title: Mutation independent activation of the Notch pathway is associated with Lapatinib resistance in Her2+ breast cancer cell lines

city: San

first: Mia

last: Grifford

state: CA

address: 1156 High Street

institute: UCSC

summary: We compared untreated HCC1419 cells with Lapatinib resistant HCC1419 cells that were either treated with Lapatinib for only 9 days before harvesting (drug tolerant persisters, DTPs) or were growing in the presence of Lapatinib (>70 days) (drug tolerant expanded persisters, DTEPs). We show that the Notch pathway is significantly over-expressed in DTEPs when compared to untreated cells.

overall_design: RNA-Seq of HCC1419 cells either untreated, treated with 1μM Lapatinib for 9 days or treated with 1μM Lapatinib for greater than 70 days

first: Melanie

last: Plastini

first: Sol

last: Katzman

first: Sofie

last: Salama

first: David

last: Haussler


title: Analysis of wildtype and Csnk2b-deficient Foxp3+ regulatory T cells transcriptomes

status: Public on Jan 13 2015

city: Mainz

first: Tobias

last: Bopp

address: Langenbeckstrasse 1

department: Institute for Immunology

country: Germany

institute: University Medical Center Mainz

type: Expression profiling by high throughput sequencing

summary: The goal of this study was to compare the transcriptional changes evoked by Csnk2b-deficiency in Foxp3+ regulatory T cells

overall_design: Messenger RNA profiles of CD4+Foxp3+ regulatory T cells isolated from 8 week old wild type (WT) and Csnk2b-deficient mice were generated by deep sequencing in duplicate, using Illumina MiSeq

first: Alexander

last: Ulges


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and cardiac-specific Bmi1 deletion [mouse]

city: Madrid

first: Manuel

middle: J

last: Gomez

state: Madrid

address: Melchor Fernández Almagro, 3

country: Spain

institute: CNIC

summary: To explore the primary cause of Dilated Cardiomyopathy in Bmi1-null mice, we set out to identify differentially expressed genes by massively parallel sequencing of heart samples from Bmi1f/f;αMHCTM-Cretg/+ mice versus αMHCTM-Cretg/+ control mice (17 weeks postinduction).

overall_design: Methods: Heart mRNA profiles of 17-weeks post-induction Bmi1f/f;  MHCTM-Cretg/+ mice and   MHCTM-Cretg/+ control mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. Sequence reads were pre-processed with Cutadapt 1.2.1, to remove TruSeq adapters and mapped on the mouse transcriptome (Ensembl gene-build GRCm38.v70) using RSEM v1.2.3. The Bioconductor package EdgeR was used to normalize data with TMM and to test for differential expression of genes using GLM.

first: Susana

last: Gonzalez

first: Ileana

last: Gonzalez-Valdez


title: Expression of microRNAs in rat kidney treated with a carcinogenic dose of Aristolochic Acid

status: Public on Jan 15 2015

city: Dalian

first: Zhiguang

last: Li

state: Liaoning

address: 9 Lvshun Road South

department: Institute of Cancer Stem Cell, Cancer Center

country: China

institute: Dalian Medical University

type: Non-coding RNA profiling by high throughput sequencing

summary: Aristolochic Acid (AA), a natural component of Aristolochia plants found in a variety of herbal remedies and health supplements, is classified as a group 1 carcinogen by the International Agency for Research on Cancer. In order to search for miRNA regulation in AA-induced carcinogenesis, we treated rats with 10 mg/kg AA and vehicle control for 12 weeks and eight kidney samples (4 for the treatment and 4 for the control) were used for examining miRNA by deep sequencing.

overall_design: Profiliing of miRNA in 4 AA-treated rats and 4 control

first: Taichun

last: Qin

first: Michael

last: Hackenberg

first: Jian

last: Yan

first: Kejian

last: Wang

first: Yuan

last: Gao

first: Lirong

last: Yu

first: Leming

last: Shi

first: Tao

last: Chen


title: Enhancer Sequence Variants and Transcription Factor Deregulation Synergize to Construct Pathogenic Regulatory Circuits in B Cell Lymphoma

city: St. Louis

first: Jacqueline

last: Payton

state: MO

address: 660 S. Euclid Ave

department: Pathology and Immunology

country: USA

institute: Washington University School 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: Detained introns are novel, widespread class of posttranscriptionally-spliced introns

city: Rochester

first: Paul

middle: L.

last: Boutz

state: NY

address: 601 Elmwood Ave.

department: Biochemistry & Biophysics

institute: University of Rochester School of Medicine

summary: Removal of introns by pre-mRNA splicing is a critical and in some cases rate-limiting step in mammalian gene expression.  Deep sequencing of mouse embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within poly(A) selected  transcripts; we classify these as “detained” introns (DIs). We identified thousands of DIs flanking both constitutive and alternatively spliced exons in human and mouse cell lines. Drug inhibition of Clk SR-protein kinase activity triggered rapid splicing changes in a specific set of DIs, about half of which showed increased splicing and half increased intron detention, altering the transcript pool of over 300 genes.  These data suggest a widespread mechanism by which a nuclear detained pool of mostly processed pre-mRNAs can be rapidly mobilized in response to stress or homeostatic autoregulation.

overall_design: v6.5 mouse embryonic stem cells were untreated, treated with the Clk kinase inhibitor KH-CB19, or treated with DMSO as a negative control.  Untreated cells were harvested and a single replicate was sequenced using a custom, ligation-based, stranded library preparation protocol.  Treated cells were harvested at time 0 and at 2 hours post-treatment, and poly(A)-selected RNA-seq libraries were made from biological duplicates for each treatment/time, barcoded, and sequenced by strand-specific, paired-end sequencing using the Illumina TruSeq kit.

middle: L

first: Phillip

middle: A

last: Sharp


title: Global mapping of ribonucleotides embedded in DNA

city: Aurora

first: Jay

middle: R.

last: Hesselberth

state: CO

department: Biochemistry and Molecular Gentetics

institute: University of Colorado School of Medicine

summary: Capture and massively parallel DNA sequencing of ribonucleotides embedded in S. cerevisiae genomic DNA

overall_design: We developed a new method to map the positions of ribonucleotides embedded in DNA using the unique specificity of A. thaliana tRNA ligase. Ribonucleotides were generated in budding yeasts of different genetic backgrounds and mapped to single nucleotide resolution using the new method.

first: Francesca

last: Storici


title: Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation

status: Public on Jan 14 2015

city: Saint Louis

first: Ezekiel

middle: John

last: Maier

address: 4444 Forest Park Ave

department: Computer Science

institute: Washington University

summary:
Purpose: Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying TFs involved in a specific process and used it to map regulation of the key virulence factor of Cryptococcus neoformans, its capsule.
Results: The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, mid-level, and “foreman” TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of 41 TF deletion mutants revealed complex relationships among virulence factors and virulence in mice.
Conclusions: These data resources and analyses provide the first integrated, systems level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to labs focused on specific physiological processes.

overall_design: A total of 288 expression profiles were generated by RNA-Seq analysis of Mutant and wildtype cells grown in capsule inducing conditions for 90 minutes. In addition, 3 replicate expression profiles were generated for wildtype cells grown in : a capsule non-inducing condition, a capsule inducing condition for 180 minutes, a capsule inducing condition for 480 minutes, and a capsule inducing condition for 1440 minutes.  In addition, ChIP-seq of NRG1(CNAG_05222) in capsule inducing conditions, and USV101(CNAG_05420) in both capsule inducing and non-inducing conditions were generated.

first: Brian

middle: C

last: Haynes

first: Stacey

middle: R

last: Gish

first: Zhuo

last: Skowyra

first: Alyssa

last: Marulli

first: Tamara

last: Doering

last: Brent


title: RNA-seq analysis of gene expression during and after exposure to hydroxyurea in WT and mec1 cells in Saccharomyces cerevisiae

status: Public on Jan 12 2015

city: Syracuse

first: Wenyi

last: Feng

address: 750 East Adams Street

department: Biochemistry and Molecular Biology

institute: SUNY Upstate Medical University

summary: We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase.  Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation.  Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure.  We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution.  We show that DSBs preferentially occur at genes transcriptionally induced by HU.  Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU.  Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors.  We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription.  Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage?  We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs.

overall_design: We queried the yeast genome for gene expression after cells were treated with 200 mM hydroxyurea during S phase.  Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 30 and 60 min after the 1 h exposure to HU.  These samples are referred to as G1, HU 1h, R30, and R60, respectively.   The strains from which the samples were collected are indicated before the time point, e.g. mec1_G1 or MEC1_R30.  Stranded mRNA libraries were prepared according to manufacturer's suggestion and sequenced on Illumina MiSeq with paired-end reads of 75 bp.

last: Haarer


title: Break-Seq of DNA double stranded breaks in Saccharomyces cerevisiae WT and mec1 cells after exposure to hydroxyurea

overall_design: We queried the yeast genome for DSBs after cells were treated with 200 mM hydroxyurea during S phase.  Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 1 h after the 1 h exposure to HU.  These samples are referred to as G1, HU 1h, and R 1h, respectively.   The strains from which the samples were collected are indicated following the time point, e.g. G1_MEC1 or R 1h_mec1.  The experiment with mec1 was done twice (Experiments A and B) and that with MEC1 was done once (Experiment C).  In addition, a control experiment of in vitro digestion with BamHI using the G1_mec1 sample (G1_BamHI) was performed.

