title: Genome-wide gene expression analysis of Fusarium graminearum isolate PH-1 in spores and mycelium

city: Wageningen

first: Theo

last: van der Lee

address: Building no.107 Droevendaalsesteeg 1

institute: Wageningen university

summary: Using RNA-Seq data from the mycelium of Fusarium graminearum, we found weakly expressed regions on all of the four chromosomes that exactly matched with non-conserved regions. Comparison of gene expression between two different developmental stages (spores and mycelium) showed that the expression of genes in conserved regions is stable, while gene expression in non-conserved regions is much more influenced by developmental stage.

overall_design: Examination of gene expression in two developmental stages (spores and mycelium). Three biological replicates were performed for each stage.

first: Chunzhao

last: Zhao


title: Nucleosomes are context-specific, H2A.Z modulated barriers to RNA polymerase

status: Public on Mar 06 2014

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

country: USA

institute: Fred Hutchinson Cancer Research Center

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Nucleosomes are barriers to transcription in vitro, however, their effects on RNA polymerase in vivo are unknown. Here we describe a simple and general strategy to comprehensively map the positions of elongating and arrested RNA Polymerase II (RNAPII) at nucleotide resolution. Our results suggest that nucleosomes present significant, context-specific barriers to RNAPII in vivo that can be tuned by the incorporation of H2A.Z.

overall_design: 8 sets of two replicates each of paired-end and 3 sets of two replicates each of single-end samples were sequenced and analyzed.

first: Christopher

middle: M

last: Weber

first: Srinivas

last: Ramachandran

first: Steven


title: Functional Evolution of the Yap/Yorkie Proto-oncogene and Elucidation of its Core Transcriptional Program

status: Public on Mar 01 2014

city: Kansas City

first: Chris

middle: W

last: Seidel

state: MO

department: Genomics

institute: Stowers Institute

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

overall_design: Refer to individual Series


title: Genome-wide analysis of Yki and Sd localization in imaginal disks of Drosophila eye by ChIP Seq

summary:
Throughout Metazoa, developmental processes are controlled by a surprisingly limited number of conserved signaling pathways.  Precisely how these signaling cassettes were assembled in early animal evolution remains poorly understood, as do the molecular transitions that potentiated the acquisition of their myriad developmental functions.  Here we analyze the molecular evolution of the proto-oncogene YAP/Yorkie, a key effector of the Hippo signaling pathway that controls organ size in both Drosophila and mammals. Based on heterologous functional analysis of evolutionarily distant Yap/Yorkie orthologs, we demonstrate that a structurally distinct interaction interface between Yap/Yorkie and its partner TEAD/Scalloped became fixed in the eumetazoan common ancestor.  We then combine transcriptional profiling of tissues expressing phylogenetically diverse forms of Yap/Yorkie with ChIP-seq validation in order to identify a common downstream gene expression program underlying the control of tissue growth in Drosophila.  Intriguingly, a subset of the newly-identified Yorkie target genes are also induced by Yap in mammalian tissues, thus revealing a conserved Yap-dependent gene expression signature likely to mediate organ size control throughout bilaterian animals.  Combined, these experiments provide new mechanistic insights while revealing the ancient evolutionary history of Hippo signaling.
We sought to determine Yki and Sd target genes in Drosophila by immunoprecipitation of Yki, sd and RNA polymerase II.

overall_design: Chromatin immunoprecipitation of Yki, sd, and RNA polymerase II from eye disks was performed.

first: Aissam

last: Ikmi

first: Bjoern

last: Gaertner

first: Mansi

last: Srivastava

first: Julia

last: Zeitlinger

first: Matthew

middle: C

last: Gibson


title: Detailed analysis of the genetic and epigenetic signature of iPS cell-derived mesodiencephalic dopaminergic neurons

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

country: United Kingdom

institute: Altos Labs

type: Methylation profiling by high throughput sequencing

summary: Induced pluripotent stem cells (iPSCs) harbor great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson’s disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterization of such neurons is still lacking. The goal of this study is to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS-purified mdDA (Pitx3gfp/+) neurons derived from mouse iPSCs and primary mdDA (Pitx3gfp/+) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopt characteristics of their in-vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed as they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in-vitro disease modeling or cell-based therapy.

overall_design: RRBS methylation maps were generated for iPSCs cells, dopaminergic neurons derived from iPSCs and primary mesodiencephalic dopaminergic neurons

first: Reinhard

last: Roessler

first: Sebastien

middle: A

last: Smallwood

first: Jesse

middle: V

last: Veenvliet

first: Petros

last: Pechlivanoglou

first: Suping

last: Peng

first: Koushik

last: Chakrabarty

first: Marian

last: Groot-Koerkamp

first: R

middle: J

last: Pasterkamp

first: Evelyn

last: Wesseling

first: Gavin

last: Kelsey

first: Erik

last: Boddeke

first: Marten

middle: P

last: Smidt

first: Sjef

last: Copray


title: Global transcriptional Analysis of Yap/Yki Orthologs in Drosophila melanogaster by RNA Seq

first: Chris

last: Seidel

summary:
Throughout Metazoa, developmental processes are controlled by a surprisingly limited number of conserved signaling pathways.  Precisely how these signaling cassettes were assembled in early animal evolution remains poorly understood, as do the molecular transitions that potentiated the acquisition of their myriad developmental functions.  Here we analyze the molecular evolution of the proto-oncogene YAP/Yorkie, a key effector of the Hippo signaling pathway that controls organ size in both Drosophila and mammals. Based on heterologous functional analysis of evolutionarily distant Yap/Yorkie orthologs, we demonstrate that a structurally distinct interaction interface between Yap/Yorkie and its partner TEAD/Scalloped became fixed in the eumetazoan common ancestor.  We then combine transcriptional profiling of tissues expressing phylogenetically diverse forms of Yap/Yorkie with ChIP-seq validation in order to identify a common downstream gene expression program underlying the control of tissue growth in Drosophila.  Intriguingly, a subset of the newly-identified Yorkie target genes are also induced by Yap in mammalian tissues, thus revealing a conserved Yap-dependent gene expression signature likely to mediate organ size control throughout bilaterian animals.  Combined, these experiments provide new mechanistic insights while revealing the ancient evolutionary history of Hippo signaling.
We sought to define the downstream target genes of selected Yap variants by performing RNA sequencing analysis (RNA-seq) on total RNA isolated from GMR-Gal4>Yap eye discs.

overall_design: Transcriptional profiles were generated in triplicate from eye imaginal disks with either endogenous Yki, or GMR-Gal4 over-expressed Yki, Trichoplax Yap, Monosiga Yap, or Monisiga Yap+TEAD domain, using deep sequencing via Illumina Hi Seq.

first: Aissam

last: Ikmi

first: Bjoern

last: Gaertner

first: Mansi

last: Srivastava


title: Changes in transcript abundance for cuticular proteins and other genes three hours after a blood meal in Anopheles gambiae.

city: Athens

first: Judy

last: Willis

state: GA

address: 302 Biological Sciences Building

department: Cell Biology

institute: University of Georgia

summary: Numerous studies have examined changes in transcript levels after Anopheles gambiae takes a blood meal. Marinotti et al. (2006) used microarrays and reported massive changes in transcript levels 3 h after feeding (BF3h) compared to non-blood fed (NBF).  We were intrigued by the number of transcripts for structural cuticular proteins (CPs) that showed such major differences in levels and employed paired-end (50 bp) RNA-seq technology to compare whole body transcriptomes from 5-day-old females NBF and BF3h.  We detected transcripts for the majority of CPs (164/243) but levels of only 12 were significantly altered by the blood meal. While relative transcript levels of NBF females were somewhat similar to the microarray data, there were major differences in BF3h animals, resulting in levels of many transcripts, both for CPs and other genes changing in the opposite direction. We compared our data also to other studies done with both microarrays and RNA-seq. Findings were consistent that a small number of CP genes have transcripts that persist even in 5-day-old adults. Some of these transcripts showed diurnal rhythms (Rund et al., 2013; Rinker et al., 2013).  In situ hybridization revealed that transcripts for several of these CP genes were found exclusively or predominantly in the eye. Transcripts other than for CPs that changed in response to blood-feeding were predominantly expressed in midgut and Malpighian tubules.  Even in these tissues, genes responsible for proteins with similar functions, such as immunity or digestion, responded differently, with transcript levels for some rising and others falling. These data demonstrate that genes coding for some CPs are dynamic in expression even in adults and that the response to a blood meal is rapid and precisely orchestrated.

overall_design: Five females 3h after the blood meal (BF3h) and non-blood fed (NBF, controls) were collected for each RNA extraction. RNA preparations were sent to HudsonAlpha where eight libraries were prepared (four replicates for each treatments) with poly-A selection and preparation of 200 bp fragments using the Illumina True-Seq RNA Sample Prep kit and analyzed with HiSEQ paired-end reads of 50 nt.

first: Laura

last: Vannini

first: William

last: Dunn

first: Tyler

last: Reed

first: Judith

middle: H


title: Developmental Timecourses total-RNA sequencing [Ribosome repleted total RNA]

city: Basel

first: Dimos

last: Gaidatzis

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute

summary: We report the presence of extensive, transcriptionally controlled oscillations in the C. elegans, developmental transcriptome. Furthermore, using ribosome profiling, we show that these oscillating transcripts are actively translated.

overall_design: Examination of two timecourses that were collected over C. elegans development and analyzed by RNA-seq of "RiboMinus" libraries

first: Gert-Jan

last: Hendriks

first: Florian

last: Aeschimann

first: Helge

last: Großhans


title: System analysis of microRNAs in the development and aluminium stress responses of the maize root system.

city: Jinan

first: Xiangpei

last: Kong

address: 27 Shanda South Road

country: China

institute: Shandong University

type: Non-coding RNA profiling by high throughput sequencing

summary: Four sRNA libraries were generated and sequenced from the early developmental stage of primary roots (PRY), the later developmental stage of maize primary roots (PRO), seminal roots (SR), and crown roots (CR). Through integrative analysis, we identified 501 miRNAs (246 conserved and 255 novel ones) and found that the expression patterns of miRNAs differed dramatically in different maize roots.

overall_design: we generated and sequenced four maize small RNA libraries from the early developmental stage of primary roots (PRY), the later developmental stage of maize primary roots (PRO), seminal roots (SR), and crown roots (CR) using Solexa high-throughput sequencing technology

first: Zhaojun

last: Ding


title: Targeted RNA sequencing for gene discovery and quantification

city: Sydney

first: Tim

middle: R.

