wound healing; their promoters are enriched for binding sites of the serum response (SRF) factor. Restoring SRF activity attenuates Myc-induced apoptosis in response to glutamine starvation, exposure to Trail and to DNA damage. We propose that supra-physiological levels of Myc engage Miz1 in repressive DNA binding complexes and suppress transcriptional progress.

overall_design: MIZ1, MYC-ER and MYC-ERVD ChIP-Seq with 10E2 and HC20 anti-ERalpha antibodies in MCF10A cells, performed on an Illumina IIx Genome Analyzer. Input sample is accessioned as GSM1423726.

first: Katrin

last: Wiese


title: Amydala transcriptome changes in germ-free mice

status: Public on Jul 09 2015

city: Cork

first: Roman

last: Stilling

address: Western Road

department: Anatomy and Neuroscience

country: Ireland

institute: University College Cork

summary: We sequenced mRNA from 12 samples extracted from mouse amygdala tissue to generate the first amygdala-specific murine transcriptome for germ-free mice (GF), conventionally raised controls (CON) and germ-free mice that have been colonized with normal microbiota from postnatal day  21 (exGF).

overall_design: Equal amounts of RNA from two to three animals were pooled to yield 4 samples per group (CON, GF, and exGF). Pairwise comparisons for CONvsGF, CONvsexGF, GFvsexGF were performed using DESeq2.

first: Feargal

last: Ryan

first: Alan

last: Hoban

first: Gerard

last: Clarke

first: Marcus

last: Claesson

first: John

middle: F

last: Cryan

first: Timoth

last: Dinan


title: Epoxyeicosatrienoic Acids Enhance Haematopoietic Stem and Progenitor Cell Specification and Engraftment

city: Boston

first: Leonard

last: Zon

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions including leukemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here, we developed a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We used this system to conduct a chemical screen and identified epoxyeicosatrienoic acids (EET) as a family of lipids that enhance HSPC engraftment.  EETs’ pro-haematopoietic effects are conserved in the developing zebrafish, where this molecule promotes HSPC specification through activating a unique AP-1/runx1 transcription program autonomous to the haemogenic endothelium. This effect requires the activation of PI3K pathway, specifically PI3Kg. In adult HSPCs, EETs induce transcriptional programs including AP-1 activation, modulating multiple cellular processes, such as migration, to promote engraftment. Finally, we demonstrated that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study established a novel method to explore the molecular mechanisms of HSPC engraftment, and discovered a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.

overall_design: To analyze the effect of 11,12-EET on gene expression of human blood cells, we treated human CD34+ cells (positively selected from cord blood) and the human leukemic cell line U937 with 5uM 11,12-EET for 2 hrs. Control treatment was done with DMSO.

first: Vera

last: Binder

first: Song

last: Yang


title: Sex comb on midleg (Scm) is a functional link between PcG repressive complexes in Drosophila

first: Peter

last: Park

address: 10 Shattuck St

department: Center for Biomedical Informatics

institute: Harvard Medical School

summary: We profiled Sex comb on midleg (Scm), Pc and E(z) in fly embryos and S2 based on BioTAP-XL ChIP-seq. ChIP-seq revealed that Scm is co-localized with PRC1, PRC2, and H3K27me3 in both S2 cells and embryos.

overall_design: Genomic binding/occupancy profiling of Scm, Pc and E(z) by high throughput sequencing

first: Mitzi

middle: I

last: Kuroda

first: Hyuckjoon

last: Kang


title: Impact of bariatric surgery on RNA-seq gene expression profiles of peripheral monocytes in humans

city: Paris

first: Laurence

last: Tiret

address: 91 Bd de l'Hôpital

country: France

institute: INSERM

summary: Genome-wide expression profiles in peripheral monocytes (PM) from 19 obese women before and 3 months after bariatric surgery using the RNA-seq technology. This dataset is linked to the dataset GSE65540 providing expression profiles in subcutaneous adipose tissue (SAT) in the same population. Due to exclusion of some individuals for technical reasons, the overlap between the 2 datasets is of 18 women.