first: Elizabeth

last: Hoffman

first: Andrew

last: McCulley


title: Break-Seq and RNA-seq analysis of gene expression during and after exposure to hydroxyurea in WT and mec1 cells in Saccharomyces cerevisiae


title: Combinatorial activities of SHORT VEGETATIVE PHASE and FLOWERING LOCUS C define distinct modes of flowering regulation in Arabidopsis

status: Public on Jan 11 2015

city: Cologne

first: Julieta

last: Mateos

address: Carl-von-Linné-Weg 10

department: Department of Plant Developmental Biology

institute: Max Planck Institute for Plant Breeding Research

summary: The MADS-box transcription factors FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) are major transcriptional repressors controlling flowering time. They enhance responses to environmental cues such as winter temperatures, high ambient temperatures and photoperiod, acting at least in part by blocking transcription of the floral pathway integrators. As other MADS-box transcription factors, FLC and SVP can interact in vivo forming multimeric complexes. Mutations in either FLC or SVP lead to similar early flowering, suggesting that FLC-SVP interaction might be the major control unit. Here we have analyzed the coordinated regulatory modes of these two key transcription factors at a genome-wide level through ChIP-seq and gene expression microarrays. Genome-wide identification of SVP and FLC DNA-binding occupancy events revealed that their binding scenarios are strongly yet differently affected by the presence of the cognate partner both at a quantitative as well as a qualitative level. Also, we identified a subgroup of genes whose regulation exclusively depends on the combinatorial binding of these two proteins, strengthening the role of the SVP-FLC complex. Some of these genes are involved in the control of flowering through direct and indirect regulation of Gibberellin-related genes such as GA2ox8, DDF1 and TEM1. Interestingly, we identified cis-regulatory elements enriched uniquely at complex-bound sites. This study decoded the regulatory code mediated by the major flowering repressors SVP and FLC.

overall_design: ChIP followed by high-throughput sequencing for two MADS-box transcription factors in Arabidopsis seedlings (ecotype: Col-0 with active FRI)

first: Pedro

last: Madrigal


title: Genome-wide map of H3K4me3 and PRDM9-binding in 12 day post-partum mouse spermatocytes

city: Bar Harbor

first: Christopher

middle: Lee

last: Baker

state: ME

address: 600 Main Street

department: Research

institute: The Jackson Laboratory

summary: PRDM9, a histone methyltransferase, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we compared activity of the Prdm9Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced. Comparing these two strains, we find that haplotype differences at hotspots leads to qualitative and quantitative changes in PRDM9 binding and activity.  Most variants affecting PRDM9Cst binding arose and were fixed in M.m castaneus, suppressing hotspot activity. Furthermore, M.m castaneus x M.m domesticus F1 hybrids exhibit novel hotspots, representing sites of historic evolutionary erosion. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.

overall_design: Identify position of meiotic H3K4me3 from various sub-species of mice and F1 hybrids from crosses between subspecies. In addition, perform ChIP-seq analysis on the meiosis-specific methyltransferase PRDM9.

first: Kenneth

last: Paigen


title: Adiponectin improves local lipid metabolism in regenerating pancreatic islets after β-cell ablation

status: Public on Jan 09 2015

city: Dallas

first: Philipp

middle: E

last: Scherer

state: TX

address: 5323 Harry Hines Boulevard

department: Touchstone Diabetes Center

institute: The University of Texas Southwestern Medical Center

summary: Pathways that stimulate β-cell regeneration remain of great clinical interest, yet effective therapeutic avenues that promote survival or reconstitution of β-cell mass remain elusive. Utilizing a mouse model with inducible β-cell apoptosis followed by adiponectin-mediated regeneration, we aimed to identify key molecules boosting β-cell viability. Within the regenerating pancreatic islets, we examined changes within the transcriptome, and observed an extensive upregulation of genes encoding proteins involved in lipid transport and metabolism. The most prominent targets were further confirmed by quantitative PCR and immunofluorescence. Among the upstream regulators predicted by pathway analysis of the transcriptome, we detected enhanced levels of two key transcription factors, HNF4α and PPARα. Enhanced leptin levels in circulation may also contribute to the anti-lipotoxic program in islets. In summary, our data suggest that improving local lipid metabolism as an important anti-lipotoxic phenomenon to boost β-cell regeneration, primarily mediated by adiponectin’s action on the β-cells directly as well as on the adipocyte.

overall_design: RNA profiles of pancreatic islets isolated from PANIC-ATTAT mice crossed with adiponectin wild-type (P-Adn+/+) or the overexpressing transgene (P-AdnTg/+) at 5 weeks after initial dimerizer administration.

first: Risheng

last: Ye


title: Noncoding RNA transcriptome analysis during cellular reprogramming

city: Pasadena

first: Diane

last: Trout

address: 1200 E California Blvd M/C 156-29

department: Biology

institute: California Institute of Technology

summary: We report the application of single-cell and bulk RNA sequencing technology to examine the noncoding transcriptome of cells undergoing reprogramming to the pluripotent state.

overall_design: Examination of noncoding RNAs in reprogrammming cells.  We examined iPS cells grown in standard ES cell culture conditions, as well as iPS cells grown in "2i" conditions (small molecule inhibition of Mek and Gsk3).  We also compared our iPS samples to male and female ES cells (mES, fES).

first: Daniel

last: Kim

first: Georgi

last: Marinov


title: Type I interferon regulates the expression of long non-coding RNAs [RNA-seq]

city: Pamplona

first: Victor

last: Segura

state: Navarra

address: Avda. Pío XII, 55

department: Unit of Proteomics, Genomics and Bioinformatics

institute: FIMA

summary: Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response.

overall_design: HuH7 cells were treated with 10000 units/ml of IFN a2 and RNA was isolated 3 days post-treatment

first: Elena

last: Carnero

first: Marina

last: Barriocanal

first: Puri

last: Fortes


title: Human Herpesvirus 6A encoded miRNAs: A role in viral lytic replication

city: Cleveland

first: Eain

middle: Anthony

last: Murphy

state: OH

department: Molecular Genetics

institute: Cleveland Clinic Foundation

summary: We report the application of size selection of small RNA species isolated from Jjhan cells harboring the human herpesvirus 6A genome. We ammassed >3.4million reads of sequence from three different sources: Normal Brain cell total RNA, Jjhan total RNA and HHV-6A BAC transfected Jjhan total RNA.  Sequences were mapped to the HHV-6A Uganda 1102 strain genome (GenBank: X83413.1) with no less than 100% match for reads >20nt and <23nt. The resulting pool of candidates was mapped to the HHV-6A genome.

overall_design: Single pass 36nt sequencing of samples either with or without HHV-6a genomes present.


title: Functional heterogeneity of human memory T cell clones primed by pathogens or vaccines

city: Bellinzona

first: Mathilde

last: Foglierini

address: Via Vincenzo Vela 6

country: Switzerland

institute: Institute for Research in Biomedicine

summary: It is unclear to what extent T helper cells with a different phenotype (chemokine receptor and cytokine production profile) are generated in response to pathogen challenge or vaccination, and whether such heterogeneity can arise among the progeny of a single precursor. We combined cellular methods to isolate antigen specific T cells from different T helper memory subsets of human donors, and next generation sequencing of their TCR. This approach revealed a high level of heterogeneity in the phenotype of human T cells responding to different pathogens/vaccines also at a clonal level, as we could characterize cells bearing an identical T cell receptor with divergent effector properties.

overall_design: NGS-based analysis of T cell receptor repertoire of T helper memory cells identical gross specificity (i.e. responding to a given pathogen or vaccine antigen) belonging to different heper subsets (Th1, Th2, Th17)

first: Federica

last: Sallusto

first: Simone

last: Becattini


title: Type I interferon regulates the expression of long non-coding RNAs


title: Identification of apple miRNAs and their potentialrole in Fire Blight resistance

city: Poznan

first: Michał

last: Szcześniak

address: Uniwersytetu Poznanskiego 6

department: Institute of Human Biology and Evolution

country: Poland

institute: Adam Mickiewicz University

summary: miRNAs are key players in multiple biological processes, therefore analysis and characterization of these small regulatory RNAs is a critical step towards better understanding of animal and plant biology. In apple (Malus domestica) two hundred microRNAs are known, which most probably represents only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species.  We performed deep sequencing of twelve small RNA libraries obtained for fire blight resistant and fire blight sensitive trees. In the sequencing results we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop qPCR and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs.  In this study we considerably expand the apple miRNAome by identifying and characterizing dozens of novel microRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance.

overall_design: Actively-growing shoot tip tissue samples were collected from twelve apple trees, which includes three biological replicates of each following scion-rootstock combinations: B.9, G.30, M.111 and M.27.

first: Elzbieta

last: Kaja

first: Michal

middle: W

last: Szczesniak

first: Philip

last: Jensen

first: Michael

last: Axtell

first: Timothy

last: McNellis

first: Izabela

last: Makalowska


title: Chromatin and signaling pathways in reprogramming

city: Madison

first: Rupa

last: Sridharan

state: WI

address: 330 N. Orchard Street

department: Department of Regenerative Biology

institute: University of Wisconsin

summary: Reprogramming intermediates (pre-iPSCs) were subjected to control DMSO, ascorbic acid (AA), 2i ( MAP kinase inhibitor + GSKinhibitor) or AA+2i conditions to assess conversion to the iinduced pluripotent stem cell state (iPSC) after 10days.

overall_design: Pre-iPSC cells  exposed to control DMSO, AA, 2i or AA+2i were harvested on day 2 for RNA sequencing after treatment, in biological triplicate.