last: Mercer

state: New South Wales

address: 384 Victoria Street Darlinghurst NSW 2010

country: Australia

institute: Garvan

summary: Due to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes, RNA sequencing samples the majority of expressed genes infrequently, resulting in sparse sequencing coverage that can hinder robust isoform assembly and quantification. Targeted RNA sequencing addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for focused sequencing. This enhanced sequencing coverage confers sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of targeted RNA sequencing, from initial probe design considerations, capture of targeted genes, to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than a day, while the central experimental capture stage requires ~7 days.

overall_design: Targetted RNA sequencing of long noncoding RNAs

middle: R


title: Genome-wide chromatin maps of T-cell acute lymphoblastic leukemia (T-ALL)

city: Boston

first: Jiang

last: Zhu

state: MA

address: 185 Cambridge Street

institute: Massachusetts General Hospital, Harvard Medical School and Broad Institute

first: Birgit

last: Knoechel

first: Bradley

middle: E

last: Bernstein


title: Genome-wide chromatin maps of T-cell acute lymphoblastic leukemia (T-ALL) [ChIP-seq]

summary: Here we modeled T-ALL resistance to Notch inhibition, identifying ‘persister’ cells that readily expand in the presence of gamma secretase inhibitor (GSI) and the absence of Notch signaling. Rare persister cells are already present in naïve T-ALL populations, and the reversibility of the phenotype is suggestive of an epigenetic mechanism. Relative to GSI-sensitive cells, persisters activate distinct signaling and gene expression programs, and exhibit global chromatin compaction. A shRNA screen identified chromatin regulators whose depletion preferentially impairs persister cell viability, including BRD4, an acetyl-histone reader. BRD4 is up-regulated in the persisters and binds enhancers near genes with critical functions in T-ALL, including MYC and BCL2. Treatment of persisters with the BRD4 inhibitor JQ1 down-regulates these targets and induces growth arrest and apoptosis, at doses well tolerated by GSI-sensitive cells. Prompted by these findings, we examined and established the efficacy of GSI – JQ1 combination therapy against primary human leukemias in vivo. Our findings establish a role for epigenetic heterogeneity in leukemia drug resistance and suggest the potential of combination therapies that include epigenetic modulators to prevent and treat resistant disease.

overall_design: Examination of 5 different histone modifications and BRD4 in the T cell leukemia cell lines DND-41 and KOPT-K1 after chronic treatment with gamma Secretase inhibitor (Compound E, 1 uM, EMD4 Bioscience; persister) or vehicle (naïve).


title: RNA-Seq global gene expression of four Dendritic cells subpopulations from lung (CD8+ and CD8- pDCs, CD103-CD11b+ and CD103+CD11b- cDCs) of influenza infected mice at four time points following infection

status: Public on Mar 03 2014

city: Tel-Aviv

first: Irit

last: Gat-Viks

address: Tel-Aviv

department: Department of Cell Research and Immunology

country: Israel

institute: Tel-Aviv University

summary: Hundreds of immune cell types work in coordination to maintain tissue homeostasis. Upon infection, dramatic changes occur with the localization, migration and proliferation of the immune cells to first alert the body of the danger, confine it to limit spreading, and finally extinguish the threat and bring the tissue back to homeostasis. Since current technologies can follow the dynamics of only a limited number of cell types, we have yet to grasp the full complexity of global in vivo cell dynamics in normal developmental processes and disease. Here we devise a computational method, digital cell quantification (DCQ), which combines genomewide gene expression data with an immune cell compendium to infer in vivo dynamical changes in the quantities of 213 immune cell subpopulations. DCQ was applied to study global immune cell dynamics in mice lungs at ten time points during a 7-day time course of flu infection. We find dramatic changes in quantities of 70 immune cell types, including various innate, adaptive and progenitor immune cells. We focus on the previously unreported dynamics of four immune dendritic cell subtypes, and suggest a specific role for CD103+CD11b- cDCs in early stages of disease and CD8+ pDC in late stages of flu infection.

overall_design: To better understand the physiological role of these differential dynamic changes in the DCs, we measured the genome-wide RNA expression of all four DC subpopulations from lung of influenza infected mice at four time points following infections (two mice per time-point). For sorting dendritic cells from lungs, the lungs from infected and control uninfected C57BL/6J mice were immersed in cold PBS, cut into small pieces in 5 ml DMEM media containing 10% Bovine Fetal Serum, the cell suspensions were grinded using 1ml syringe cup on a 70 μm cell  strainers (BD Falcon). The cells were washed with ice cold PBS. Remaining red blood cells were  lysed using ammonium chloride solution (Sigma). Cells were harvested, immersed 1ml FACS  buffer [PBS+2% FBS, 1mM EDTA], Fc receptors were blocked with anti-mouse CD16/CD32, washed with FACS buffer and divided into two tubes for sorting cDC and pDC cells.

first: Mandelboim

last: Michal

first: Gat-Viks

last: Irit

first: Amit

last: Ido


title: Genome-wide Transcriptional Profiling of Skin and Dorsal Root Ganglia after Ultraviolet-B-Induced Inflammation

city: Malaga

first: James

last: Perkins

address: Pl. Hosp. Civil

department: Hospital Civil

country: Spain

institute: Hospital Carlos Haya

summary: Ultraviolet-B (UVB) irradiation of the skin was performed on rat (skin and DRG) and human (skin) tissue. The resulting changes in gene expression were then profiled by using RNA-seq to compare gene expression between irradiated and non-irradiated samples

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: Poly(A) selected RNA was sequenced for 5 irradiated and 4 non-irradiated humans (skin), and for 6 irradiated and 6 non-irradiated rats (DRG and skin)

first: John

last: Dawes

first: Ana

last: Antunes-Martins

first: Kathryn

last: Paterson

first: Ramona

last: Schmid

first: Werner

last: Rust

first: Tobias

last: Hildebrandt

first: Gerd

last: Giesslinger

first: Christine

last: Orengo

first: David

middle: L

last: Bennett

first: Steven

middle: B

last: McMahon


title: Genomewide identification of LHY binding targets.

city: Coventry

first: Nigel

middle: Paul

last: Dyer

address: Gibbet Hill Road

institute: University of Warwick

summary: The plant circadian enables plants to adjust their physiology and modulate their developmental responses in anticipation of diurnal and seasonal changes in environmental conditions. Thus, it makes an important contribution to overall fitness. The complex regulatory network underlying the oscillatory mechanism of the clock is now well characterised, but we still know relatively little about how downstream processes are controlled. In order to investigate this question we identified genes that are under direct control of one of the oscillator components, a MYB transcription factor known as LATE ELONGATED HYPOCOTYL (LHY).

overall_design: 2-week-old wild-type (Ws) and LHY knock-out (lhy-21) seedlings were harvested 2 h after dawn. Tissue was  cross-linked using 1% formaldehyde for 10 min. Chromatin was extracted and used for immunoprecipitation using an antibody to the full-length LHY protein. The immunoprecipitated DNA was sequenced using Illumina technology. LHY binding regions were identified as genomic regions that showed over-representation of reads in the wild-type sample as compared to the LHY knock-out sample.

first: Isabelle

last: Carre

first: Sally

last: Adams

first: Siren

last: Veflingstad


title: Extensive oscillatory gene expression during C. elegans larval development [Ribosome footprinting]

first: Dimos

last: Gaidatzis

overall_design: Ribosome-profiling analysis of a timecourse that was collected over C. elegans development

first: Florian

last: Aeschimann

first: Helge

last: Großhans


title: Clustering of CpG islands constitutes an important determinant of the interphase chromosome 3D organization [4C-Seq]

city: Vienna

first: Artem

last: Artemov

address: Spitalgasse 4

department: Center for Brain Research

country: Austria

institute: Medical University of Vienna

summary: Using 4C-Seq experimental procedure we have characterized, in cultured chicken lymphoid and erythroid cells, genome-wide patterns of spatial contacts of several CpG islands scattered along the chromosome 14. A clear tendency for interaction of CpG islands present within the same and different chromosomes has been observed. Accordingly, preferential spatial contacts between Sp1 binding motifs, and other GC-rich genomic elements including DNA sequence motifs capable to form G-quadruplexes were demonstrated. On the other hand, an anchor placed in gene/CpG islands-poor area was found to form spatial contacts with other gene/CpG islands-poor areas within chromosome 14 and other chromosomes. These results corroborate the two compartments model of interphase chromosome spatial organization and suggest that clustering of CpG islands harboring promoters and origins of DNA replication constitutes an important determinant of the 3D organization of eukaryotic genome in the cell nucleus. Using ChIP-Seq experimental procedure we have mapped genome-wide the CTCF deposition sites in chicken lymphoid and erythroid cells subjected to the 4C analysis. A good correlation between the density of these sites and the level of 4C signals was observed for the anchors located in CpG islands. It is thus possible that CTCF contributes to the clustering of CpG islands revealed in our experiments.

overall_design:
We applied 4C-seq to map long-range interactions of a CpG island harboring promoter of a housekeeping gene NPRL3 on a genome-wide scale in DT40 and HD3 cell lines in chicken (Gallus gallus). Two replicates per cell line were sequenced in paired-end mode with a depth of 45-64 million reads. We next performed additional 4C experiments on HD3 cells with different 4C anchors. In three experiments anchors were placed on different CpG island on the chromosome 14 that have demonstrated a strong interaction with the NPRL3 anchor (anchors near TSR3, TRAP1, PPL genes). In the forth experiment (anchor GENE-Des) the anchor was placed in a gene-poor area that did not interact with the NPRL3 promoter. The libraries for all anchors were pooled and sequenced in paired-end mode with a total depth of 75 million reads.
Anchor NPRL3: two replicates for HD3 and two replicates for DT40 cell lines. Other anchors (TSR3, GENE-Des, TRAP1, PPL): two replicates, pooled library

first: Ekaterina

middle: S

last: Gushchanskaya

first: Sergey

last: Ulyanov

first: Maria

middle: D

last: Logacheva

first: Aleksey

last: Penin

first: Alexey

last: Gavrilov

last: Razin


title: Genome-wide maps of histone H2A.Z and its chaperone.

city: Beijing

first: Zhuqiang

last: Zhang

address: 15 Datun Road, Chaoyang District.