overall_design: mRNA sequencing of peripheral monocyte (PM) samples from 19 obese women before and 3 months after bariatric surgery

first: Claire

last: Perret

first: François

last: Mathieu

first: Vinh

last: Truong

first: Hervé

last: Durand

first: Ewa

last: Ninio

first: Christine

last: Poitou

first: Karine

last: Clément

first: Rohia

last: Alili

first: Nadjim

last: Chelghoum

first: Adriana

last: Torcivia


title: Next Generation Sequencing of tomato WT, M82 and pro∆GRAS mutant treated with Gibberellin

city: Rehovot

first: Gilgi

last: Friedlander

address: Hertzel st

country: Israel

institute: Weizmann Institute of Science

summary: The goal of this study was to search for DELLA-independent GA-regulated genes in tomato. RNA deep sequencing (RNA-seq) was performed to GA-treated M82 and pro∆GRAS mutant. Using a two fold increased or decreased cutoff we identified 81 GA-up regulated and 15 GA-down regulated genes, from which five genes were DELLA-independent.

overall_design: M82 and pro∆GRAS seedlings were treated with Paclobutrazol (10mg/l) once a day for three days followed by a single GA3 application (100 µM). Three hours after the GA treatment, young leaves were collected and RNA was extracted for Illumina HiSeq 2500 sequencing. A total of eight samples were analyzed, each treatment had two biological replicates.

first: Sivan

last: Livne

first: Vai

middle: S

last: Lor

first: Ido

last: Nir

first: Natanella

last: Eliaz

first: Asaph

last: Aharoni

first: Neil

last: Olszewski

first: Yuval

last: Eshed

first: David

last: Weiss


title: Analysis of MYC and MIZ1 binding and MIZ1-dependent gene expression changes in MCF10A cells

first: Martin

last: Eilers

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

overall_design: Refer to individual Series


title: Analysis of MOF under stress conditions

city: Freiburg im Breisgau

first: Thomas

last: Manke

address: Stuebeweg 51

institute: Max-Planck-Institute for Immunobiology and Epigenetics

summary: We analyzed the role of MOF in primary MEFs and differentiated podocytes in response to Adriamycin. Mof was deleted in MEFs using the Cre-ERT2 trasgene, while Mof was knockdown in podocytes using shRNA infection. Samples were treated with Adriamycin for 24 hours and gene expression changes analysed.

overall_design: Analysis of gene expression changes upon Mof depletion in two cell lines, MEFs and podocytes, with and without Adriamycin

first: Bilal

middle: N

last: Sheikh

first: Abdullah

middle: H

last: Sahyoun

first: Vivek

last: Bhardwaj

first: Asifa

last: Akhtar


title: RNA elements in precursors instruct directional and processive primary piRNA biogenesis

city: Gneveva

first: Ramesh

last: Pillai

address: 30, Quai Ernest-Ansermet

department: Department of Molecular Biology

country: Switzerland

institute: University of Geneva

summary: PIWI-interacting RNAs (piRNAs) are animal gonad-specific small RNAs that control the activity of transposable elements. Long single stranded RNAs from a variety of sources are substrates for the nebulous primary processing pathway that converts these into thousands of 24-30 nucleotide (nt) piRNAs. How these transcripts are selected as precursors is not known. Here we show that targeting a transcript with PIWI slicer activity of cysosolic Ago3 is sufficient to trigger ~30-nt waves of non-overlapping primary piRNAs in the fly ovarian germline. The generated primary piRNAs are almost exclusively loaded into the nuclear PIWI protein, Piwi. In the fly ovarian somatic environment we find that an RNA fragment from the 5′ end of a piRNA cluster is able to direct a heterologous sequence into primary processing. This piRNA trigger sequence (PTS) element drives generation of overlapping piRNAs from the transcript. Both mechanisms proceed with general 5′-3′ directionality. We propose that the former pathway serves to link cytoplasmic silencing of a target to nuclear transcriptional repression, while the latter extracts silencing information from a wide variety of genomic sources including piRNA clusters, select protein coding and transposon transcripts.

sample_organism:
Bombyx mori
Drosophila melanogaster
Mus musculus

overall_design: Total or immunoprecipitated small RNAs were purified from transfected BmN4 cells, Drosophila ovarian somatic cells (OSC) and from fly ovaries and high-throughput sequencing libraries were prepared. The mouse testicular RNAs were purified after ribozero treatment.