title: X-chromosome dynamics revealed by the RNA interactome and chromosomal binding of CTCF

city: Boston

first: Johnny

middle: T

last: Kung

state: MA

address: 185 Cambridge St

department: Molecular Biology

institute: HHMI, Massachusetts General Hospital, Harvard Medical School

summary: CTCF is a master regulator that plays a role in genome architecture and gene expression. A key aspect of CTCF’s mechanism involves bringing together distant genetic elements for intra- and inter-chromosomal interactions. Evidence from epigenetic processes, such as X-chromosome inactivation (XCI), suggests that CTCF may carry out its functions through interacting RNAs. Using genome-wide approaches to investigate the relationship between CTCF’s RNA interactome and its epigenomic landscape, here we report that CTCF interacts with thousands of transcripts in mouse embryonic stem cells (mESC), many in close proximity to CTCF’s genomic binding sites. Biochemical analysis demonstrates that CTCF is a high-affinity RNA binding protein that contacts RNA directly and specifically. In the XCI model, CTCF binds the active and inactive X-chromosomes allele-specifically. At the X-inactivation center, Tsix RNA binds CTCF and targets CTCF to a region associated with X-chromosome pairing. Our work implicates CTCF-RNA interactions in long-range chromosomal interactions in trans and adds a new layer of complexity to CTCF regulation. The genome-wide datasets reported here will provide a useful resource for further study of CTCF-mediated epigenomic regulation.

overall_design: CTCF RNA interactome was identified by UV-crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq), and was compared to CTCF's epigenomic landscape as obtained by chromatin immunoprecipitation (ChIP-seq).

first: Barry

last: Kesner

first: Jeannie

last: Lee


title: The porcine microRNA transcriptome response to transmissible gastroenteritis virus infection

status: Public on Jan 08 2015

city: Ya’an

first: Xiao

last: Liu

state: Sichuan Province

address: 46# Xinkang Road

department: College of Veterinary Medicine

institute: Sichuan Agricultural University

summary: Transmissible gastroenteritis virus (TGEV; Coronaviridae family) causes huge economic losses to the swine industry. MicroRNAs (miRNAs) play a regulatory role in viral infection and may be involved in the mammalian immune response. Here, we report a comprehensive analysis of host miRNA expression in TGEV-infected swine testis (ST) cells. Deep sequencing generated 3,704,353 and 2,763,665 reads from uninfected ST cells and infected ST cells, respectively. The reads were aligned to known Sus scrofa pre-miRNAs in miRBase 19, identifying 284 annotated miRNAs.   Certain miRNAs were differentially regulated during TGEV infection, which confirmed the hypothesis that specific miRNAs play a regulatory role in virus-host interactions. 59 unique miRNAs displayed significant differentially expression between the normal and TGEV-infected ST cell samples: 15 miRNAs were significantly up-regulated and 44 were significantly down-regulated. Stem-loop RT-PCR was carried out to determine the expression levels of specific miRNAs in the two samples, and the results were consistent with those of sequencing. Gene ontology enrichment analysis of host target genes demonstrated that the differentially expressed miRNAs are involved in regulatory networks, including cellular process, metabolic process, immune system process.  This is the first report of the identification of ST cell miRNAs and the comprehensive analysis of the miRNA regulatory mechanism during TGEV infection, which revealed the miRNA molecular regulatory mechanisms for the viral infection, expression of viral genes and the expression of immune-related genes. The results presented here will aid research on the prevention and treatment of viral diseases.

overall_design: 2 ST(Porcine testicular cells) cell samples, ST: normal ST cell sample (contro sample), TGEV: TGEV (Transmissible gastroenteritis virus) infected ST cell samples


title: Interferon lambda restricts West Nile virus neuroinvasion by enhancing integrity of the blood-brain barrier

last: Diamond

address: McDonnell Pediatric Research Building, Room 7264

department: Division of Infectious Diseases

institute: Washington University in St. Louis

summary: Although type III interferons (IFN), also known as IFN-λ or IL28/IL-29, restrict infection by several viruses, their mechanism of inhibitory action has remained uncertain. We used recombinant IFN-λ and mice lacking the IFN-λ receptor (IFNLR1) to evaluate the effect of IFN-λ on infection with West Nile virus (WNV), an encephalitic flavivirus. Cell culture studies in keratinocytes and dendritic cells showed no direct antiviral effect of exogenous IFN-λ even though ISGs were induced. Correspondingly, we observed no differences in WNV burden between wild-type and Ifnlr1-/- mice in the draining lymph node, spleen, and blood. However, we detected earlier dissemination and increased WNV infection in the brain and spinal cord of Ifnlr1-/- mice, yet this was not associated with a direct antiviral effect on infection of neurons. Instead, an increase in blood-brain barrier (BBB) permeability was observed in Ifnlr1-/- mice. Accordingly, treatment of mice with pegylated IFN-λ2 resulted in decreased BBB permeability, reduced WNV infection in the brain without impacting viremia, and improved survival against lethal virus challenge. An in vitro model of the BBB showed that IFN-λ signaling in brain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movement across the barrier, and modulated tight junction protein localization in a protein synthesis- and STAT1-independent manner. Our data establish a novel indirect antiviral function of IFN-λ in which non-canonical signaling through IFNLR1 tightens the BBB and restricts viral neuroinvasion and pathogenesis. This finding suggests new clinical applications for IFN-λ in treating viral or autoimmune diseases.

overall_design: Transcriptome profiling of bone-marrow derived Dendritic cells(BMDCs), treated with either Serum Free Media(Mock), interferon beta(IFNb), or interferon lambda(IFNL) for 6 hours.

first: Helen

middle: M

last: Lazear

first: Brian

middle: P

last: Daniel

first: Amelia

middle: K

last: Pinto

first: Albert

last: Huang

first: Sarah

last: Vick

first: Sean

last: Doyle

last: Gale Jr

first: Robyn

middle: S

last: Klein


title: High Resolution N6-Methyladenosine (m6A) Map Using Photo-crosslinking-Assisted m6A Sequencing

city: Chicago

first: zhike

last: lu

state: IL

address: 5801 South Ellis Avenue

department: department of chemistry

institute: University of Chicago

summary: We present here a photo-crosslinking-assisted m6A sequencing strategy to more accurately define sites with m6A modifications. Using this strategy, we obtained a high-resolution map of m6A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m6A sites at base resolution. Our results provide insights into the relationship between the methylation regions and the binding sites of RNA-binding proteins.

overall_design: Anti-m6A antibody capture and enrichment based photo-crosslinking-assisted m6A sequencing strategy

first: Kai

last: Chen

first: Zhike

last: Lu


title: Defining soma-specific/enriched and germline-specific/enriched transcripts

city: Basel

first: Dimos

last: Gaidatzis

address: Maulbeerstrasse 66

institute: Friedrich Miescher Institute

summary: To define what genes are predominantly or specifically expressed in either soma or germline in C. elegans adults, total RNA was extracted from germline-less glp-4 mutant animals or from dissected gonads, respectively.

overall_design: Total RNA sequencing was peformed in duplicates. Four samples in total.

first: Jane

last: Wright

first: Rafal

last: Ciosk


title: Functional characterization of C. elegans Y-box binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes (RNA-Seq)

summary: We present a basic characterization of the function of Y-box binding proteins in C. elegans. Besides playing an important role for fertility in the germline (all four CEY proteins), we found that the presence of CEY-1 and CEY-4 is essential for the assembly of larger polysomes in the soma. We therefore performed ribosome-profiling in combination with total RNA sequencing in wild type and cey-1,-4 double mutant animals to globally compare mRNA levels and their translation status.

overall_design: Total RNA sequencing was peformed on RNA extacted from wild type and cey-1,-4 mutant animals in duplicates. Four samples in total.

first: Andreas

last: Arnold


title: Stretch responsive miRNAs in human aortic valve interstitial cells

city: Seattle

first: Vishal

last: Nigam

state: WA

address: 1900 9th Ave

department: Pediatrics

institute: Seattle Childrens Research Institute

summary: miRNA-Sequencing was performed on human aortic valve interestitial cells (AVICs) exposed to 14% stretch at 1 hz or static conditions for 24h.

overall_design: Six static control and six samples exposed to cyclic stretch 14% for 24h


title: Functional characterization of C. elegans Y-box binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes (ribosome)

overall_design: Ribosome-profiling analysis was peformed on wild type and cey-1,-4 mutant animals in duplicates. Four samples in total.


title: RNA-Seq Based Reconstruction of the Phylogenetic Tree of the Genus Flaveria (Asteraceae)

city: shanghai

first: Ming

middle: Ju

state: shanghai

address: 320 Yueyang Road, Xuhui Area, Shanghai, China

department: Partner Institute for Computational Biology, Shanghai Institutes for Biologic Sciences, CAS

institute: Shanghai Institutes for Biological Sciences, CAS

summary: The genus Flaveria has been extensively used as a model to study the evolution of C4 photosynthesis as it contains both C3 and C4 species as well as a number of species that exhibit intermediate types of photosynthesis. The current phylogenetic tree of the Flaveria genus contains 21 of the 23 known Flaveria species and has been constructed using a combination of morphologicial data and three non-coding DNA sequences (nuclear encoded ETS, ITS and chloroplast encoded trnl-F). However, recent studies have suggested that phylogenetic trees inferred using a small number of molecular sequences may often be incorrect. Moreover, studies in other genera have often shown substantial differences between trees inferred using morphological data and those using molecular sequence. To provide new insight into the phylogeny of the genus Flaveria we utilize RNA-Seq data to construct a multi-gene concatenated phylogenetic tree of 17 Flaveria species. Furthermore, we use this new data to identify 14 C4 specific non-synonymous mutation sites, 12 of which (86%) can be independently verified by public sequence data. We propose that the data collection method provided in this study can be used as a generic method for facilitating phylogenetic tree reconstruction in the absence of reference genomes for the target species.

sample_organism:
Flaveria bidentis
Flaveria pringlei
Flaveria trinervia
Flaveria cronquistii
Flaveria brownii
Flaveria palmeri
Flaveria pubescens
Tragopogon dubius
Tanacetum parthenium
Helenium autumnale
Flaveria angustifolia
Flaveria vaginata
Flaveria kochiana
Flaveria sonorensis

overall_design: 18 Flaveria sample including 11 species are sequenced, other three samples were also sequenced as out-group. In all, 21 samples.