institute: Institute of Biophysics, CAS

summary: We use ChIP-Seq technology to profile the occupancy of histone H2A.Z and its chaperone in HeLa cells genome wide to check if they are co-colocalized.

overall_design: H2A.Z is ChIPed by antibody and its chaperone is tagged by Flag and ChIPed by M2 beads against Flag. ChIPed fragments are sequenced by Illumina HiSeq 2000 platform.

first: Zhuo

last: Mao

first: Pan

last: Lu

first: Weixiang

last: Wang

first: Jian

last: Sun

first: Shan

last: Shan

first: Qiang

last: Dong

first: Xiaoping

last: Liang

first: Xiaojun

last: Ding

first: She

last: Chen

first: Bing

last: Zhu

first: Zheng

last: Zhou


title: Multi-Tissue Omics Analyses Reveals Molecular Regulatory Networks for Puberty in Composite Beef Cattle

status: Public on Feb 28 2014

city: Guelph

first: Angela

last: Cánovas

state: ON

address: 50 Stone Road East

department: Animal Biosciences

country: Canada

institute: University of Guelph

summary: Puberty is a complex physiological event measured by various indicator traits in genetic improvement programs of beef cattle; thus, developing a more complete understanding of the genes and regulatory pathways and networks involved in puberty will provide knowledge to help improve genetic selection strategies. Herein, we characterized the transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to growth and metabolism needed for cattle to achieve puberty (i.e., longissimus dorsi muscle, fat, and liver). These tissues were collected from pre (PRE)- and post (POST)-pubertal Brangus (3/8 Brahman; Bos indicus x 5/8 Angus; Bos taurus) heifers derived from a population of cattle used to identify QTL associated with fertility traits. In order to exploit the power of complementary omics analyses, PRE and POST puberty co-expression gene networks were constructed by combining the results from RNA-Seq, GWAS, and bovine transcription factors. RNA-Seq of 8 tissues among PRE and POST Brangus heifers revealed 1515 differentiallyexpressed and 943 tissue-specific genes within the 17,832 genes confirmed by metrics of RNA-Seq analysis. Combining the results from RNA-Seq and GWAS indentified a total of 25 QTL associated to heifer fertility. The hypothalamus experienced the most notable up-regulation of genes via puberty. Complementary omics procedures revealed 2,450 co-expressed genes across the 8 tissues relative to puberty. The PRE network had 372,861 connections whereas the POST network had 328,357 connections. A sub-network from this process revealed key transcriptional regulators (i.e., PITX2, FOXA1, TSG1D1, DACH2, LHX4, PROP1 and SIX6).  Results from multiples sources of omics data will facilitate the design of breeding strategies to improve fertility in Bos indicus-influenced composite cattle.

overall_design: Sixty-one samples from PRE and POST pubertal composite beef heifers were analyzed with RNA-Seq. The transcriptome of five reproductive tissues (i.e. hypothalamus, pituitary gland, ovary, uterus, and endometrium) as well as tissues known to be relevant to metabolism andbody morphometrics needed for cattle to achieve puberty (i.e.,) was characterized. These tissues were collected from pre (PRE)- and post (POST)-pubertal Brangus (3/8 Brahman x 5/8 Angus) heifers derived from a population of cattle used to identify QTL associated with fertility. Total RNA was purified using a Trizol protocol (Invitrogen, Carlsbad, CA). Sequencing libraries were made using TruSeq RNA Sample Preparation kit of Illumina (San Diego, CA).

first: Antonio

last: Reverter

first: Kasey

last: DeAtley

first: Ryan

last: Ashley

first: Michelle

last: Colgrave

first: Marina

last: Fortes

first: Alma

last: Islas-Trejo

first: Sigrid

last: Lehnert

first: Laercio

last: Porto-Neto

first: Gonzalo

last: Rincón

first: Gail

last: Silver

first: Warren

last: Snelling

first: Juan

middle: F

last: Medrano

first: Milton

middle: G

last: Thomas


title: Clustering of CpG islands constitutes an important determinant of the interphase chromosome 3D organization [ChIP-Seq]

first: Artem

last: Artemov

overall_design:
Using ChIP-Seq experimental procedure we have mapped genome-wide the CTCF deposition sites in chicken lymphoid and erythroid cells subjected to the 4C analysis.
CTCF deposition sites in chicken lymphoid and erythroid (induced and non-induced) cells.

first: Elena

last: Kotova

first: Sergey

last: Akopov

first: Lev

last: Nikolaev

first: Eugene

last: Sverdlov

first: Olga

last: Iarovaia

last: Razin


title: Clustering of CpG islands constitutes an important determinant of the interphase chromosome 3D organization

overall_design: Refer to individual Series


title: Poly(A) tail length change of miRNA targets in HeLa cells

city: Seoul

first: Hyeshik

last: Chang

state: South Korea

address: Building 203 Room 525, School of Biological Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu

department: School of Biological Sciences

country: South Korea

institute: Seoul National University

summary: MicroRNA induces deadenylation of its targets according to the current model in the scientific community, but this model is based on the studies of a few individual genes. We tested the model by examining the global effect of miRNA on poly(A) tail of the targets. Synthetic miR-1 mimic was transfected into HeLa cells, and the poly(A) length was measured by TAIL-seq. Deadenylation of miR-1 targets was evident as early as 3 hours post-transfection without a significant change in mRNA level. After 6 or 9 hours, target mRNA level was substantially downregulated. Therefore, although there are some exceptions, our result confirms that, in general, miRNA indeed induces deadenylation. Furthermore, our kinetic global analysis confirms that deadenylation precedes mRNA decay.

overall_design: Four culture dishes of HeLa cells were treated with miR-1 all together, then collected and prepared for sequencing after different time of incubation (0, 3, 6, and 9 hours post-transfection)

first: Minju

last: Ha

first: V

middle: N

last: Kim


title: Contribution of natural antisense transcription to an endogenous siRNA signature in human cells

city: Newcastle

first: Simon

last: Cockell

address: Framlington Place

institute: Newcastle University

summary:
Background: Eukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs).

Methods/Results: We used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPolII or bound to Argonaute (AGO1  by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with PolII occupancy with an average density of 130 (relative units); this decreased to 65%/20 in middle exons and 54%/12 in the last exon. CLASH reads mapping to multi-exon genes showed little distribution bias with an average of about 5 CLASH reads overlapping with 60% of the endo-siRNA reads. However, endo-siRNAs (21-25 nt) intersecting with CLASH reads were enriched at the 5'end and decreased towards the 3'end. We then investigated the 378 genes with particular focus on features indicative for short RNA production; however, found that endo-siRNA numbers did not correlate with gene structures that favor convergent transcription. In contrast, our gene set was found notably over-represented in the NATsDB sense/antisense group as compared to non-overlapping and non-bidirectional groups.  Moreover, read counts showed no correlation with the steady-state levels of the related mRNAs and the pattern of endo-siRNAs proved reproducible after an induced mutagenic insult.

Conclusions: Our results suggest that antisense transcripts contribute to low levels of endo-siRNAs in fully differentiated human cells. A characteristic endo-siRNA footprint is being produced at sites of RNAPolII transcription which is also related to AGO1. This endo-siRNA signature represents an intriguing finding and its reproducibility suggests that the production of endo-siRNAs is a regulated process with potential homoeostatic impact.

overall_design: Size selected RNASeq of 3 human embryonic kidney cell (HEK293) samples. 1 control and 2 samples exposed to 100 μg/ml ethyl methanesulfonate for 24 hrs.

first: Andreas

last: Werner

first: Jane

last: Falconer

first: Mark

last: Carlile

first: Sammer

last: Alnumeir

first: John

last: Robinson


title: Differentially expressed genes in Reamuria soongorica leaf under multiple stresses.

city: Lanzhou

first: Ruiqing

last: Zhu

state: Gansu

address: DingxiSouth Road 197

department: Instituye of biology

institute: Gansu Academy of Sciences

summary: In this study, the DEGs in leaves of R. soongorica in response to PEG-induced drought stress, UV-B radiation, combined stress by UV-B radiation and PEG-induced drought, and combined stresses by UV-B radiation, PEG-induced drought and NaCl in contrast to control group were examined, respectively, using DGE tag profiling technology. Analysis of gene expression related to stress response should provide further insight into the molecular mechanisms of stress tolerance in R. soongorica. Based on the putative functions of the identified genes, some important genes may be cloned. Moreover, the cloning of stress tolerance genes and determination of their expression patterns may offer some attractive candidate genes and valuable information for improving stress tolerance of plants through genetic engineering.

overall_design: Analysis of differentially expressed genes under stresses in Reaumuria soongorica

first: Meiling

last: Liu

first: Yubing


title: The distinct transcription patterns correlate with the differential growth responses to salt stress in maize roots

first: Maolin

last: Zhang

state: Shandong

address: No.27 Shanda South Road

summary: We found that primary root (PR) is more resistant to salt stress compared with crown roots (CR) and seminal roots (SR). To understand better salt stress responses in maize roots, six RNA libraries were generated and sequenced from primary root (PR), primary roots under salt stress (PR-salt) , seminal roots (SR), seminal roots under salt stress (SR-salt), crown roots (CR), and crown roots under salt stress (CR-salt). Through integrative analysis, we identified 444 genes regulated by salt stress in maize roots, and found that the expression patterns of some genes and enzymes involved in important pathway under salt stress, such as reactive oxygen species scavenging, plant hormone signal perception and transduction, and compatible solutes synthesis differed dramatically in different maize roots. 16 of differentially expressed genes were selected for further validation with quantitative real time RT-PCR (qRT-PCR).We demonstrate that the expression patterns of differentially expressed genes are highly diversified in different maize roots. The differentially expressed genes are correlated with the differential growth responses to salt stress in maize roots. Our studies provide deeper insight into the molecular mechanisms about the differential growth responses of different root types in response to environmental stimuli in planta.

overall_design: Examination of three  root types of maize under salt treatment for understanding the different responding mechenism to salt stress.