last: Homolka

first: Radha

last: Pandey

first: Coline

last: Goriaux

first: Emilie

last: Brasset

first: Chantal

last: Vaury

first: Marie-Odile

last: Fauvarque-Marras


title: MIWI2 and MILI have differential effects on piRNA biogenesis and DNA methylation

city: Pasadena

first: Sergei

last: Manakov

address: 1200 E California blvd, MC 147-75

department: Biology and Biological Engeneering

institute: California Institute of Technology

summary: In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use piRNA guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2-deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 suggesting that the proteins have independent roles in establishing DNA methylation patterns.

overall_design: Small RNA profiles (19-30 nt range) of embryonic (E16.5) and post-natal (P10) testis of Miwi2 mutant mice and matched heterozygote controls. mRNA profiles of embryonic testis (E16.5) of heterozygote control mice and of postnatal testis (P10) of Miwi2 and Mili mutants and heterozygote controls. CpG methylation BS-seq profile of postnatal (P10) spermatocytes of Miwi2 mutant mice and matched heterozygote controls.

first: Dubravka

last: Pezic

first: William

last: Pastor

first: Georgi

last: Marinov

first: Ravi

last: Sachidanandam

first: Alexei

last: Aravin


title: miRNA alterations are an important mechanism in the adaptation of maize to a low-phosphate environment

status: Public on Jul 08 2015

city: jinan

first: juren

last: zhang

address: shanda nanlv 27

institute: shandong university

summary: Maize is a globally important food and feed crop, and a low-phosphate (Pi) supply in the soil frequently limits maize yield in many areas. MicroRNAs (miRNAs) play important roles in the development and adaptation of plants to the environment. In this study, the spatio-temporal miRNA transcript profiling of the maize inbred line Q319 root and leaf in response to low Pi was analyzed with high-throughput sequencing technologies, and the expression patterns of certain target genes were detected by real-time RT-PCR. Complex small RNA populations were detected after low-Pi culture and displayed different patterns in the root and leaf. miRNAs identified as responding to Pi deficiency can be grouped into ‘early’ miRNAs that respond rapidly, and often non-specifically, to Pi deficiency, and ‘late’ miRNAs that alter the morphology, physiology or metabolism of plants upon prolonged Pi deficiency. The miR827-Nitrogen limitation adaptation (NLA)-mediated post-transcriptional pathway was conserved in response to Pi availability of maize, but the miR399-mediated post-transcriptional pathway was different from Arabidopsis. Abiotic stress-related miRNAs engaged in interactions of different signaling and/or metabolic pathways. Auxin-related miRNAs (zma-miR393, zma-miR160a/b/c, zma-miR160d/e/g, zma-miR167a/b/c/d and zma-miR164a/b/c/d/g) and their targets play important roles in promoting primary root growth, inhibiting lateral root development and retarding upland growth of maize when subjected to low Pi. The changes in expression of miRNAs and their target genes suggest that the miRNA regulation/alterations compose an important mechanism in the adaptation of maize to a low-Pi environment; certain miRNAs participate in root architecture modification via the regulation of auxin signaling. A complex regulatory mechanism of miRNAs in response to a low-Pi environment exists in maize, revealing obvious differences from that in Arabidopsis.