first: Ming-Ju

last: Lv

last: Covshoff

first: Xin-Guang

last: Zhu

first: Gane

last: Wong


title: Expression data from RhoA-null and wildtype keratinocytes

status: Public on Jan 07 2015

city: Copenhagen

first: Alberto

last: Garcia Mariscal

address: Ole Maaløes Vej 5

department: BRIC

country: Denmark

institute: University of Copenhagen

summary: Using mice with a keratinocyte-restricted deletion of the RhoA gene, we explored its role in the regulation of gene expression in vivo and in vitro

overall_design: RNA was isolated from RhoA-null or wildtype keratinocytes, in two different conditions, freshly isolated (in vivo) or cultured (in vitro), using the RNeasy kit  (Qiagen, Germany). Hybridization to GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA) was carried out at the Copenhagen University Hospital Microarray Center using standard protocols. 2 biological replicates were analyzed for each in vivo genotype (2 samples from RhoA-null, 2 from wildtype), while 3 replicates for each in vitro genotype.

first: Karine

last: Peyrollier

middle: G

last: Mariscal


title: Transcriptomic changes in aquaporin PIP1;2 deficient Arabodposis plants

status: Public on Jan 10 2015

city: Darmstadt

first: Marlies

last: Heckwolf

address: Schnittspahnstraße 10

institute: Technische Universität Darmstadt

summary:
The leaf transcriptome of the Arabidopsis thaliana aquaporin gene PIP1;2 T-DNA insertion line was compared to that of control plants. In total 730 genes were found to be differentially regulated. This regulation pattern was compared to mild drought stress and low CO2 Affymetrix data to elucidate whether loss of the aquaporin resembles transcriptomic changes of drought stress or lack of CO2 supply.
Mild drought stress data were obtained from Harb A, Krishnan A, Ambavaram MMR, Pereira A (2010) Molecular and Physiological Analysis of Drought Stress in Arabidopsis Reveals Early Responses Leading to Acclimation in Plant Growth. Plant Physiology 154: 1254-1271 (GSE24177).
Low CO2 data were obtained from Oliver E. Bläsing, Yves Gibon, Manuela Günther, Melanie Höhne, Rosa Morcuende, Daniel Osuna, Oliver Thimm, Björn Usadel, Wolf-Rüdiger Scheible, and Mark Stitt (2005) Sugars and Circadian Regulation Make Major Contributions to the Global Regulation of Diurnal Gene Expression in Arabidopsis. The Plant Cell, Vol. 17, 3257-3281 (GSE3423).

overall_design: 2 samples examined: wildtype and atpip1;2-1 mutant

first: Ralf

last: Kaldenhoff


title: De Novo Transcriptome Sequencing for Tetramorium bicarinatum: The first comprehensive venom gland transcriptome analysis from an ant species.

city: Albi

first: Wafa

last: BOUZID

address: place de verdun

country: France

institute: centre universitaire JFC

summary: In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium bicarinatum and to unveil the potential of its products, high-throughput expressed sequence tags were generated using Illumina paired-end sequencing technology. A total of 212,371,758 pairs of quality-filtered, 100-base-pair Illumina reads were obtained. The de novo assemblies yielded 36,042 contigs for which 27,873 have at least one predicted ORF among which 59.77% produce significant hits in the available databases. The investigation of the reads mapping toxin class revealed a high diversification with the major part consistent with the classical hymenopteran venom protein signature represented by venom allergen (33.3%) followed by a diverse toxin-expression profile including several distinct isoforms of phospholipase A1 and A2, venom serine protease, hyaluronidase, protease inhibitor and secapin. Moreover, our results revealed for the first time the presence of toxin-like peptides that have been previously identified from unrelated venomous animals such as waprin-like (snakes) and agatoxins (spiders and conus).

overall_design: 300 ant specimens from the species Tetramorium bicarinatum were dissected in order to extract the RNA from their venom gland, The whole ant body was used as a reference,

last: Bouzid

first: Angélique

last: Vétillard

first: Christophe

last: Klopp

first: Marion

last: Verdenau

first: Frédéric

last: Ducancel


title: A systems approach identifies networks and genes linking sleep and stress: implications for neuropathology

city: new york

first: Bojan

last: Losic

address: 1425 Madison Avenue

department: Genetics and Genomics

institute: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI

summary: Sleep dysfunction and stress susceptibility are co-morbid complex traits, which often precede and predispose patients to a variety of neuropsychiatric diseases. Here, we demonstrate multi-level organizations of genetic landscape, candidate genes, and molecular networks associated with 328 stress and sleep traits in a chronically stressed population of 338 (C57BL/6J×A/J) F2 mice. We constructed striatal gene co-expression networks, revealing functionally and cell-type-specific gene co-regulations important for stress and sleep. Using a composite ranking system, we identified network modules most relevant for 15 independent phenotypic categories, highlighting a mitochondria/synaptic module that links sleep and stress. The key network regulators of this module are overrepresented with genes implicated in neuropsychiatric diseases. Our work suggests the interplay between sleep, stress, and neuropathology emerge from genetic influences on gene expression and their collective organization through complex molecular networks, providing a framework to interrogate the mechanisms underlying sleep, stress susceptibility, and related neuropathology.

overall_design: Examination of genomic and transcriptomic networks in a random subset of 100 (B6xA/J)F2 mice modeling the natural spectrum of stress susceptibility.

first: Joseph

last: Scarpa

first: Peng

last: Jiang


title: Low-cost, low-input RNA-seq protocols perform nearly as well as high-input protocols

city: Stanford

first: Peter

middle: Acuña

last: Combs

address: Gilbert Rm 309, 371 Serra Mall

institute: Stanford University

summary: We sequenced mRNA according to several library prep protocols with known mixtures of two species of Drosophila in order to establish linear response in each protocol.

sample_organism:
Drosophila melanogaster
Drosophila virilis

overall_design: For each library prep protocol, mixtures with 0%, 5%, 10%, and 20% D. virilis total RNA was prepared, then libraries prepared according to instructions.

first: P

first: M

middle: B

last: Eisen


title: Altering cancer transcriptomes using epigenomic inhibitors

city: Burlington

first: Seth

last: Frietze

state: VT

address: 149 Beaumont Avenue

department: Biomedical and Health Sciences

institute: University of Vermont

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

overall_design: Refer to individual Series


title: Altering cancer transcriptomes using epigenomic inhibitors [RNA-Seq]

summary: We have compared the genome-wide effects on the transcriptome after treatment with ICG-001 (the specific CBP inhibitor) versus C646, a compound that competes with acetyl-coA for the Lys-coA binding pocket of both CBP and p300. We found that both drugs cause large-scale changes in the transcriptome of HCT116 colon cancer cells and PANC1 pancreatic cancer cells, and reverse some tumor-specific changes in gene expression. Interestingly, although the epigenetic inhibitors affect cell cycle pathways in both the colon and pancreatic cancer cell lines, the WNT signaling pathway was affected only in the colon cancer cells. Notably, WNT target genes were similarly down-regulated after treatment of HCT116 with C646 as with ICG-001.

overall_design: To identify genes affected by direct targeting of a component of the transcriptional complex implicated in WNT regulation, we used siRNAs to knockdown TCF7L2 in PANC1 cells. Cells were treated with control siRNAs or siRNAs specific for TCF7L2 and RNA was analyzed by RNA-seq.

first: Peggy

last: Farnham


title: Detecting differential peaks in ChIP-seq signals with ODIN

city: Aachen

first: Martin

last: Zenke

state: NRW

address: Universitatsklinikum Aachen, RWTH

department: Cell Biology

institute: Institute for Biomedical Engineering

summary: Motivation: Detection of changes in DNA-protein interactions from ChIP-seq data is a crucial step in unraveling the regulatory networks behind biological processes. The simplest variation of this problem is the differential peak calling problem. Here one has to find genomic regions with ChIP-seq signal changes between two cellular conditions in the interaction of a protein with DNA. The great majority of peak calling methods can only analyse one ChIP-seq signal at a time and are unable to perform differential peak calling. Recently, a few approaches based on the combination of these peak callers with statistical tests for detecting differential digital expression have been proposed. However, these methods fail to detect detailed changes of protein-DNA interactions. Results: We propose ODIN; an HMM-based approach to detect and analyse differential peaks in pairs of ChIP-seq data. ODIN performs genomic signal processing, peak calling and p-value calculation in an integrated framework. We also propose an evaluation methodology to compare ODIN with competing methods. The evaluation method is based on the association of differential peaks with expression changes in the same cellular conditions. Our empirical study based on several ChIP-seq experiments from transcription factors, histone modifications and simulated data shows that ODIN outperforms considered competing methods in most scenarios.

overall_design: H3K4me1 and PU.1 occupancy in MPP, CDP, cDC and pDC

first: Manuel

last: Allhoff

first: Kristin

last: Sere

first: Heike

last: Chauvistre

first: Qiong

last: Lin

first: Ivan

last: Costa


title: Functional characterization of C. elegans Y-box binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes

first: Dimos

last: Gaidatzis

type: Expression profiling by genome tiling array


title: Altering cancer transcriptomes using epigenomic inhibitors [Illumina beadchip]

overall_design: Total RNA obtained from isolated HCT116 or PANC1 cell lines were treated with 10uM ICG-001, 10uM C646, or 0.05% DMSO and collected after 12 or 96 hours.

first: Frietze

last: Seth

first: Gaddis

last: Malaina

first: Farnham

last: Peggy


title: Next-Generation Sequencing Identifies Differentially Expressed Genes in Embryonic Hearts Following Caffeine Treatment

city: Gainesville

first: Xiefan

last: Fang

state: Florida

address: 1200 Newell Drive, ARB R1-148

institute: University of Florida

summary:
Purpose: This study aimed to identify differentially expressed genes including alternative splice variants in embryonic ventricles following in utero caffeine treatment.