first: Zhaojun

last: Ding


title: The 3′ extremity of mRNA unveiled by TAIL-seq

summary: Global investigation of the 3′ extremity of mRNA, despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. By developing a new methodology, TAIL-seq, we find a number of unforeseen features at the 3′ termini of mRNA. Our analyses reveal the transcriptome-wide distribution of poly(A) tail and estimate the median poly(A) length as 55-60 nt in mammalian cells, which is substantially shorter than generally conceived. Poly(A) length correlates with mRNA half-life but not with translation efficiency. Surprisingly, we find widespread uridylation and guanylation at the downstream of poly(A) tail. The U-tails are generally attached to short poly(A) tails (<25 nt) while the G-tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. In addition, TAIL-seq identifies, with a single nucleotide resolution, numerous nucleolytic events involved in microRNA processing and mRNA cleavage.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Single replicate without any special experimental treatment for each of mouse fibroblast cell line NIH3T3 and human cervical cell line HeLa.

first: Jaechul

last: Lim


title: SOLiD sequencing of Noccaea (Thlaspi) caerulescens root transcriptomes from three contrasting ecotypes

city: Kuopio

first: Pauliina

last: Halimaa

address: Yliopisonranta 1 E

department: Department of Biology

country: Finland

institute: University of Eastern Finland

summary: Root transcriptomes were sequenced using SOLiD 4 chemistry. The three accessions LC, GA and MP differed significantly in the expression of genes that contribute to metal ion transport and detoxification, stress tolerance and secondary metabolite biosynthesis. These data provide comprehensive genome-wide view to the root processes in several N. caerulescens accessions and highlight differences that potentially facilitate adaptation to various metalliferous soils.

overall_design: Comparison of three N. caerulescens accessions with contrasting metal accumulation and tolerance traits

first: Asta

last: Laiho


title: Global effects of the CSR-1 RNA interference pathway on transcriptional landscape

city: New York

first: Germano

last: Cecere

state: NY

address: 701 West 168th

department: Biochemistry and Molecular Biophysics

institute: Columbia University

summary: Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions.

overall_design: GRO-seq (Global Run-On sequencing) for detection of nascent transcripts. Two biological replicates were generated for csr-1 hypomorphic mutant and the corresponding WT samples using ~300,000 worms for each experiment, and two biological replicates were prepared for drh-3 (ne4253) mutant and the corresponding WT samples using ~100,000 worms for each experiment. The GRO-seq were performed in late L3/early L4 stage worms. (8 samples total)

first: Sebastian

last: Hoersch

first: Sean

last: O'Keeffe

first: Ravi

last: Sachidanandam

first: Alla

last: Grishok


title: Transcription and enhancer profiling in human monocyte subsets

city: Regensburg

first: Michael

last: Rehli

address: F.-J.-Strauss-Allee 11

department: Internal Med III

country: Germany

institute: University Hospital Regensburg

summary: Human blood monocytes comprise at least three subpopulations that differ in phenotype and function. Here we generated global maps of H3K4me1 and H3K27ac deposition for classical and nonclassical monocytes defining enhanceosomes of the two major subsets. In combination with HeliScopeCAGE data for all three subpopulations we identify differential regulatory elements (including promoters and putative enhancers) that were associated with subset-specific motif signatures corresponding to different transcription factor activities and exemplarily validate a novel downstream enhancer of the CD14 locus.

overall_design: ChIP-seq of 2 histone marks in "classical" CD14++CD16- and "nonclassical" CD14dimCD16+ human blood monocytes

first: Christian

last: Schmidl


title: F1 Interspecies Hybrid Analysis Reveals Parent-Of-Origin Bias in the Mouse Placenta

city: Stanford

first: Elizabeth

middle: Havener

last: Finn

state: California

address: 300 Pasteur Drive, Alway M311

institute: Stanford University

summary: To investigate the epigenetic landscape at the interface between mother and fetus, we provide a comprehensive analysis of parent of origin bias in the placenta.  Using F1 interspecies hybrids, we sequenced RNA from 23 individual midgestation placentas, five late stage placentas, and two yolk sac samples and then used SNPs to determine whether transcripts were preferentially generated from the maternal or paternal allele.  In the placenta, we find 103 genes that show significant and reproducible parent-of-origin bias, of which 78 are novel candidates. Most (96%) show a strong maternal bias which, using multiple models, we demonstrate is not due to maternal decidual contamination.  Analysis of the X chromosome also reveals paternal expression of Xist and several genes that escape inactivation, most significantly Rps4x, Fhl1, and Slc38a5. Finally, sequencing individual placentas allowed us to reveal notable expression similarity between littermates.  In all, we observe a striking preference for maternal transcription in the midgestation mouse placenta and a dynamic imprinting landscape in extraembryonic tissues, reflecting the complex nature of epigenetic pathways in the placenta.

overall_design: 3'-end Sequencing for Expression Quantification (3SEQ) and SNP Analysis to observe parent-of-origin bias in 28 placental samples at two time points and 2 yolk sac samples

first: Cheryl

last: Smith

first: Jesse

last: Rodriguez

first: Arend

last: Sidow

first: Julie

middle: C

last: Baker


title: Regulation of the KEAP1/NRF2 oxidative stress response pathway by BRD4 in prostate and colorectal cancer

city: Berlin

first: Michal

middle: Ruth

last: Schweiger

address: Ihnestrasse 63-73

department: Lehrach

institute: Max Planck Institute For Molecular Genetics

summary: To identify genes regulated by BRD4 and to provide insight into new mechanisms de-regulated by BRD4, such as the response to oxidative stress, we integrated BRD4-binding regions with BRD4 gene expression data. For this analysis we performed BRD4 chromatin immunoprecipitation  experiments and BRD4 knock down experiments followed by RNA-Seq analyses. By integration of both gene lists we identified top candidate genes regulated by BRD4.

overall_design: HEK cells have been investigated for genomewide BRD4 binding sites and expression changes after knock down of BRD4. Illumina sequencing was used to gather data of the type ChIP Seq and mRNA Seq.

first: Michelle

last: Hussong

first: Stefan

middle: T

last: Boerno

first: Andrea

last: Wunderlich

first: Martin

last: Kerick

first: Alexandra

last: Franz

first: Bernd

last: Timmermann

first: Monica

last: Hirsch-Kauffmann


title: Role of DNA methylation in modulating transcription factor occupancy

first: Matthew

last: Maurano

address: 435 East 30th Street

department: Institute for Systems Genetics

institute: NYU School of Medicine

summary: This data includes regulatory factor profiling using DNase and ChIP-seq and methylation profiling using bisulfite-seq.

overall_design: We investigated CTCF occupancy in the context of reduced methylation by performing genome-wide profiling with chromatin immunoprecipitation (ChIP-seq) in HCT116 cells and DNMT1 and DNMT3B double knockout (DKO) HCT116 cells. We also profiled HCT116 and DKO using DNaseI-seq and ChIP-seq for trimethylation of histone 3 lysine 4 (H3K4me3) and acetylation of histone 3 lysine 27 (H3K27ac), Finally, we performed ChIP-seq on 3 replicates of mock-treated and 2 replicates of 5-aza-CdR-treated K562 cells.

first: Hao

last: Wang

first: John

middle: A

last: Stamatoyannopoulos


title: Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion

status: Public on Feb 27 2014

city: Vancouver

first: Scott

last: Tebbutt

state: BC

address: 1190 Hornby Street

institute: PROOF Centre of Excellence

summary: In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. We compared technical and biological replicates having undergone globin depletion or not and found that globin depletion removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures.

overall_design: Peripheral whole blood transcriptome assessed by RNA-Seq on Illumina HiSeq 2000 in 6 healthy individuals and 6 pooled samples, either globin depleted or not.

first: Casey

middle: P

last: Shannon

middle: J


title: An atlas of in vivo transcribed enhancers in human primary cells

first: Michael

last: Rehli

summary: Enhancers are powerful regulatory regions, important for development and the maintenance of differentiated cells and tissues. Here, we generate global maps for two enhancer-associated histone marks, H3K4me1 and H3K27ac for a number of major human blood cell types. This data was generated to show that capped RNAs transcribed bidirectionally can identify known and novel enhancers in vivo.

overall_design: ChIP-seq of 2 histone marks in human blood monocytes, CD19+ B cells, CD8+ T cells, CD4+CD25-CD45RA+ naive T cells, & NK cells

first: Claudia

last: Gebhard

first: Christian

last: Schmidl


title: Novel primate miRNAs co-evolved with ancient target genes in germinal zone specific expression patterns [RISC trap immunoprecipitation]

city: Santa Barbara

first: Kenneth

middle: S.

last: Kosik

address: UC Santa Barbara

department: NRI

institute: UCSB

summary: Major non primate-primate differences in corticogenesis include the dimensions, precursor lineages and developmental timing of the germinal zones (GZ). microRNAs (miRNAs) of laser dissected GZ compartments and cortical plate (CP) from embryonic E80 macaque visual cortex were deep sequenced. The CP and the GZ including Ventricular Zone (VZ), outer and inner subcompartments of the Outer SubVentricular Zone (OSVZ) in area 17 displayed unique miRNA profiles. miRNAs present in primate, but absent in rodent, contributed disproportionately to the differential expression between GZ sub-regions. Prominent among the validated targets of these miRNAs were cell-cycle and neurogenesis regulators. Co-evolution between the emergent miRNAs and their targets suggested that novel miRNAs became integrated into ancient gene circuitry to exert additional control over proliferation. We conclude that multiple cell-cycle regulatory events contribute to the emergence of primate-specific cortical features, including the OSVZ, generated enlarged supragranular layers, largely responsible for the increased primate cortex computational abilities.