overall_design: Maize (Zea mays L.) inbred line Q319 was used in this study. Seeds of the maize inbred line Q319 were surface sterilized and held at 28°C in darkness. Seedlings (4 days old) were transferred to a sufficient phosphate (SP, 1,000 μM KH2PO4) solution (Ca(NO3)2.4H2O 2 mM, NH4NO3 1.25 mM, KCl 0.1 mM, K2SO4 0.65 mM, MgSO4 0.65 mM, H3BO3 10.0 mM, (NH4)6Mo7O24 0.5 mM, MnSO4 1.0 mM, CuSO4.5H2O 0.1 mM, ZnSO4.7H2O 1.0 mM, Fe-EDTA 0.1 mM), allowed to grow for 4 days (plants with 2–3 leaves). After 2-3 days of re-culturing in SP nutrient solutions, half of the seedlings were transplanted into a low phosphate (LP, same composition as the SP solution, except that 5 μM KH2PO4 and 1 mM KH2PO4 were replaced with 1 mM KCl) nutrient solution. The plants were grown under a 32°C/25°C (day/night) temperature regime at a photon flux density (PFD) of 700 μmol m-2 s-1 with a 14 h/10 h light/dark cycle in a greenhouse with approximately 65% relative humidity. The roots and leaves were then harvested at 0, 1, 2, 4, 8 days and 8 days and cultured in SP solution (as a normal growth control) for small RNA analysis. The samples were frozen in liquid nitrogen immediately and stored at -80℃ for further analysis. Each biological repeat contains segments from 15~20 plants. Total RNA was extracted as previously described in Molecular Cloning (Sambrook and Russell David, 1989) and was then subjected to two additional chloroform washes prior to nucleic acid precipitation. The small RNA digitalization analysis based on HiSeq high-throughput sequencing takes the SBS-sequencing by synthesis. Then the 50nt sequence tags from HiSeq sequencing will go through the data cleaning first, which includes getting rid of the low quality tags and several kinds of contaminants from the 50nt tags. Length distribution of clean tags are then summarized. Afterwards, the standard bioinformatics analysis will annotate the clean tags into different categories and take those which can not be annotated to any category to predict the novel miRNA and base edit of potential known miRNA. Compare the known miRNA expression between two samples to find out the differentially expressed miRNA. The procedures are shown as below:   (1)Normalize the expression of miRNA in two samples (control and treatment) to get the expression of transcript per million (TPM).       Normalization formula: Normalized expression = Actual miRNA count/Total count of clean reads*1000000   (2)Calculate fold-change and P-value from the normalized expression. Then generate the log2ratio plot and scatter plot.

first: Zhaoxia

last: Li

first: Xinrui

last: Zhang

first: Xiuxia

last: Liu

first: Yajie

last: Zhao

first: Baomei

last: Wang

first: Juren


title: Prostate Cancer Risk SNPs in Androgen Receptor Target Sites

city: Innsbruck

first: Klocker

last: Helmut

address: Anichstrasse 35

department: Department of Urology

country: Austria

institute: Medical University of Innsbruck

summary: We profiled androgen receptor (AR) genomic targets using high-throughput sequencing of chromatin-immunoprecipitated (ChIP) DNA from TMPRSS2-ERG fusion gene positive DUCaP prostate cancer cells. ChIp-seq and microarray gene expression profiling datasets were integrated with the NHGRI GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor binding sites (ARBSs). Eighty GWAS index or linked SNPs were found to be localized in ARBSs. Among these rs11891426:T>G in the 7th intron of the melanophilin gene was found located within a novel putative auxiliary AR binding motif, which we found enriched in the neighborhood of canonical androgen responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay of prostate cancer cell models. It went also in line with decreased melanophilin protein level in primary prostate tumors with G allele.These results unravel a hidden link between androgen receptor and a functional PCa risk SNP, whose allele alteration affects androgen regulation of its host gene melanophilin .

overall_design: Genomic profile of androgen receptor binding sites of androgen or vehicle treated DUCaP cells using ChIP-seq. IgG precipiated DNAs from both treatments served as controls.

first: Helmut

last: Klocker


title: Sperm RNA: A window to idiopathic infertile couples

city: Detroit

first: Stephen

last: Krawetz

state: MI

address: 275 E. Hancock

department: Center for Molecular Medicine and Genetics

institute: Wayne State University School of Medicine

summary: We examine how NGS sequencing of sperm can provide a window as to how particular perturbations of the sperm RNA profile from baseline may be indicative of male factor infertility, and may thus provide direction as to proper course of infertility treatment for couple.