Methods: Pregnant CD-1 mice were injected with 20 mg/kg of caffeine or vehicle control daily from embryonic day (E) 6.5-9.5. On E10.5, total RNA was isolated from embryonic ventricles and used for transcriptomic RNA sequencing with Illumina HiSeq 2000 (1X75bp). RNA-seq reads were aligned to the mouse genome (build mm10) with the Tophat for Illumina tool in the PSU galaxy platform. Counting and annotation of RNA-seq reads as well as alternative splicing analysis were performed with Partek Genomics Suite version 6.11. Differential expression of gene and transcript reads between treatments was analyzed with R package EdgeR. Genes/transcripts with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Differentially expressed genes were defined as genes with altered expression at either gene or transcript level. Unique differentially expressed genes were identified by combining the results from annotations with the RefSeq Transcripts (2013-05-10) or Ensembl Transcripts release 71 databases.

Results: Differential expression analysis revealed that 59 genes and 451 transcripts were significantly up-regulated, and 65 genes and 398 transcripts were down-regulated by prenatal caffeine treatment (fold change >1.5 or <-1.5; p-value with FDR<0.05). In total, 900 unique genes were identified to have altered expression either at the gene or transcription level. Further analysis with Partek GS revealed that 183 genes had abnormal alternative splicing at the exon level after in utero caffeine treatment.

Conclusions: In utero caffeine exposure caused gene expression changes in embryonic ventricles and these changes may lead to long-term effects on cardiac morphology and function.

overall_design: mRNA profiles in E10.5 heart ventricles treated with caffeine were generated by deep sequencing (n=2 for vehicle, n=3 for caffeine), using Illumina HiSeq 2000.

first: Scott

middle: A

last: Rivkees

first: Christopher

last: Wendler


title: Next Generation Sequencing identifying the dosage compensation state in human endometrial carcinoma and adjacent tissues

city: Suzhou

first: Yifeng

last: Zhou

state: Jiangsu

address: Ren Ai road #199

institute: Soochow University

summary: Mammals have evolved an XY sex chromosome system, resulting in dosage imbalance not only between sexes, but also between X-chromosome and autosome.

overall_design: mRNA profiles of 9 pairs of human endometrial carcinoma and adjacent tissues were generated by Illumina 100-nucleotide paired-end sequencing


title: Functional characterization of C. elegans Y-box binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes (RIP-Seq)

summary: We present a basic characterization of the function of Y-box binding proteins (YBPs) in C. elegans. YBPs in other organisms are known RNA-binding proteins. Our global analysis of associated mRNAs supports a role of YBPs as general mRNA binders in C.elegans.

overall_design: FLAG IPs were performed on transgenic animals expressing FLAG-tagged versions of either CEY-1, CEY-2, or CEY-4. MYC IPs served as controls. All IPs were performed in duplicates. 12 samples in total.

first: Andreas

last: Arnold

first: Rafal

last: Ciosk


title: Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function [Dnase-Seq]

first: Ann

last: Harris

state: Illinois

address: 2430 N Halsted

institute: Northwestern University

summary: The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human fetal epididymis epithelial cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in silico analysis identified transcription factor binding sites (TFBS) that were over-represented in the HEE open chromatin, include the motif for paired box 2 (PAX2). PAX2 is a critical transcriptional regulator of urogenital tract development, which is well studied in the kidney but is unexplored in the epididymis. Due to the limited lifespan of primary HEE cells in culture we investigated the role of PAX2 in an immortalized HEE cell line (REP). First, REP cells were evaluated by DNase-seq and their open chromatin map overlapped that of primary HEE cells at ~ 65% of sites. Moreover, the PAX2-binding motif was again identified as an overrepresented TFBS within intergenic open chromatin, though on fewer chromosomes than in the primary HEE cells. To identify PAX2-target genes in REP cells, RNA-seq analysis was performed after siRNA-mediated depletion of PAX2 in comparison to a non-targeting siRNA. In response to PAX2-represssion, 3142 transcripts were differentially expressed (1334 up-regulated and 1808 down-regulated). Novel PAX2 targets included multiple genes encoding proteins with a predicted function in the epididymis epithelium.

overall_design: examination of open chromatin region in REP cells with 2 replicates

first: James

last: Browne

first: Rui

last: Yang


title: Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function [Rnase-Seq]

overall_design: mRNA profile of control and PAX2 knockdown REP cells


title: Localization and Abundance Analysis of Human lncRNAs at Single Cell and Single Molecule Resolution

city: Cambridge

first: Nataly

middle: Moran

last: Cabili

address: 7  Cambridge Center

institute: Broad Institute ;  Harvard University

summary: Long noncoding RNAs (lncRNAs) have emerged as key players in different cellular processes and are required for diverse functions in vivo. However, fundamental aspects of lncRNA biology remain poorly characterized, including their subcellular localization, abundance and variation at a single cell resolution. Here, we used single molecule, single-cell RNA fluorescence in situ hybridization (RNA FISH) to survey 61 lncRNAs, chosen by properties such as conservation, tissue specific expression, and expression abundance, and to catalog their abundance and cellular localization patterns in three human cell types. Our lncRNAs displayed diverse sub-cellular localization patterns ranging from strictly nuclear localization to almost exclusive cytoplasmic localization, with the majority localized primarily in the nucleus. The low abundance of these lncRNAs as measured in bulk cell populations cannot be explained by high expression in a small subset of 'jackpot' cells. Simultaneous analysis of lncRNAs and mRNAs from corresponding divergently transcribed loci showed that divergent lncRNAs do not present a distinct localization pattern and are not always co-regulated with their neighbor. Overall, our study highlights important differences and similarities between lncRNAs and mRNAs. The rich set of localization patterns we observe are consistent with a broad range of potential functions for lncRNA, and assists in hypothesis generation for mechanistic studies. Here we provide the RNA-Seq expression matrix, as well as RNA-Seq raw data, which we used for comparison with RNA FISH molecule counts.

overall_design:
We estimate FPKM of coding genes and lncRNAs across HeLa, human lung fibroblasts and human foreskin.

This study includes data from human foreskin fibroblasts (hFF), human lung fibroblasts (hLF), and HeLa cells. An hFF sample (GSM1376178) and the hLF samples (GSM1376175-GSM1376177) were previously submitted and are available in GSE30554 as GSM759893 and GSM759890-GSM759892, respectively. The HeLa samples (GSM591670-GSM591671) were previously submitted and are available in GSE23316.

The complete dataset representing: (1) the hFF Samples, including the re-analysis of the hFF Sample from GSE30554, (2) the re-analysis of the hLF Samples from GSE30554, and (3) the re-analysis of the HeLa Samples from GSE23316, is linked below as a supplementary file.

first: Moran

middle: N

first: David

last: Hendrickson

first: John

middle: L

last: Rinn

first: Arjun

last: Raj

first: Aviv

last: Regev


title: TRIM28 silences endogenous retroviruses in neural progenitor cells

city: Lausanne

first: Adamandia

last: Kapopoulou

address: Station 15

department: Sciences de la Vie

institute: EPFL

summary: Here we show that in neural progenitor cells (NPCs) TRIM28 silences transcription of two groups of endogenous retroviruses (ERVs): IAP1 and MMERVK10C. Derepression of ERVs in Trim28-deficient NPCs was associated with a loss of H3K9me3 and resulted in transcriptional upregulation and reverse transcription. These findings demonstrate a unique dynamic transcriptional regulation of ERVs in NPCs.

overall_design: Analysis of upregulation of ERVs in Trim28-deficient NPCs

first: Liana

last: Fasching

first: Rohit

last: Sachdeva

first: R

last: Petri

first: M

last: Jönsson

first: Christian

last: Männe

first: Priscilla

last: Turelli

first: Patric

last: Jern

first: Florence

last: Cammas

first: Didier

last: Trono

first: Johan

last: Jakobsson


title: RNA-seq analysis of differential gene expression in muscle from angus beef steers undergoing diet restriction and compensatory growth

city: Dunsany

first: Anthony

middle: Gerard

last: Doran

state: Co Meath

address: Grange

department: Animal and Bioscience Department

country: Ireland

institute: Teagasc

summary: The objective of this study was to examine changes in muscle gene expression of growing steers during a period of dietary energy restriction followed by a period of realimentation. Crossbred Aberdeen Angus x Holstein Friesian (n = 24) steers were assigned to one of two feeding treatments. Over a 99 d period, 1 group (n=12) was offered a high energy control diet consisting of concentrates ad libitum and 7 kg of grass silage per head daily. The second group (n=12) was offered an energy restricted diet consisting of grass silage ad libitum plus 0.5 kg of concentrate per head per day. From the end of the differential feeding period (99 d), both groups of animals were offered a total mixed ration (grass silage:concentrate ratio of 80:20). This period, which lasted 200 d, was known as the realimentation period. All animals were slaughtered on d 299 of the study. Muscle biopsies were collected at 2 time points (end of the differential feeding period (d 99) and during the realimentation period (d131). RNA was extracted and muscle gene expression was examined using RNA-seq technology and bioinformatic analysis. During the differential feeding period, 17 over-represented pathways were identified, including the peroxisome proliferator activated receptor signalling, glycolysis/ gluconeogenesis and metabolic pathways controlling the metabolism of lipids and lipoproteins which indicate reduced energy intake and fat tissue accumulation occurring in muscle tissue during the restriction phase. During the realimentation period, 164 differentially expressed genes were annotated to 9 over-represented pathways including starch and sucrose metabolism, carbohydrate digestion and absorption and TGF-β signalling pathway. It is hypothesised that the signalling effects of the TGF-β pathway were reduced thereby promoting accelerated cell growth and proliferation in muscle tissue of animals experiencing compensatory growth. This information can be exploited in genomic breeding programmes to assist selection of cattle with a greater ability to compensate following a period dietary restriction.