overall_design: target mRNAs for selected miRNAs were detected with RISC trap immunoprecipitation

first: Mary

last: Arcila

first: Marion

last: Betizeau

first: Xiaolu

last: Cambronne

first: Elmer

last: Guzman

first: Nathalie

last: Doerflinger

first: Frantz

last: Bouhallier

first: Hongjun

last: Zhou

first: Bian

last: Wu

first: Cyril

last: Huissoud

first: Neha

last: Rani

first: Dani

last: Bassett

first: Ugo

last: Borello

first: Richard

middle: H

last: Goodman

first: Colette

last: Dehay


title: Classifying leukemia types with chromatin conformation data

city: Montreal

first: Josée

last: Dostie

state: QC

address: 3655 Promenade Sir-William-Osler

department: Biochemistry

institute: McGill University

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


title: Novel primate miRNAs co-evolved with ancient target genes in germinal zone specific expression patterns

sample_organism:
Macaca fascicularis
Homo sapiens

platform_organism:
Macaca fascicularis
Homo sapiens


title: Novel primate miRNAs co-evolved with ancient target genes in germinal zone specific expression patterns [miRNA-Seq]

overall_design: Seven brain regions (VZ17, OSVZ17int, OSVZ17ext, CP17, VZ18, OSVZ18, CP18). OSVZ17int corresponds to the inner (apical) third of OSVZ 17 and OSVZ17ext to the most outer (basal) third of OSVZ 17, located immediately below the Outer Fiber Layer (Smart et al., 2002; Betizeau et al., 2013)

middle: L


title: LRH-1 governs vital transcriptional programs in endocrine sensitive and resistant breast cancer cells

city: Sherbrooke

first: Nicolas

last: Gévry

state: Québec

address: 2500 boulevard de l'université

institute: Université de Sherbrooke


title: The transcriptome landscape of Prochlorococcus MED4 and the factors for stabilizing the core genome

city: Tianjin

first: Bang

last: Wang

address: 32 Xi Qi Dao, Tianjin Airport Economic Area

institute: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

summary: RNA-Seq technique was used to obtain the transcriptome map of Prochlorococcus MED4, including operons, untranslated regions, non-coding RNAs, and novel genes. Genome-wide expression profiles revealed that three factors contribute to core genome stabilization. First, a negative correlation between gene expression levels and protein evolutionary rates was observed. Highly expressed genes were overrepresented in the core genome but not in the flexible genome. Gene necessity was determined as a second powerful constraint on genome evolution through functional enrichment analysis. Third, quick mRNA turnover may increase corresponding proteins’ fidelity among genes that were abundantly expressed. Together, these factors influence core genome stabilization during MED4 genome evolution.

sample_organism: Prochlorococcus marinus subsp. pastoris str. CCMP1986

overall_design: The cells were cultured on Pro99 medium for a certain time course (1, 3, 4, 8, and 10 days); or transferred to AMP (Artificial Medium for Prochlorococcus) with 6 mM or 24 mM sodium bicarbonate for 5 or 10 hours.

first: Chaoguang

last: Tian


title: Dynamic Reorganization of the Cardiomyocyte Transcriptome in Response to TNFα-induced Proinflammatory Signaling [ChIP-Seq]

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: Inflammation is associated with many cardiovascular pathologies, but the underlying mechanisms remain unclear.  To explore this in more detail, we characterized the transcriptome of an immortalized adult human ventricular cardiomyocyte cell line (AC16) in response to tumor necrosis factor (TNFa).  Using a combination of genomic approaches, including global nuclear run-on sequencing (GRO-seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we identified ~30,000 transcribed regions in AC16 cells, which includes a set of RNA polymerases I and III (Pol I and Pol III) transcribed regions revealed in the presence of α-amanitin.  The set of transcribed regions produces both protein-coding and non-coding RNAs, many of which have not been annotated previously, including enhancer RNAs originating from NF-κB binding sites.  In addition, we observed that AC16 cells rapidly and dynamically reorganize their transcriptomes in response to TNFa stimulation in an NF-κB-dependent manner, switching from a basal state to a proinflammatory state affecting a spectrum of cardiac-associated protein-coding and non-coding genes.  Moreover, we observed distinct Pol II dynamics for up- and downregulated genes, with a rapid release of Pol II into productive elongation for TNFa-stimulated genes.    Our studies shed new light on the regulation of the cardiomyocyte transcriptome in response to a proinflammatory signal and help to clarify the link between inflammation and cardiomyocyte function at the transcriptional level.

overall_design: Using GRO-seq and ChIP-seq (p65 and RNA Pol II) over a time course of TNFα signaling in AC16 human cardiomyocytes.

first: Xin

last: Luo

first: Minho

last: Chae

first: Raga

last: Krishnakumar

first: Charles

last: Danko

first: W


title: Dynamic Reorganization of the Cardiomyocyte Transcriptome in Response to TNFα-induced Proinflammatory Signaling

overall_design: Refer to individual Series


title: Genome-wide profiling of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings and in wt seedlings

city: shenzhen

first: Lei

last: Gao

state: guangdong

address: Nanhai Ave 3688

department: College of Life Sciences

institute: shenzhen university

summary: By using ChIP-seq, we found that loss of BRM activity in developing seedlings leads to ectopic and increased H3K27me3 deposition at several hundred genes, indicating the critical role of BRM in preventing the inappropriate deposition of this histone mark. Unexpectedly, BRM also facilitates PcG function at some PcG targets, suggesting the requirement for BRM in the deposition of this mark at certain PcG target genes.

overall_design: Examination of H3K27me3 in 14-day-old wt and brm seedlings. Two biological replicates for each one.

first: Chenlong

last: Li

first: Yuhai

last: Cui


title: LRH-1 governs vital transcriptional programs in endocrine sensitive and resistant breast cancer cells: LRH-1 ChIP-seq

summary: Tumor characteristics are decisive in the determination of treatment strategy for breast cancer patients. Patients with estrogen receptor a(ERa)-positive breast cancer can benefit from long-term hormonal treatment. Nonetheless, the majority of patients will develop resistance to these therapies. Here, we investigated the role of the nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in anti-estrogen (AE) sensitive and resistant breast cancer cells. We identified genome-wide LRH-1 binding sites using ChIP-seq, uncovering preferential binding to regions distal to transcriptional start sites (TSS). We further characterized these LRH-1 binding sites by integrating overlapping layers of specific chromatin marks, revealing that many LRH-1 binding sites are active and could be involved in long-range enhancer-promoter looping. Combined with transcriptome analysis of LRH-1 depleted cells, these results show that LRH-1 regulates specific subsets of genes involved in cell proliferation in AE-sensitive and AE-resistant breast cancer cells. Furthermore, the LRH-1 transcriptional program is highly associated with a signature of poor outcome and high-grade breast cancer tumors in vivo. Herein we report the genome-wide location and molecular function of LRH-1 in breast cancer cells and reveal its therapeutic potential for the treatment of breast cancers, notably for tumors resistant to treatments currently used in therapies.

overall_design: ChIP-seq examination of LRH-1 binding sites with specific chromatin marks in MCF7 breast cancer cells.

first: Stephanie

last: Bianco

last: Gevry


title: Zic3 interacts with distant regulatory elements to regulate zebrafish developmental genes

city: Singapore

first: Vibhor

last: Kumar

address: 60 Biopolis Street, Genome, #02-01

country: Singapore

institute: Genome Institute of Singapore

summary: Zic3 regulates early embryonic patterning in vertebrates. Its loss-of-function disrupts gastrulation, left-right patterning, and neurogenesis. We use the zebrafish as a model to study the developmental role of Zic3 in vivo. Using a combination of two genomics approaches – ChIP-seq and microarray, we identified Zic3 targets, which include genes from the Nodal and Wnt pathways, and show for the first time cis-regulation of these genes by Zic3 using in vivo enhancer assay. We uncovered a previously unrecognized link between Zic3 and the non-canonical Wnt pathway in gastrulation and left-right patterning, and identified neural pre-pattern genes as Zic3 targets during the early steps of neural induction. Zic3 preferably binds to distal intergenic regions, some of which contain evolutionarily conserved functional enhancers. Our study establishes the zebrafish as an excellent model for genome-wide study of a transcription factor in vivo.

overall_design: Zic3 ChIP of wild type and sqet33 transgenic

first: Cecillia

last: Winata

first: Sinnakarupan

last: Mathavan


title: Dynamic Reorganization of the Cardiomyocyte Transcriptome in Response to TNFα-induced Proinflammatory Signaling [GRO-Seq]


title: Obesity-associated variants within FTO form long-range functional connections with IRX3

status: Public on Feb 24 2014

city: Sevilla

first: Juan

middle: J.

last: Tena

address: Univ. Pablo Olavide, Ctra. Utrera km1

institute: CABD/CSIC

summary: Genome-wide association studies in diverse populations have reproducibly associated variants within introns of FTO with increased risk for obesity and type-2 diabetes.While the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. Yet, no direct connection between the obesity-associated intronic variants and FTO expression or function has been made. We show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, primarily with the homeobox gene IRX3, rather than with FTO.

sample_organism:
Danio rerio
Mus musculus

platform_organism:
Danio rerio
Mus musculus

overall_design: 4C-seq samples for Fto/fto and Irx3/irx3a genes promoters in different samples: adult mouse brain, E9.5 mouse embryos and 24hpf zebrafish embryos.

first: Scott

last: Smemo

first: Silvia

last: Naranjo

first: Rafael

last: Domínguez-Acemel

first: José

last: Gómez-Skarmeta

first: Marcelo

last: Nobrega


title: Classifying leukemia types with chromatin conformation data (ChIP-Seq)

first: Josée

last: Dostie

summary:
Background: Although genetic or epigenetic alterations have shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed.
Results: Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data.
Conclusions: Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes.

overall_design: Examination of CTCF and RAD21 binding sites in THP-1 cell.

first: Mathieu

last: Rousseau

first: Maria

last: Ferraiuolo

first: Jennifer

last: Crutchley

first: Xue

middle: Q

last: Wang

first: Hisashi

last: Miura

last: Blanchette


title: Nucleosome loss leads to global transcriptional upregulation and genomic instability during yeast replicative aging

status: Public on Feb 26 2014

city: Houston

first: kaifu

last: chen

address: 1 Baylor plaza, suite 450A

institute: Baylor College of Medicine

summary: All eukaryotic cells divide a finite number of times, termed replicative aging, but the reason for this is not clear. Consistent with the decreased total histone protein levels in aged Saccharomyces cerevisiae, which is a cause of aging (1), we find that nucleosome occupancy decreases 50% across the whole genome during replicative aging by spike-in controlled MNase sequencing.  Nucleosomes become fuzzier or move to sequences predicted to better accommodate histone octamers. All yeast genes are induced during aging. Genes that are repressed in young cells are most induced, accompanied by nucleosome loss from their promoters that have unique chromatin organization. Contrary to the loss of mitochondrial function during aging, mitochondrial DNA content increases and unprecedented levels of large-scale chromosomal alterations and increased retrotransposition are observed.

overall_design: Mnase-Seq experiments were carried out for young yeast, old yeast, and old yeast with histone over expression, 3 replicates were done for each category. RNA-Seq were carried out for the same categories of yeast cells but with 2 replicates for each. Genome-Seq were done for the young and old yeast with 2 replicates for each.