overall_design: NGS RNA-seq of 72 sperm samples from male partner of couples undergoing fertility treatment

first: Meritxell

last: Jodar

first: Edward

middle: D

last: Sendler

first: Sergey

last: Moskovtsev


title: Widespread Occurrence of m6A in bacterial mRNA

city: Hong Kong

first: Xin

last: Deng

address: To Yuen Building

department: Biomedical Sciences

country: Hong Kong

institute: City University of Hong Kong

summary: N6-methyladenosine (m6A) is one of the most abundant modifications in eukaryotic RNA. Recent mapping of m6A methylomes in mammals, yeast, and plants as well as characterization of m6A methyltransferases, demethylases, and binding proteins have revealed regulatory functions of this dynamic RNA modification. In bacteria, although m6A is present in ribosomal RNA (rRNA), its occurrence in messenger RNA (mRNA) still remains elusive. Here, we used liquid chromatography-mass spectrometry (LC-MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved distinct m6A pattern that is significantly different from that in eukaryotes. Most m6A peaks are located inside open reading frames (ORF), and carry a unique consensus motif (GCCAU). Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified transcripts are associated with respiration, amino acids metabolism, stress response, and small RNAs genes, suggesting potential regulatory roles of m6A in these pathways.

sample_organism:
Escherichia coli K-12
Pseudomonas aeruginosa PAO1

overall_design: m6A profiling in E.coli and P.aeruginosa mRNA


title: Dynamics and Function of Distal Regulatory Elements during Neurogenesis and Neuroplasticity

city: Cambridge

first: Sudhir

last: Thakurela

address: 7 Divinity Avenue

institute: HSCRB

summary: Gene regulation in mammals involves a complex interplay between promoter and distal regulatory elements that function in concert to drive precise spatio-temporal gene expression programs. However, the dynamics of distal gene regulatory elements and its function in transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here we use a combinatorial analysis of genomewide datasets for chromatin accessibility (FAIRE-Seq) and enhancer mark H3K27ac to reveal a highly dynamic nature of chromatin accessibility during neurogenesis that gets restricted to certain genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further reveal that the distal open regions serve as target sites of distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. A prolonged NMDA-driven neural activity results in epigenetic reprogramming at a large number of distal regulatory elements as well as dramatic reorganization of super-enhancers that in turn mediate critical transcriptional responses. Taken together, these findings reveal dynamics of distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate transcriptome underlying neuronal development and function.

overall_design: FAIRE-Seq and H3K27ac profiles for three stages on neuronal differentation viz. neuronal progenitors, day 1 neurons and day 10 neurons,  were generated to understand the dynamics of accessible and ehancer chromatin landscape. Along with this we also generated RNASeq and H3K27ac profiles for day 10 neurons upon control and NMDA treatment.

first: Sanjeen

last: Sahu

first: Angela

last: Garding

first: Vijay

last: Tiwari


title: Mapping active promoters by ChIP-seq profiling of H3K4me3 in cichlid fish - a first step to uncover cis-regulatory elements in ecological model teleosts

status: Public on Jul 07 2015

city: Konstanz

first: Claudius

last: Kratochwil

address: Universitaetsstr. 10

department: Biology

institute: University of Konstanz

summary: Evolutionary alterations to cis-regulatory sequences are likely to cause adaptive phenotypic complexity, through orchestrating changes in cellular proliferation, identity and communication. For non-model organisms with adaptive key-innovations, patterns of regulatory evolution have been predominantly limited to targeted sequence-based analyses. Chromatin-immunoprecipitation with high-throughput sequencing (ChIP-seq) is a technology that has only been used in genetic model systems and is a powerful experimental tool to screen for active cis-regulatory elements. Here, we show that it can also be used in ecological model systems and permits genome-wide functional exploration of cis-regulatory elements. As a proof of concept, we use ChIP-seq technology in adult fin tissue of the cichlid fish Oreochromis niloticus to map active promoter elements, as indicated by occupancy of trimethylated Histone H3 Lysine 4 (H3K4me3). The fact that cichlids are one of the most phenotypically diverse and species-rich families of vertebrates could make them a perfect model system for the further in-depth analysis of the evolution of transcriptional regulation.

overall_design: examination of H3K4me3 in adult fin tissue of the Nile tilapia (Oreochromis niloticus)

first: Axel

last: Meyer


title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

city: Bethesda

first: Peng

last: Li

state: MD

address: 9000 Rockville Pike

department: NHLBI

institute: NIH

summary: The canonical pathway for IL-1β production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1β.  Here we show that IL-21 unexpectedly induces IL-1β production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4+ T cells, with differential histone marks present in these cells versus cDCs.  IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1β expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with Pneumonia Virus of Mice. These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.