overall_design: 24 muscle RNA samples were analysed in total. 6 samples were from muscle biopsies collected at the end of a period of dietary restriction (d99) and 6 samples were from muscle biopsies collected at the peak of compensatory growth (d131). In addition, RNA was also analysed from 6 samples collected from animals fed ad libitum at each of these two timepoints.

first: Sarah

last: Keady

last: Kenny

first: Chris

last: Creevey

first: Sinead

last: Waters


title: Hepatitis C virus functionally sequesters miR-122

status: Public on Jan 06 2015

city: New York

first: Joseph

last: Luna

state: NY

address: 1230 York Ave #64

institute: The Rockefeller University


title: Hepatitis C virus functionally sequesters miR-122 [RNA-Seq]

summary: Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5’UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect could be relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and number of sites. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.

overall_design: mRNA-seq libraries were generated from mock or J6/JFH1 Clone2 infected Huh7.5 cells. Cells were infected at an MOI of 1-2 and harvested at 72 hours and 96 hours post-infection for CLIP. Libraries were generated using Illumina Truseq technology.

first: Troels

last: Scheel

first: Tal

last: Danino

first: Katharina

last: Shaw

first: Aldo

last: Mele

first: John

middle: J

last: Fak

first: Eiko

last: Nishiuchi

first: Constantin

last: Takacs

first: Maria

last: Catanese

first: Ype

last: de Jong

first: Ira

last: Jacobson

first: Charles

last: Rice

first: Robert

last: Darnell


title: Hepatitis C virus functionally sequesters miR-122 [miR-122 KO vs Control CLIP]

overall_design: AGO HITS-CLIP libraries were generated from single cell clones of miR-122 deleted Huh7.5 cells using CRISPR (KO), unedited controls (WT), or cells transfected with GFP instead of CRISPR. Libraries were generated with a 4nt index read, a common priming sequence, followed by a 5nt degenerate barcode terminiating in a G. Files have been demultiplexed such that the 5nt degenerate barcode has been appended as the first 5 nucleotides of the read.


title: Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function

city: Chicago

first: Ann

last: Harris


title: ATAC-seq data in mouse and zebrafish limbs

city: Sevilla

first: Juan

middle: J.

last: Tena

address: Univ. Pablo Olavide, Ctra. Utrera km1

institute: CABD/CSIC

summary: Assay for Transposable Accessible Chromatin (ATAC) reveals a genome wide view of areas of open chromatin at very high resolution, which are often associated with regulatory activity.

sample_organism:
Danio rerio
Mus musculus

platform_organism:
Danio rerio
Mus musculus

overall_design: The ATAC-seq technology uses a Tn5 transposase loaded with nex-generation sequencing primers in order to simultaneously fragment areas of open chromatin and ligate adapters.

first: Andrew

last: Gehrke

first: Igor

last: Schneider

first: Carlos

last: Gómez-Marín

first: Tetsuya

last: Nakamura

first: Mayuri

last: Chandran

first: Elisa

last: de la Calle-Mustienes

first: Ingo

last: Braasch

last: Postlethwait

first: José

last: Gómez-Skarmeta

first: Neil

last: Shubin


title: Hepatitis C virus functionally sequesters miR-122 [LNAs vs Mock CLIP]

first: Joseph

last: Luna

overall_design: AGO HITS-CLIP libraries were generated from mock, LNA or miravirsen treated, or from U3 virus electroporated Huh7.5 cells. Cells were harvested for CLIP 48 hours after small RNA treatment and 17 days after virus electroporation. LNA treatments for U3 virus infected cells was carried out for 48hours. Libraries were generated with a 4nt index read, a common priming sequence, followed by a 5nt degenerate barcode terminiating in a G. Files have been demultiplexed such that the 5nt degenerate barcode has been appended as the first 5 nucleotides of the read.

first: Troels

last: Scheel

first: Tal

last: Danino

first: Katharina

last: Shaw

first: Aldo

last: Mele

first: John

last: Fak

first: Eiko

last: Nishiuchi

first: Constantin

last: Takacs

last: Catanese


title: Long Non-coding RNAs Control Hematopoietic Stem Cell (HSC) Function

city: Houston

first: deqiang

last: sun

address: 2121 W Holcombe Blvd

institute: Texas A&M University


title: Chromatin structure dynamics of hoxa13a, hoxa13b and hoxd13a loci during zebrafish development.

first: Juan

last: Tena

summary: We designed 4C-Seq experiments for hoxa13a, hoxa13b and hoxd13a promoters in three developmental stages in zebrafish embryos: 24, 48 and 60 hpf.

overall_design: 4C-seq samples for three different hox13 genes promoters in three developmental stages in zebrafish embryos: 24, 48 and 60 hpf.

first: Andrew

last: Gehrke

first: Igor

last: Schneider

last: Gómez-Marín

last: Nakamura


title: Hepatitis C virus functionally sequesters miR-122 [HCV vs Mock CLIP]

overall_design: AGO HITS-CLIP libraries were generated from mock or J6/JFH1 Clone2 infected Huh7.5 cells. Cells were infected at an MOI of 1-2 and harvested 72hours post-infection for CLIP. Libraries were generated with a 4nt index read, a common priming sequence, followed by a 5nt degenerate barcode terminiating in a G. Files have been demultiplexed such that the 5nt degenerate barcode has been appended as the first 5 nucleotides of the read.

first: Maria

first: Ype

last: de Jong

first: Ira

last: Jacobson

first: Charles

last: Rice

first: Robert

last: Darnell


title: A Cell-based Systems Biology Assessment of Human Blood to Monitor Immune Responses After Influenza Vaccination

city: Nashville

last: Link

state: Tennessee

address: 1161 21st Ave South

department: Pathology, Microbiology and Immunology

institute: Vanderbilt University School of Medicine

summary: Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination.  Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.

overall_design: PBMC and six purified cell types from two vaccinated donors were isolated prior to (d0) and at days 1, 3, and 7 post-TIV vaccination for RNA-seq analysis

first: Kristen

last: Hoek


title: MethylC-Seq of ago6-2, ago4-6 and ago4-6 ago6-2 double mutants

city: West Lafayette

first: Jian-Kang

last: Zhu

state: IN

address: 625 Agriculture Mall Dr.

department: Department of Horticulture and Landscape Architecture

institute: Purdue University

type: Methylation profiling by high throughput sequencing

summary: Using MethylC-Seq to provide single-base resolution of DNA methylation status in  ago6-2, ago4-6 and  ago4-6 ago6-2 double mutants

overall_design: MethylC-Seq: 3 mutants examined, ago6-2, ago4-6 single mutant and  ago4-6 ago6-2 double mutants

first: Cheng-Guo

last: Duan

first: Huiming

last: Zhang

first: Kai

last: Tang


title: miRNome of endometriotic lesion

city: Tartu

first: Merli

last: Saare

address: Puusepa 8

department: Obstetrics and Gynecology

country: Estonia

institute: University of Tartu

summary: miRNA high-throughput sequencing was used to investigate endometriosis lesion-specific miRNA expression profiles by comparing a set of paired samples of peritoneal endometriotic lesions and matched healthy surrounding tissue together with eutopic endometrium of the same patients. We found that miRNAs of surrounding peritoneal tissue mask most of the miRNA expression differences that could originate from endometriotic tissue and thus only miRNAs with significantly different levels in the endometriotic lesions compared to peritoneal tissue were detected. According to the results of this study, two miRNAs – miR-34c and miR-449a showed remarkably higher expression in lesions compared to healthy tissue.

overall_design: Eleven tissue samples (two endometria, five peritoneal lesions and four matched adjacent normal-appearing tissues) were analysed from two patients with a histologically confirmed diagnosis of moderate-severe endometriosis (III-IV stage)

first: M

last: Peters


title: Genome wide mapping of NFAT1 binding sites in dendritic cells and of chromatin state in steady and dectin-1 activated dendritic cell line D1

status: Public on Jan 05 2015

city: Farmington

first: Marina

last: Yurieva

state: Connecticut

address: 10 Discovery dr

department: Computational Sciences

institute: Jackson Laboratory

summary: We generated genome-wide chromatin-state maps of mouse dendritic cells in steady and dectin-1 activated with or without NFAT inhibitor FK506. We found the differential H3K4me3 signatures in these cells. This study provides the epigenetic signatures of steady, dectin-1activated with or without NFAT inhibition. We also generated stable dendritic cell line D1 expressing a V5 tagging NFAT1. This cell line was used to map the global binding sites of NFAT1 upon dectin-1 activation with 10ug/ml curdlan for 30 minutes.

overall_design: examine NFAT1-v5 genome wide binding sites and differential H3K4me3 epigenetic modification in steady, dectinn-1 activated with or without NFAT inhibiton.

first: Hong-Bing

last: Yu


title: Translatome analyses of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation

status: Public on Jan 04 2015

city: Haifa

first: Sigal

last: Savaldi-Goldstein

address: Technion City

country: Israel

institute: Technion

summary: The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone, is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the inner-most tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR, revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, while BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth.

overall_design: 24 samples of polyribosome-associated mRNA (following 0, 3, and 8 hours of BR treatment, in two biological repetitions), were collected. RNA samples were sequenced on an Illumina HiSeq 2500, to obtain 50 bp single-end reads. These reads were aligned to the TAIR10 assembly of the Arabidopsis thaliana genome, using Tophat2 v2.0.9. Transcript coordinates from the TAIR10 reference set were used to guide the alignment process. After alignment, the numbers of reads per transcript were estimated using HTSeq v0.5.3p3, and DESeq2 v1.2.8 was used to normalize read counts and call differentially expressed genes.