first: Zheng

last: Hu

first: Kaifu

last: Chen

last: Xia

first: Myrriah

last: Chavez

first: Ja-Hwan

last: Seol

first: Wei

last: Li

first: Jessica

middle: K

last: Tyler


title: RNA-Seq analysis of KLF3 knockout E14.5 TER119+ (erythroid) murine fetal liver cells

status: Public on Feb 23 2014

city: Sydney

first: Jon

last: Burdach

state: NSW

address: UNSW

department: BABS

institute: University of New South Wales

summary: The aim of this study was to analyze the transcriptome of TER119+ fetal liver cells in the absence of the transcription factor KLF3 at murine embryonic day E14.5

overall_design: Three wildtype (WT; Klf3+/+) and three knockout (KO; Klf3-/-) samples

first: Alister

last: Funnell

first: Merlin

last: Crossley


title: Histone variant H3.3-specific readout of H3K36 trimethylation by ZMYND11 links transcription elongation control to tumour suppression

first: Yuanxin

last: Xi

address: 1400 Pressler St

department: Bioinformatics and Computational Biology

institute: The University of Texas MD Anderson Cancer Center

summary: Recognition of modified histones by “reader” proteins plays a critical role in the regulation of transcription1. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions following RNA polymerase II (Pol II) elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin at an appropriate state to suppress cryptic transcription2,3. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies4. Here we show that the candidate tumor suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates Pol II elongation. Structural studies reveal that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific “Ser31” residue in a composite pocket formed by the tandem bromo-PWWP domains of ZMYND11. ChIP-sequencing analysis reveal a genome-wide colocalization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription corepressor via modulating the transition of the promoter-proximal paused Pol II to elongation. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumor cell growth; higher expression of ZMYND11 is observed in triple-negative breast cancer patients with better prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth and tumor formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone variant-mediated transcription elongation control to tumor suppression.

overall_design: ChIP-seq analysis of ZMYND11, H3K36me3 in U2OS cells and ZMYND11 knockdown cells; ChIP-seq of H3.3 in Flag-H3.3 stable U2OS cells; RNA-seq of ZNYMD11 depleted U2OS cells.

first: Hong

last: Wen

first: Xiaobing

last: Shi


title: Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain

status: Public on Feb 22 2014

city: Beijing

first: Xianlong

address: 5, Yiheyuan Rd., Haidian District

department: Biodynamic Optical Imaging Center, College of Life Sciences

institute: Peking University

summary: 5-Hydroxymethylcytosine (hmC) is particularly abundant in mammalian brains with yetto be revealed functions. Here, we present genome-wide and single-base-resolutionmaps of hmC and mC in the human brain. We demonstrated that hmCs increasemarkedly from the fetal to the adult stage, and in the adult brain, 13.4% of all CpGs arehighly hydroxymethylated with strong enrichment at genic regions and distal regulatoryelements. Notably, hmC peaks were identified at the 5' splicing sites at the exon-intronboundary, suggesting a mechanistic link between hmC and splicing. We also report asurprising transcription-correlated hmC bias toward the sense strand and an mC biastoward the antisense strand of gene bodies. Furthermore, hmC is negatively correlatedwith H3K27me3-marked repressive genomic regions, and is more enriched in poisedenhancers than active enhancers. Our results provide insights into understanding themultiple potential functions of hmC in the human brain.

overall_design: A cell type specific hydroxymethylome sample of NeuN+ neurons in frontal lobe from the same adult individual, whose TAB-Seq data was deposited in GSE46710

first: Lu

first: Liying

last: Yan

first: Yuexi

last: Tan

first: Fuchou

last: Tang

first: Jie

last: Qiao


title: Acinetobacter oleivorans DR1 : Wild-type cell harboring plasmid (pAST2) in nutrient media

city: Seoul

first: Hyerim

last: Hong

address: Korea Univ. Anam Campus, Anam-dong 5-ga, Seongbuk-gu

department: Environmental Science and Ecological Engineering

institute: Korea University

summary: Transcriptional profiling of A. oleivorans DR1 cells harboring pAST2. Plasmid pAST2 is a tetracycline-resistance plasmid which was isolated from activated sludge (Hong et al., 2014). The complete plasmid sequence was deposited in the National Center for Biotechnology Information (NCBI) GenBank under accession number KC734561 [PMID: 24337108].

overall_design: Acquisition of TC resistance through plasmid uptake is related to loss of biological fitness and affected host gene expression in oil-degrading soil bacterium, Acinetobacter oleivorans DR1. To identify genes in A. oleivorans DR1 harboring pAST2, the cells were grown to exponential phase (OD600 ~0.4) and total RNA was extracted using an RNeasy Mini kit (Qiagen, USA) following the manufacturer's instructions.


title: RNA-seq  from paired human CRC/control mucosa samples

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

institute: Fudan University

summary: We have used RNA-seq to examine gene expression from paired human CRC/control mucosa samples and identified CRC-specific expressed long noncoding RNAs

overall_design: To identify CRC-specific expressed genes, including long noncoding RNAs

first: Jian-Feng

last: Xiang

first: Qing-Fei

last: Yin

first: Ling-Ling

last: Chen


title: 3C-seq of HT29 cells with CCAT1-L knockdown or scramble knockdown

summary: CCAT1-L is a highly expressed long noncoding RNA located in the colorectal cancer specific super enhance region about 500 kb upstream of MYC gene. Knockdown of CCAT1-L significantly down-regulated interaction frequency between CCAT1 and MYC locus and repress MYC expression, suggesting a long-range chromatin interaction between CCAT1-L and MYC locus maintained by CCAT1-L underlie the MYC regulation. To further validate this hypothesis, multiplexed 3C sequencing (3C-seq) was employed to evaluate chromatin interaction strength between CCAT1-L and MYC locus in CCAT1-L knockdown and scramble knockdown (Scr) HT29 cells.

overall_design: The 3C-Seq design and data analysis were performed according to Stadhouders et al, Nat Protoc. 2013, 8:509-524. A series of bait sequences accommodating different locus around CCAT1-L and MYC were selected. Through integrating with specific sample barcodes, bait-specific primer sets were designed to construct relevant 3C-seq libraries in CCAT1-L knockdown and scramble knockdown (Scr) HT29 samples. All of the 3C sample libraries from different treatment, including CCAT1-L knockdown and scramble knockdown (Scr), were then pooled together for high-throughput sequencing. Two technical 3C-seq were performed (CCAT1_myc_3C_1.txt.gz and CCAT1_myc_3C_2.txt.gz) and then combined together to get enough reads for further analysis. 3C-seq reads from different samples were divided according to sample barcodes (CCAT1-L knockdown: ATGTCA; Scr: GCCAAT) and different bait sequences, and then mapped to human reference genome to assess chromatin interaction strength between CCAT1-L and MYC locus in different treatments. In our study, one representative bait-specific sequencing data (CTTCTACTGATTGGCCCTAAACACTTTTCCAAAGCTT) was select to generate bedgraph files and upload to UCSC for visualization to show the chromatin interaction between CCAT1-L and Myc locus in CCAT1-L knockdown (CCAT1-L_knockdown_out.bedgraph) and scramble knockdown (Scr_out.bedgraph) samples.

first: Xiao-Ou

last: Zhang


title: Widespread Use of Non-Productive Alternative Splice Sites in Saccharomyces cerevisiae

status: Public on Feb 21 2014

city: Los Angeles

first: Stephen

last: Douglass

state: CA

address: 5128 TLSB Terasaki Life Sciences Building, 610 Charles Young Drive East

department: Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

summary: Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many Saccharomyces cerevisiae intron-containing genes exhibit usage of alternative splice sites, but most transcripts generated by splicing from these sites are non-functional because they introduce premature termination codons leading to transcript degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3’ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3’-splice site. The use of non-productive alternative splice sites can limit the expression of some transcripts and can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results reveal that alternative splicing is frequent in S.cerevisiae but masked by RNA degradation and that the use of alternative splice sites is mostly aimed at controlling transcript levels rather than increasing proteome diversity.

overall_design: mRNA-Seq profiling of 3 mutants in the nonsense-mediated mRNA decay pathway and wildtype yeast

first: Tadashi

middle: R

last: Kawasima

middle: M

first: Matteo

last: Pellegrini

first: Guillaume

last: Chanfreau


title: Discovery of novel and conserved microRNAs in Spirodela polyrhiza by deep sequencing

city: Shenzhen

state: Guangdong

address: Rm. E118, PKU Campus, Shenzhen University Town

institute: School of Environment and Energy, Peking University Shenzhen Graduate School

summary: MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs, which play key roles in a variety of developmental and physiological processes by regulating gene expressions at post-transcriptional or translational levels. The objective of our work was to explore conserved and novel miRNAs in Spirodela at the whole-genome level, and further characterize their potential roles in gene regulation.

overall_design: 1 sample

first: Ying

first: Yingying

first: Jiong

last: Ma


title: RNA sequence reveals mouse retinal transcriptome changes early after axonal injury

city: Sendai

first: Toru

last: Nakazawa

address: 1-1 Seiryo

department: Ophthalmology

country: Japan

institute: Tohoku University

summary:
Purpose: The purpose of this study was　to use RNA-seq to investigate the molecular mechanisms of damage in the early stages of the response to axonal injury, before the onset of RGC death.
Methods: 12-week-old wild-type (WT) mice were used in this study. The experiment group underwent an optic nerve crush (ONC) procedure to induce axonal injury in the right eye, and the control group underwent a sham procedure. Retinal mRNA profiles were generated by deep sequencing, in triplicate, using IlluminaHiseq2000. The sequence reads were analyzed by CLC genomics workbench and R software. qRT–PCR validation was performed using TaqMan assays.
Results: Using an optimized data analysis workflow, we mapped about 66 million sequence reads per sample to the mouse genome (build mm9). Differential gene expression analysis showed that endoplasmic reticulum stress-related genes and antioxidative response-related genes have been shown to be significantly upregulated 2 days after ONC.
Conclusions: Our study represents the first detailed analysis of retinal transcriptomes in the early stages after axonal injury. Our results indicated that ER stress plays a key role under these conditions. Furthermore, the antioxidative defense and immune responses occurred concurrently in the early stages after axonal injury. We believe that our study will lead to a better understanding of and insight into the molecular mechanisms underlying RGC death after axonal injury.

overall_design: Retinal mRNA profiles of 12 week-old wild type (WT) after ONC or sham were generated by deep sequencing, in triplicate, using Illumina Hiseq2000.