overall_design: Genome-wide transcription factors mapping and binding of STAT3, H3K4me3, H3K27me, H3K4me1, H3K27ac in mouse CD4+ T cells and dendritic cells in WT and Stat3-/- mice. RNA-Seq is performed in mouse CD4+ T cells and dendritic cells in WT mice, with or without indicated cytokines.

first: Chi-Keung

last: Wan

first: Rosanne

last: Spolski

first: Jangsuk

last: Oh

first: Allison

last: Andraski

first: Ning

last: Du

first: Zu-Xi

last: Yu

first: Christopher

middle: P

last: Dillon

first: Douglas

last: Green

first: Warren

last: Leonard


title: Analysis of the senescent transcriptome upon expression of a ZFP36L1 phosphomutant

city: London

first: Gopuraja

last: Dharmalingam

address: Faculty of Medicine, Imperial College, Hammersmith Hospital Campus

department: Epigenetics section

institute: MRC London Institute of Medical Sciences

summary: We expressed either a wt or a phosphomutant version of ZFP36L1 in IMR90 ER:RAS cells. 7 days upon RAS induction (when the cells reach a fully senescent phenotype) we collected the RNA. ZFP36L1 is a RNA binding protein that binds to AU-rich elements in the 3’UTR of mRNAs and triggers their degradation. Our previous experiments showed that the activity of ZFP36L1 was key in the regulation of the senescent phenotype.By performing RNAseq we have uncovered the effect of expressing a constitutively active mutant of ZFP36L1 within the senescent transcriptome.

overall_design: 4 samples examined: Non-senescent cells (EV - 4OHT), Senescent cells (EV + 4OHT), Senescent cells expressing ZFP36L1wt and Senescent cells expressing ZFP36L1mut

first: Nicolas

last: Herranz

first: Jesus

last: Gil


title: Functional and Transcriptomic Characterization of iPSC-derived Macrophages and their Application in Modeling Mendelian Disease

city: New York

first: Chenyi

last: Xue

institute: Columbia University

summary: Using RNA-Seq, we reported novel findings in the comparison of transcriptome profiles of isogenic HMDM and IPSDM during differentiation and polarization. First, IPSDM lost expression of pluripotency markers, had remarkably distinct gene expression profiles relative to precursor iPSCs, and had largely similar gene expression as HMDM. Second, macrophage polarization to M1 was associated with a dramatic change in the transcriptome; expression profiles of IPSDM- and HMDM-derived M1 lines were highly correlated with each other but much less so with their respective IPSDM and HMDM precursors. Third, M2-HMDM lines had limited difference in gene expression compared to their non-polarized precursors, likely due to the known M2-like phenotype of M-CSF differentiated macrophages and their similarity to the IL-4 derived M2 phenotype Finally, through RNA-Seq we identified many new genes modulated during polarization in both HMDM and IPSDM thus providing novel, and potentially regulatory, candidates that warrant further study.

overall_design: iPS, IPSDM (including M1/M2) and HMDM (including M1/M2)cells were sequenced by Illumina HiSeq 2000 with poly-A selection

first: Hanrui

last: Zhang


title: RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation

city: Davis

first: James

middle: Lee

last: Chitwood

address: 1 Shields Avenue

department: Animal Science

institute: University of California, Davis

summary: Polyadenylated RNA from individual germinal vesicle and metaphase II ooyctes was amplified and processed for Illumina sequencing. Increases in polyadenylated transcript abundance were observed and are likely due to cytoplasmic polyadenylation.

overall_design: Transcriptomic analysis of GV and MII bovine oocytes.

middle: L

first: Pablo

last: Ross


title: Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity

status: Public on Jul 06 2015

city: La Jolla

first: Gene

last: Yeo

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD


title: Transcriptional profiling of zebrafish embryos developmentally exposed to oxygenated PAHs 1,9-benz-10-anthrone and benzanthracene-7,12-dione

city: Corvallis

middle: Michael

last: Sullivan

address: Sinnhuber Aquatic Research Laboratory, 28645 East HWY 34

department: Environmental & Molecular Toxicology

institute: Oregon State University

summary: The purpose of this study was to identify transcripts differentially expressed in zebrafish embryos exposed to two oxygenated PAHs, 1,9-benz-10-anthrone and benzanthracene-7,12-dione, which cause abnormal development.