first: Kristina

last: Vragovic

first: Elizabeth

last: Bartom


title: Hepatitis C virus functionally sequesters miR-122 [m15 vs Mock CLIP]

first: Joseph

last: Luna

overall_design: AGO HITS-CLIP libraries were generated from mock or m15 J6/JFH1 Clone2 infected Huh7.5 cells. Cells were infected at an MOI of 1-2 and harvested 72 hours post-infection for CLIP. Libraries were generated with a 4nt index read, a common priming sequence, followed by a 5nt degenerate barcode terminiating in a G. Files have been demultiplexed such that the 5nt degenerate barcode has been appended as the first 5 nucleotides of the read

first: Troels

last: Scheel

first: Tal

last: Danino

first: Katharina

last: Shaw

first: Aldo

last: Mele

first: John

last: Fak

first: Eiko

last: Nishiuchi

first: Constantin

last: Takacs

first: Maria

last: Catanese

last: de Jong


title: Causal signals between codon bias, mRNA structure and the efficiency of translation and elongation

city: Stanford

first: Cristina

last: Pop

state: CA

address: 353 Serra Mall

department: Computer Science

institute: Stanford University

summary: Ribosome profiling data reports on the distribution of translating ribosomes, at steady-state, with codon-level resolution. We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate-reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation.

overall_design: We test whether tRNA abundance affects elongation or translation efficiency by changing the tRNA levels through deletion or over expression and measuring the ribosomal dwell time at each codon using a robust statistical method that accounts for flow conservation.

first: Silvi

last: Rouskin

first: Nicholas

last: Ingolia

first: Lu

last: Han

first: Eric

last: Phizicky

first: Jonathan

last: Weissman

first: Daphne

last: Koller


title: H2A.Z marks antisense promoters and has positive effects on antisense transcript levels in budding yeast

first: Muxin

last: Gu

email: muxingu@gmail.com, mg445@cam.ac.uk

address: Jeffrey Cheah Biomedical Centre, Puddicombe Way

department: Haematology

country: United Kingdom

institute: University of Cambridge

summary: The histone variant H2A.Z, which has been reported to have both activating and repressive effects on gene expression, is known to occupy nucleosomes at the 5’ ends of protein-coding genes. We now find that H2A.Z is also significantly enriched in gene coding regions and at the 3’ ends of genes in budding yeast, where it co-localises with histone marks associated with active promoters. By comparing H2A.Z binding to global gene expression in budding yeast strains engineered so that normally unstable transcripts are abundant, we show that H2A.Z is required for normal levels of antisense transcripts as well as sense ones. High levels of H2A.Z at antisense promoters are associated with decreased antisense transcript levels when H2A.Z is deleted, indicating that H2A.Z has an activating effect on antisense transcripts. Decreases in antisense transcripts affected by H2A.Z are accompanied by increased levels of paired sense transcripts. Therefore, the effect of H2A.Z on protein coding gene expression is a reflection of its importance for normal levels of both sense and antisense transcripts.

overall_design: Htz1 ChIP-seq in wild-type (WT) and rrp6Δ yeast, along with negative control ChIP-seq in htz1Δ and input control. Strand-specific transcriptomic profiles of WT, htz1Δ, rrp6Δ and htz1Δrrp6Δ. Replicates are present for all samples except the negative and input control ChIP samples.

first: Yanin

last: Naiyachit

first: Thomas

last: Wood

first: Catherine

last: Millar


title: Oxymetholone therapy of Fanconi anemia suppresses osteopontin transcription and induces hematopoietic stem cell cycling

city: Portland

first: Qingshuo

last: Zhang

state: OR

address: 3181 SW Sam Jackson Park Road

department: Department of Pediatrics

institute: Oregon Health & Science University

summary:
We used Fancd2-/- mice to understand its mechanism of action. Transcriptome analysis of cKit+ Sca1+ Lin- (KSL) cells discovered that only four genes changed their expression levels significantly after chronic OXM administration in both Fancd2−/− and wild-type mice: mKi67 and Cenpf were up-regualted by 1.4 fold; Spp1 and Oasl2  were significantly down-regulated by 10.5 and 1.5 fold, respectively. Both mKi67 and Cenpf genes are cell cycle-regulated genes and proliferation markers. Their up-regulation was consistent with our observation in flow cytometry analysis that oxymetholone stimulated the proliferation of hematopoietic stem and progenitor cells. RNAseq analysis showed no effects on mTert mRNA expression with chronic androgen therapy, but instead suggested down-regulation of Spp1 and Oasl2 as an important mechanism for the drug’s action.

Our RNAseq analysis also revealed that Fancd2−/− KSL cells showed clear changes in mRNA expression profiles compared to wild-type controls: 430 genes were down-regulated by more than 1.5 fold, whereas 159 genes were up-regulated. Gene ontology analysis revealed key pathways to be significantly altered in Fancd2−/− KSL cells. Besides the abnormal cell cycle status expected from our earlier flow cytometry analysis, surprisingly we noticed that a group of genes involved in immune responses and inflammation, comprising Cfp (Properdin), Socs2, Ccr1, Ccr2, Ccr5, Chga (Chromogranin A), Ifi30 (Interferon Gamma-Inducible Protein 30), Lgmn, Txn, and Sell (selectin L), were up-regulated in Fancd2−/− KSL cells. We therefore hypothesize that some genes up-regulated in FA HSPCs may be part of an innate immune response to DNA damage.

In addition, whole bone marrow cells were also analyzed in parallel with KSL cells. As compared to whole bone marrow cells, the genes enriched in KSL cells in wild-type mice were listed in details in the corresponding publication. This information can be a good resource for the future gene expression analysis of HSPCs.

Finally, we compared the gene expression profiles of early progenitors between OXM-treated and placebo-treated mice. There were no significant differences at all in gene expression between OXM-treated wild-type erythroid progenitors and their placebo-treated wild-type counterparts, with no genes displaying an expression change higher than 1.2 fold. Importantly, no up-regulation of EPO-inducible genes such as Socs1, Socs2, Socs3, and Cish was seen in wild-type mice treated with OXM. Furthermore, there was no differential expression of the well-known EPO target transferrin receptor or any other major players of the Epo-R signaling network such as Bcl2l1, Cdc25a, Btg3, Ccnd2, Lyl1, Pim3, and Tnfrsf13c. These results indicate that EPO might not play a role in the action of OXM in the erythroid lineage.

overall_design:
The goal of this study is to investigate gene expression changes in Fancd2 knockout mice in response to oxymetholone treatment. The study focuses on two bone marrow cell populations: cKit+ Sca1+ Lin- cells (representing hematopoietic stem and progenitor cells) and Ter119+/CD71high/FSChigh cells (representing proerythroblasts and basophilic erythroblasts). Both populations were sorted twice by FACS to ensure the purity. Cells of interest were collected in Trizol and RNA was isolated using RNAeasy mini prep kit. mRNAs were positively selected using oligo(dT)- Dynobeads and treated with DNase I. RNAseq libraries were then constructed using Illumina TruSeq RNA Sample Prep Kit and sequenced as 51 base-length reads on an Illumina HiSeq 2000 genome analyzer. For KSL libraries, each sample represented total mRNA isolated from pooled KSL cells of 5 individual mice; for basophilic erythroblast libraries, each library represented total mRNA isolated from basophilic erythroblasts of one individual mouse; for whole bone marrow libraries, each sample represented a combined library originally from 5 individual mice.

All reads were mapped to the mouse reference genome (version mm9) using Bowtie short read aligner software (http://bowtie-bio.sf.net). Most of the data analysis was performed using EdgeR GLM algorithms. For the comparison of oxymetholone KSL libraries vs placebo KSL libraries, more stringent pair-wise comparisons were used to keep a consistent flow cytometric setting among each pair. The common gene list was the one shared by all three comparisons: COM17 vs HSC_101b, HSC_13 vs HSC_18, and HSC_23 vs QZ_35 for Fancd2−/− KSL cells; HSC_3 vs QZ_36, HSC_22 vs HSC_24, and COM15 vs COM16 for wild-type KSL cells. Data-mining and pathway analysis were carried out with the MetaCore integrated software suite (Thomson Reuters, New York, USA).

first: Markus

last: Grompe


title: Hybrid dysfunction and physiological compensation in gene expression

city: La Jolla

first: Felipe

last: Barreto

state: California

address: 8750 Biological Grade

department: Marine Biology Research Division

institute: Scripps Institution of Oceanography

summary: The formation of new species is often a consequence of genetic incompatibilities accumulated between populations during allopatric divergence. When divergent taxa interbreed, these incompatibilities impact physiology and have a direct cost resulting in reduced hybrid fitness. Recent surveys of gene regulation in interspecific hybrids have revealed anomalous expression across large proportions of the genome, with 30-70% of all genes apparently misexpressed, mostly in the direction of down-regulation.  However, since most of these studies have focused on pairs of species exhibiting high degrees of reproductive isolation, the association between regulatory disruption and reduced hybrid fitness prior to species formation remains unclear.  Within the copepod species Tigriopus californicus, interpopulation hybrids show reduced fitness associated with mitochondrial dysfunction.  Here we show that in contrast to studies of interspecific hybrids, only 1.2% of the transcriptome was misexpressed in interpopulation hybrids of T. californicus, and nearly 80% of misexpressed genes were overexpressed rather than underexpressed.  Moreover, many of the misexpressed genes were components of functional pathways impacted by mitonuclear incompatibilities in hybrid T. californicus (e.g., oxidative phosphorylation and antioxidant response).  We also show that the magnitude of hybrid misregulation is not dependent on levels of protein sequence divergence, even though the latter is correlated with expression divergence between parental populations.  Our results suggest that hybrid breakdown at early stages of speciation may result from initial incompatibilities amplified by the cost of compensatory physiological responses.

overall_design:
Our experiment included nine RNA-seq samples: 3 San Diego, 2 Santa Cruz, and 4 hybrid samples.  For each sample, 400-500 copepods across all developmental stages were collected from their stock cultures.  They were transferred to fresh filtered seawater in a 50-mL Falcon tubes and immersed in a 20°C water bath for two hours.  Water was then quickly removed, 4 mL of Tri-Reagent (Sigma) added, and tissue immediately disrupted using a tissue homogenizer.  RNA was isolated following the manufacturer’s protocol.  Re-suspended RNA pellets were further purified with RNeasy Mini columns (Qiagen), and final sample integrity and quantity were assessed with an Agilent 2100 BioAnalyzer.