first: Yuji

last: Tanaka

first: Masayuki

last: Yasuda


title: The Fusarium graminearum Histone H3 K27 Methyltransferase KMT6 Regulates Development and Expression of Secondary Metabolite Gene Clusters

city: Corvallis

first: Kristina

last: Smith

state: OR

department: Biochemisty and Biophysics

institute: Oregon State University

summary: We performed ChIP-seq of histone modifications and RNA-seq in WT and kmt6 mutant Fusarium graminearum grown in high and low nitrogen conditions.

overall_design: Two replicates of RNA seq for WT and kmt6 strains at high and low nitrogen. Four histone modification ChIPs in WT and kmt6 mutant strains at high and low nitrogen, plus two histone modifications in a kmt6+ complemented strain at high nitrogen.

first: Lanelle

last: Connolly

first: Michael

last: Freitag


title: Energy Metabolism during Anchorage-Independence

city: Cincinnati

first: Mario

last: Medvedovic

state: OH

address: 3223 Eden Av. ML 56

department: Department of Environmental Health

institute: University of Cincinnati

summary: The detachment of epithelial cells, but not cancer cells, causes anoikis due to reduced energy production. Invasive tumor cells generate three splice variants of the metastasis gene osteopontin. The cancer-specific form osteopontin-c supports anchorage-independence through inducing oxidoreductases and upregulating intermediates/enzymes in the hexose monophosphate shunt, glutathione cycle, glycolysis, glycerol phosphate shuttle, and mitochondrial respiratory chain. Osteopontin-c signaling upregulates glutathione (consistent with the induction of the enzyme GPX-4), glutamine and glutamate (which can feed into the tricarboxylic acid cycle). Consecutively, the cellular ATP levels are elevated. The elevated creatine may be synthesized from serine via glycine and also supports the energy metabolism by increasing the formation of ATP. Metabolic probing with N-acetyl-L-cysteine, L-glutamate, or glycerol identified differentially regulated pathway components, with mitochondrial activity being redox dependent and the creatine pathway depending on glutamine. The effects are consistent with a stimulation of the energy metabolism that supports anti-anoikis. Our findings imply a synergism in cancer cells between osteopontin-a, which increases the cellular glucose levels, and osteopontin-c, which utilizes this glucose to generate energy.

overall_design: mRNA profiles of MCF-7 cells transfected with  osteopontin-a, osteopontin-c and vector control were generated by RNA-Seq, in triplicate, by Illumina HiSeq.

first: Georg

middle: F

last: Weber

first: Zhanquan

last: Shi


title: RNA-DNA DIFFERENCES IN NASCENT RNA

city: Ann Arbor

first: Isabel

middle: Xiaorong

last: Wang

state: Michigan

address: 210 washtenaw ave

department: Pediatrics&Genetics

institute: HHMI/University of Michigan

summary: RNA sequences are expected to be identical to their corresponding DNA sequences.  Advances in technologies have enabled deep sequencing of nucleic acids that uncovered exceptions to the one-to-one relationship between DNA and RNA sequences.  Previously in human cells, post-transcriptional RNA editing was the only known mechanism that changes RNA sequences from the underlying DNA sequences.  Here, we sequenced nascent RNA  and found all 12 types of RNA-DNA differences.  Using various experimental analyses, we validated this finding.  Our results showed that sequences of nascent RNAs within 40 nucleotides of the exit channel of RNA polymerase II already differ from the corresponding DNA sequences.  These RNA-DNA differences are mediated by RNA processing steps closely coupled with transcription and not by known deaminase-mediated RNA editing mechanisms nor during NTP incorporation by Pol II.  This finding identifies sequence substitution as part of co-transcriptional RNA processing.

overall_design:
We sequenced nascent RNA using global run-on sequencing, GRO-seq from human B-cells from two individuals and a variant of the GRO-seq procedure, known as precision run-on sequencing, PRO-seq.  The RNAs are prepared after a short run-on assay performed with isolated nuclei in the presence of Br-UTP.  The isolated RNAs are base hydrolyzed to ~100 nucleotides and affinity purified with anti-BrU beads three times at each successive step of preparing the RNAs for orientation-specific sequencing using Illumina technology.  The 5’ ~half of each sequence represents nascent RNA made in the cell and the 3’ ~half  represents sequences made in vitro during the run-on reaction.  The precision variation, PRO-seq, incorporates one or at most a few biotin-labeled nucleoside triphosphates during the run-on, and sequencing from the 3’ end of this affinity purified, nascent RNA maps the cellular location of engaged polymerases with near single nucleotide precision.  We obtained ~ 100 million 100-nucleotide uniquely mapped GRO-seq reads from B-cells of two individuals.  For one subject, we also carried out pGRO-seq and obtained 60 million uniquely mapped reads.  In addition, we sequenced ~135 million uniquely mapped RNA-seq reads, and the corresponding DNA of the two individuals to 30X and 60X coverage.
Additionally, we isolated and sequenced nascent RNA with an alternate method described by Wuarin and Schibler (1994) in order to compare chromatin-bound RNA to the very nascent RNA from PRO-seq.  We obtained ~190 million uniquely mapped reads from chormatin-bound RNA-seq.

middle: X

first: Vivian

middle: G

last: Cheung


title: Dynamics of the chili pepper transcriptome during fruit development

city: Irapuato

first: Octavio

last: Martinez de la Vega

state: Guanajuato

address: km 9.6 Libramiento Norte

department: Unidad de Genómica Avanzada (Langebio)

country: Mexico

institute: Cinvestav

summary:
Background
The set of all mRNA molecules present in a cell constitute the transcriptome. The transcriptome varies depending on cell type as well as in response to internal and external stimuli during development. Chili pepper is an economically and culturally important horticultural crop as well as a good model for the study of secondary metabolism during fruit development. Here we present a study of the changes that occur in the transcriptome of chili pepper fruit during development and ripening.
Results
RNA-Seq was used to obtain transcriptomes of whole Serrano-type chili pepper fruits (Capsicum annuum L.; 'Tampiqueno 74') collected at 10, 20, 40 and 60 days after anthesis (DAA). 15,550,468 Illumina MiSeq reads were assembled de novo into 34,066 chili genes. We classified the expression patterns of individual genes as well as genes grouped into Biological Process ontologies and Metabolic Pathway categories using statistical criteria. For the analyses of gene groups we added the weighted expression of individual genes. This method was effective in interpreting general patterns of expression changes and increased the statistical power of the analyses. Subsets of genes were expressed only at a single time point sampled (1,278, 1,596, 1,519 and 1,583 genes at 10, 20, 40 and 60 DAA, respectively). We also estimated the variation in diversity and specialization of the transcriptome during chili pepper development. Approximately 17% of genes exhibited a significant change of expression in at least one of the intervals sampled. In contrast, significant differences in approximately 63% of the Biological Processes and 80% of the Metabolic Pathways studied were detected in at least one interval. Confirming previous reports, genes related to capsaicinoid and ascorbic acid biosynthesis were significantly upregulated at 20 DAA while those related to carotenoid biosynthesis were highly expressed in the last period of fruit maturation (40-60 DAA). Our RNA-Seq data was validated by examining the expression of nine genes involved in carotenoid biosynthesis by qRT-PCR.
Conclusions
In general, more profound changes in the chili fruit transcriptome were observed in the intervals between 10 to 20 and 40 to 60 DAA. The last interval, between 40 to 60 DAA, included 49% of all significant changes detected, and was characterized predominantly by a global decrease in gene expression. This period signals the end of maturation and the beginning of senescence of chili pepper fruit. The transcriptome at 60 DAA was the most specialized and least diverse of the four states sampled.

overall_design:
RNA-seq libraries of whole chili pepper (Capsicum annuum Serrano 'Tampiqueno 74' landrace) were prepared from fruits at 10, 20, 40 and 60 days after anthesis (DAA). Two biological replicates of each fruit state were prepared (8 libraries in total). Libraries were sequenced in the Illumina MiSeq platform.  The title of the libraries are: <DAA>repbio<#> where <DAA> are the days after anthesis (10, 20, 40 or 60) and <#> is the number of biological replicate, 1 or 2. Details follow.
Biological material and RNA extraction
Capsicum annuum Serrano 'Tampiqueno 74' seeds were germinated and the seedlings were cultivated until maturity under greenhouse conditions in a completely randomized experimental design at Cinvestav-Unidad Irapuato (Guanajuato, Mexico). The plants were grown during the spring and summer, and individual flowers were tagged at anthesis. Chili pepper fruits were randomly collected from different plants at 10, 20, 40 and 60 DAA. After sampling, the fruits were cleaned with ethanol immediately frozen in liquid nitrogen and stored at -80C until further use. For total RNA extractions, 10 fruits at the 10 DAA developmental stage and 5 fruits from each of the 20, 40 and 60 DAA stages were randomly selected from the pool of all harvested fruits. Whole fruits (pericarp, placenta and seeds) were ground in liquid nitrogen with a mortar and pestle to form a fine uniform powder. Samples were mixed vigorously and 100 mg aliquots were measured for each RNA extraction. The process was repeated with different sets of fruits in order to obtain two independent biological replicates at each developmental stage. A NucleoSpin RNA Plant kit (Macherey-Nagel) was used for total RNA extraction and contaminating genomic DNA was removed by DNase I (Macherey-Nagel) treatment following the manufacturers' protocols.
Total RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Nano-Drop, Wilmington, DE, USA) and RNA quality was evaluated by gel electrophoresis on 1.0% denaturing agarose gels. In addition, aliquots of RNA were run on an Agilent 2100 Bioanalyzer using RNA 6000 chips (Agilent, Santa Clara, CA, USA) to test the RNA integrity number (RIN). All eight samples had RIN values higher than 8.7. Thirty μg of total RNA from each of the eight samples (two biological replicates of four fruit developmental stages) was used for cDNA library preparation.
Library construction and sequencing
The eight total RNA samples (two biological replicates from chili pepper fruits at 10, 20, 40 and 60 DAA) were prepared for RNA-Seq using the Illumina TruSeq RNA Sample Preparation v2 Guide following the manufacturer's instructions. Briefly, mRNA was purified from 20μg of total RNA using poly-T oligo- attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, mRNA was fragmented using divalent cations under elevated temperature and primed for cDNA synthesis. The cleaved RNA fragments were primed with random hexamers and reverse transcribed into single-stranded cDNA using reverse transcriptase. In the next step, the RNA template was removed and the complementary cDNA strand was synthesized using RNAse H and DNA polymerase I, respectively.
The overhangs that resulted from fragmentation were polished into blunt ends using an End Repair Mix (consisting of T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase). A single T nucleotide was added on the 3' end of the adapter for ligating the adapter to the cDNA fragments. Indexing adapters were ligated to the ends of the cDNAs using T4 DNA ligase, preparing them for hybridization onto a flow cell. Finally, the DNA fragments with adapter molecules at both ends were amplified by PCR to enrich the amount of DNA in the library. PCR was performed with a PCR primer cocktail that anneals to the ends of the adapters. Quantity and quality of the DNA libraries was assessed using Agilent DNA-1000 chips on an Agilent 2100 Bioanalyzer.
The 8 cDNA libraries were pooled and simultaneously sequenced from both 5’ and 3’ ends using the Illumina MiSeq System platform according to the manufacturer's instructions. We performed three sequencing runs (technical replicates) with the aim of increase the sequencing depth. 150 bp paired-end reads were obtained in each sequencing run. Fluorescent image processing, base-calling and quality value calculations for each of the three runs were performed using Illumina MiSeq Control Software. Data were deposited at the NCBI (GEO database under series record GSE54123).