overall_design: We used RNA-seq (Illumina HiSeq) to identify mRNA profiles of whole zebrafish embryos exposed to 10 μM 1,9-benz-10-anthrone, benzanthracene-7,12-dione or vehicle control (1% DMSO) from 6-48 hours post fertilization

first: Britton

middle: C

last: Goodale

first: Robert

last: Tanguay


title: The Partitioning of RNAs in sperm

first: Graham

last: Johnson

state: Michigan

department: CMMG

institute: WSUSOM

summary: A diverse pool of RNAs remain encapsulated within the transcriptionally and translationally silent spermatozoon. These transcripts persist within the male gamete despite the dramatic reduction in cellular volume achieved through expulsion of the cytoplasm and quite possibly the nucleoplasm. The precise location of RNAs retained within the sperm cell remains largely unknown. However, early evidence suggested that many are embedded within the nucleus (1). To discern the global pattern of transcript compartmentalization in sperm, total RNA was extracted from whole mouse spermatozoa and detergent demembranated nuclei fractionated through a sucrose gradient. Isolated RNAs were subjected to RNA-sequencing (RNA-seq) and their abundance used to infer localization. Transcripts enriched in the unfractionated cells were related to the production and function of mitochondria and surprisingly, exosomes. The absence of these extracellular vesicles associated RNAs within the inner-nuclear compartment was suggestive of an origin other than sperm. This contributes to the growing evidence for sperm-bound exosomes rich in RNA. In comparison, the majority of the remaining sperm RNAs were associated with the nucleus. This included the abundant fragmented ribosomal transcripts which likely persist between the nuclear envelope and the perinuclear theca. The spermatozoal inner-nuclear compartment was also enriched in repetitive transcribed sequences. This included LINE elements and simple repeat sequences both of which have been shown to contribute to chromatin structure in other cell types suggesting that they may serve parallel roles in the spermatozoon.

overall_design: RNA-seq analysis of whole mouse sperm and fractionated nuclei

first: Stephen

middle: A

last: Krawet


title: Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (RIP-Seq)

summary: RNA quality control pathways serve to get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the ATPase cycle of SF1 Helicase Upf1 is required for mRNA discrimination during Nonsense-Mediated Decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD - translation and termination codon-proximal poly(A) binding protein - depend on Upf1 ATPase to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase-dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be an underappreciated, yet widely employed, activity in target RNA discrimination.

overall_design: CLIP and RIP-seq against Wild Type and Mutant Upf1 in HEK293-T cell lines

first: Suzanne

last: Lee

first: Gabriel

last: Pratt

middle: W

first: Jens

last: Lykke-Andersen


title: Transcriptome comparison of oocytes obtained from in vitro culture and in vivo

first: Michał

last: Pasternak

state: - None -

address: Francis Crick Avenue

institute: MRC Laboratory of Molecular Biology

summary: The comparison of trancriptomes was part of the study by Pfender, Kuznetsov, Pasternak et al, titled: "Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes". The goal was to check if the oocytes cultured in vitro in follicles (for RNAi studies) correspond to real gametes obtained directly from mice (in vivo). Apart from functional experiments showing that they can be fertilized and develop into an embryo, we also compared transcriptomes of those oocytes.

overall_design: 3 samples of 50 oocytes were collected for both groups of in vitro and in vivo grown oocytes.

first: Balaji

last: Santhanam

first: Melina

last: Schuh


title: Target discrimination in nonsense-mediated mRNA decay requires Upf1 ATPase activity (CLIP-Seq)


title: Delay and restoration of persistent wound-induced retinal pigmented epithelial-to-mesenchymal transition by TGF-beta pathway inhibitors: Implications for age-related macular degeneration

city: Santa Barbara

first: Monte

middle: J.

last: Radeke

address: Neuroscience Research Institiute

department: Neuroscience Research Institute

institute: University of California, Santa Barbara

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

overall_design: Refer to individual Series