Please note that two samples (GSM1531288, GSM1531290) have been accessioned under BioProject PRJNA168170, SRA study SRP013608, while the remaining seven samples under BioProject PRJNA263967, SRA Study SRP048974.
The current records including all 9 samples (PRJNA264820/SRP049247) were re-created for the convenient retrieval of the complete raw data from SRA

first: Ricardo

last: Pereira

first: Ronald

last: Burton


title: ChIP-Seq of Histone H3K4me1 and H3K4me3 in human GIST48 cells

status: Public on Jan 01 2015

first: Yu

last: Chen

address: 1275 York Ave, Box 20

department: Human Oncology and Pathogenesis Program

institute: Memorial Sloan Kettering Cancer Center

summary: We previously mapped ETV1 using ChIP-Seq in GIST48 cells (GSE22441). Here, we map the enhancer landscape marked by histone H3K4me1 and the promoter landscape marked by histone H3K4me3 in GIST48 cells.

overall_design: Crosslink ChIP-Seq of H3K4me1 and H3K4me3 in GIST48 cells

first: Ping

last: Chi


title: Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals

city: Berlin

first: Andranik

last: Ivanov

address: Chariteplatz 1

department: Core Unit Bioinformatics

institute: Berlin Institute of Health

summary: Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human Alu repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified new circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched compared to linear controls. By scoring the presence of reverse complementary sequences in human introns we predicted and experimentally validated novel circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyper-editing events. Knockdown of the double-strand RNA editing ADAR1 enzyme significantly and specifically up-regulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA:RNA interactions of introns form larger structures which promote circularization of embedded exons, while ADAR1 antagonizes circRNA expression by melting stems within these interactions. Thus, we assign a new function to ADAR1.

overall_design: Examination of 12 samples in different stages of C.elegans development.

first: Sebastian

last: Memczak

first: Emanuel

last: Wyler

first: Francesca

last: Torti

first: Hagit

last: Porath

first: Marta

middle: R

last: Orejuela

first: Michael

last: Piechotta

first: Erez

middle: Y

last: Levanon

last: Landthaler

first: Christoph

last: Dieterich

first: Nikolaus

last: Rajewsky


title: RNA-Seq of murine GIST-like tumors after Etv1 ablation

summary: We used a mouse expressing three alleles 1) KitV558Delta/+ activating allele that develop GIST-like tumors in the cecum, 2) Etv1 flox/flox conditional knockout allele and 3) Rosa26-CreERT2 tamoxifen activated Cre allele. Mice were treated with either Tamoxifen (to delete Etv1) or corn oil (control). Cecal tumors were isolated for gene expression profiling by RNA-Seq.

overall_design: Expression profile mouse cecal GIST tumor with or without Etv1 ablation was generated by RNA-Seq


title: Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

status: Public on Jan 02 2015

city: Montreal

first: Soizik

last: Berlivet

state: Quebec

address: 110, avenue des Pins Ouest

country: Canada

institute: Institut de Recherches Cliniques de Montreal

summary: Though important for gene regulation most studies of genome organisation use either fluorescence in situ hybridisation (FISH) or chromosome conformation capture (3C) methods. FISH directly visualises the spatial relationship of sequences, but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde crosslinking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organisation, but this has not been tested extensively.  We investigate the murine HoxD locus with 5C and FISH in different developmental and activity states, and in the presence or absence of epigenetic regulators. We identify situations where the two datasets are concordant, but find other conditions where chromatin topographies extrapolated from 5C or FISH data are not compatible. We conclude that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart – influenced by nuclear environment and chromatin composition. We conclude that results obtained at high-resolution with either 3C methods or by FISH alone must be interpreted with caution and that conclusions about genome organisation should be validated by independent methods.

overall_design: 5C oligonucleotides were designed around EcoRI restriction sites following an alternative scheme

first: Iain

last: Williamson

first: Denis

last: Paquette

first: Josée

last: Dostie

first: Wendy

last: Bickmore


title: Identification of host microRNAs potentially related to SGIV infection in grouper Epinephelus coioides

city: Guangzhou

first: Yang

last: Yan

address: West Xingang Road

institute: South China Sea Institute of Oceanology Chinese Academy of Sciences

summary: microRNAs (miRNAs) are an evolutionarily conserved class of non-coding RNA molecules, which regulate kinds of biological processes at post-transcriptional level. Investigation of miRNAs expression profiles using high-throughput strategies is efficiently conductive to identify and characterize miRNAs. In this study, through Solexa deep sequencing approach, we obtained 115 orange spotted grouper (Epinephelus coioides) encoded miRNAs. Among them, 107 miRNAs shared high similarity with miRNAs encoded by zebrafish (Danio rerio) and other four vertebrates, indicating that cellular miRNAs are highly conserved between species.

overall_design: 18-26 nt small RNAs from GS cells were sequenced in one Solexa lane

first: Qiwei

last: Qin


title: Promoter hypermethylation of TERT is associated with hepatocellular carcinoma in the Han Chinese population

status: Public on Jan 03 2015

city: Shanghai

first: Hong

last: Zhang

address: Ming Dao Building  138 Yi Xue Yuan Road

department: Institute for Biomedical Sciences

institute: Fudan University

summary: A recent study by Castelo-Branco, P., et al. Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study. Lancet Oncol 2013;14:534-542 found upstream of the transcription start site (UTSS) hypermethylation of TERT is associated with tumor progression and poor prognosis in paediatric brain tumours. They interpreted that the UTSS region of telomerase reverse transcriptase (TERT) gene is a potentially accessible biomarker for various cancers. This study, we aimed to explore the role of TERT in hepatocellular carcinoma (HCC) and to investigate whether the UTSS region of TERT promoter shows the same methylation pattern in HCC.

overall_design: We analyzed a methylation assay of TERT including the UTSS region in 125 paired HCC samples using Mass Array EpiTyper (Sequenom, SanDiego, CA, USA). To obtain the relationship between TERT promoter methylation status and TERT expression level, we analysed a validation group of 12 paired HCC samples and acquired the FPKM value of TERT gene.

first: Xiaoling

last: Weng

first: Daizhan

last: Zhou

first: Yun

last: Liu


title: Intrinsic Protection From Leukemic Transformation at The Level of Hematopoietic Stem Cells

city: Lund

first: Shamit

last: Soneji

address: Solvegatan 19

country: Sweden

institute: BMC

summary: Genome wide RNA-seq from pGM and HSCs in response to expression of the MLL-ENL fusion gene

overall_design: Examination of mRNA abundance in two cell types with or without induction of the MLL-ENL fusion gene (following 48h of culture)

first: David

last: Bryder


title: An integrated transcriptome and expressed variant analysis of sepsis survival and death

city: Albuquerque

first: Raymond

last: Langley

state: New Mexico

address: 2425 Ridgecrest Dr

department: immunology

institute: LRRI

summary:
Background: Sepsis, a leading cause of morbidity and mortality, is not a homogeneous disease but rather a syndrome encompassing many heterogeneous pathophysiologies. Patient factors including genetics predispose to poor outcomes, though current clinical characterizations fail to identify those at greatest risk of progression and mortality.
Results: The Community Acquired Pneumonia and Sepsis Outcome Diagnostic study enrolled 1,152 subjects with suspected sepsis. We sequenced peripheral blood RNA of 129 representative subjects with systemic inflammatory response syndrome (SIRS) or sepsis (infection with SIRS), including 78 sepsis survivors and 28 sepsis nonsurvivors, who had previously undergone plasma proteomic and metabolomic profiling. The expression of 338 genes differed between subjects with SIRS and those with sepsis, primarily reflective of immune activation in sepsis. The expression of 1,238 genes differed with sepsis outcome: Nonsurvivors had lower expression of many immune function-related genes. Functional genetic variants associated with sepsis mortality were sought based on a common disease – rare variant hypothesis. VPS9D1, whose expression was increased in sepsis survivors, had a higher burden of missense variants in sepsis survivors, and these were associated with altered expression of 3,799 genes, primarily reflecting Golgi and endosome biology.
Conclusions: Host response in sepsis survivors – activation of immune response-related genes – was muted in sepsis nonsurvivors. The association of sepsis survival with robust immune response and presence of missense variants in VPS9D1 warrants replication and further functional studies.

overall_design: We sequenced peripheral blood RNA of 129 representative subjects with systemic inflammatory response syndrome (SIRS, n=23) or sepsis (infection with SIRS), including 78 sepsis survivors and 28 sepsis nonsurvivors, who had previously undergone plasma proteomic and metabolomic profiling.

first: Neil

last: Miller


title: ATRX tolerates activity-dependent histone H3 “methyl/phos switching” to maintain repetitive element silencing in neurons

city: New York

first: Li

last: Shen

address: 1425 Madison Ave

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

summary: ATRX is a member of the SWI2/SNF2 family of chromatin remodeling proteins and primarily functions at heterochromatic loci via its recognition of ‘repressive’ histone modifications (e.g., H3K9me3). Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, H3K9me3S10ph, in post-mitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively following periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph bound Atrx represses non-coding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

overall_design: For Atrx ChIP-seq, IPs were performed on three control vs. three KCl stimulated (all representing biological, and not technical replicates) primary cultured mouse cortical neurons at DIV 8. All samples were normalized to background input levels. For H3K9me3S10phos ChIP-seq, biological singlecates (control vs. forskolin) were analyzed against respective inputs.

first: Ian

last: Maze