last: Martínez

first: Neftalí

last: Ochoa-Alejo

first: Luis

last: Martínez-López


title: UPF2 establishes testis-specific transcriptome enriched in transcripts with shorter 3’UTRs

status: Public on Feb 20 2014

city: Reno

first: Chong

last: Tang

state: Nevada

address: 1664 N.Virginia Street MS330

department: physiology

institute: University of Nevada Reno

summary: This report not only adds a novel mechanism to the current dogma on achieving global shortening of 3'UTRs, but also unveils a novel function of the NMD pathway in establishing tissue-specific transcriptome identity

overall_design: We first generated prospermatogonia-specific Upf2 conditional knockout mice (Ddx4-Cre; Upf2 fl/Δ, hereafter called Ddx4-KO) by crossing Ddx4-Cre13 with Upf2 floxed.

first: Wei

last: Yan

first: Porse

last: Bo


title: Essential role of poly(ADP-ribosyl)ation in cocaine action

city: New York

first: Eric

last: Nestler

address: 1425 Madison Avenue

institute: Ichan School of Medicine at Mount Sinai

summary: Many of the long-term effects of cocaine on the brain's reward circuitry have been shown to be mediated by alterations in gene expression. Several chromatin modifications, including histone acetylation and methylation, have been implicated in this regulation, but the effect of other histone modifications remains poorly understood. Poly(ADP-ribose) polymerase-1 (PARP-1), a ubiquitous and abundant nuclear protein, catalyzes the synthesis of a negatively charged polymer called poly(ADP-ribose) or PAR on histones and other substrate proteins and forms transcriptional regulatory complexes with several other chromatin proteins. Here, we identify an essential role for PARP-1 in cocaine-induced molecular, neural, and behavioral plasticity. Repeated cocaine administration, including self-administration, increased global levels of PARP-1 and its mark PAR in mouse nucleus accumbens (NAc), a key brain reward region. Using PARP-1 inhibitors and viral-mediated gene transfer, we established that PARP-1induction in NAc mediates enhanced behavioral responses to cocaine, including increased self-administration of the drug. Using chromatin immunoprecipitation sequencing, we demonstrated a global, genome-wide enrichment of PARP-1 in NAc of cocaine-exposed mice and identified several PARP-1 target genes that could contribute to the lasting effects of cocaine. Specifically, we identified sidekick-1-important for synaptic connections during development-as a critical PARP-1 target gene involved in cocaine's behavioral effects as well as in its ability to induce dendritic spines on NAc neurons. These findings establish the involvement of PARP-1 and PARylation in the long-term actions of cocaine.

overall_design: c57bl/6 mice were given IP injections of chronic cocaine 20mg/kg once per day for 7 days and sacrificed 30 minutes after the final dose of cocaine. Control animals were given saline for 7 days and sacrificed 30 minutes after the final dose of saline. Nucleus accumbens (NAc) tissue was collected and then PARP-1 ChIP-seq was performed. Three sequencing replicates were performed on each group.

first: Kimberly

last: Scobie

first: Ning-Yi

last: Shao

first: Li

last: Shen


title: RNA m5C Methylation in breast cancer using MeRIP-Seq

city: San Antonio

first: Xiaodong

last: Cui

state: Texas

address: 126 Arrow Oaks

department: Electrical engineering

institute: Univ of Texas SA

summary: RNA m5C methylation profile of MCF10A and MDA486 by using MeRIP-Seq protocol

overall_design:
Immunoprecipitation of Methylated mRNA at Cytosine (m5C) residues: Affinity purified of anti-methyl cytosine (m5C) polyclonal antibody 7ug (Zymo Research, Catalog#A3001-50) was conjugated with protein-A magnetic beads for 2 h at 4°C in end to end rotator. After that, conjugated beads were extensively washed with RNA immunoprecipitation (RIP) wash buffer to remove unbound antibody.
Fragmented 25 ug polyA RNA (mRNA) was incubated with m5C conjugated beads for overnight at 4°C in in the rotating platform in RIP buffer. RIP was done using Megna RNA Immunoprecipitation kit (Millipore, Catalog#17-700).
m5C mRNA-immune bead complex was treated with proteinase K buffer to release m5C mRNA from the conjugated antibody. To isolate m5C, mRNA was treated with phenol:chloroform:isoamyl and mixed with 400 ul of chloroform, which was centrifuged at 14000 rpm for 10 minutes to separate aqueous phase. The aqueous phase was ethanol precipitated at -80°C for overnight, to get m5C mRNA.
This precipitated m5C mRNA pellet was washed twice with 70% ethanol and air dried. Finally, m5C mRNA pellet was dissolved in nuclease free Water. The m5C mRNA integrity and conentration was quantified by bioanalyzer (Agilent) and Qubit 2.0 flurometer (Invitrogen). The fragmented mRNA was used by following TruSeq RNA Sample Preparation Guide to develop RNA-Seq library for sequencing.

first: Manjeet

last: Rao

first: Yidong

last: Chen

first: Yufei

last: Huang


title: Opposing actions of IL-2 and IL-21 on Th9 differentiation correlate with their differential regulation of BCL6 expression

city: Bethesda

first: Peng

last: Li

state: MD

address: 9000 Rockville Pike

department: NHLBI

institute: NIH

summary: Interleukin 9 (IL-9) is a γc-family cytokine that is highly produced by T-helper 9 (Th9) cells and regulates a range of immune responses, including allergic inflammation. Here we show that IL-2–JAK3–STAT5 signaling is required for Th9 differentiation, with critical STAT5 binding sites in the Il9 (the gene encoding IL-9) promoter. IL-2 also inhibited B cell lymphoma 6 (BCL6) expression, and over- expression of BCL6 impaired Th9 differentiation. In contrast to IL-2, IL-21 induced BCL6 and diminished IL-9 expression in wild-type but not Bcl6−/− cells, whereas Th9 differentiation was increased in Il21−/− or Il21r−/− T cells. Interestingly, BCL6 bound in proximity to many STAT5 and STAT6 binding sites, including at the Il9 promoter. Moreover, there was increased BCL6 and decreased STAT binding at this site in cells treated with blocking antibodies to IL-2 and the IL-2 receptor, suggesting a possible BCL6–STAT5 binding competition that influences IL-9 production. BCL6 binding was also increased when cells were Th9-differentiated in the presence of IL-21. Thus, our data reveal not only direct IL-2 effects via STAT5 at the Il9 gene, but also opposing actions of IL-2 and IL-21 on BCL6 expression, with increased BCL6 expression inhibiting IL-9 production. These data suggest a model in which increasing BCL6 expression decreases efficient Th9 differentiation, indicating possible distinctive approaches for controlling this process.

overall_design: Genome-wide transcription factors mapping and binding of STAT5B and STAT6 in mouse polarized Th9 cells treated with or without blocking antibodies to IL-2 (anti-IL-2). RNA-Seq is conducted in WT and Il2-/- mice.

last: Liao

first: Rosanne

last: Spolski

first: Ning

last: Du

first: Erin

middle: E

last: West

first: Min

last: Ren

first: Suman

last: Mitra

first: Warren

last: Leonard


title: A splicing-dependent transcriptional checkpoint associated with pre-spliceosome formation

status: Public on Feb 19 2014

city: Edinburgh

first: Shaun

middle: Michael

last: Webb

address: 2.21 Swann Building, Kings Buildings

department: Wellcome Trust Centre for Cell Biology

institute: University of Edinburgh

summary: There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 is an RNA-stimulated ATPase required for pre-spliceosome formation in yeast. We demonstrate through in vivo RNA labelling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at serine 5 of the largest subunit’s heptad repeat accumulates over introns, and that this defect requires the U2 snRNP-associated Cus2p. A similar accumulation of polymerase was observed when pre-spliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with pre-spliceosome formation during co-transcriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription.

overall_design: Examining the Pol II profile in MT strain and WT strain

first: Keerthi

middle: T

last: Chathoth


title: Tet and TDG Mediate DNA Demethylation Essential for MET in Somatic Cell Reprogramming

first: Shiqing

last: Mao

address: 320 Yue Yang Road

institute: SIBS

summary: Tet-mediated DNA oxidation is a new type of epigenetic modification in mammals and its role in the regulation of cell fate transition remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capability to be reprogrammed into iPS cells. We demonstrate that these Tet-deficient MEFs cannot be reprogrammed due to a blockage in the mesenchymal-to-epithelial transition (MET). Reprogramming of MEFs deficient in TDG is similarly blocked. The blockage is caused by impaired activation of crucial microRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either miR-200c or catalytically active Tet and TDG restores reprogramming to the respective knockout MEFs. Thus, oxidative demethylation is essential for somatic cell reprogramming. These findings provide mechanistic insights into the operation of epigenetic barriers in cell lineage conversion.

overall_design: Reduced Representation Bisulfite (RRBS, MspI,~75-400bp size fraction) and Tet-Assisted RRBS (TARRBS) of MEFs & reprogramming MEFs at Day 5

first: Xianlong

first: Xiao

last: Hu

first: Lei

last: Zhang

first: Boqiang

first: Fuchou

last: Tang

first: Guo-Liang

last: Xu