title: High-throughput sequencing of small RNAs in Capsicum annuum

first: Emanuele

summary: Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different Capsicum annuum tissues (including leaves, flowers and fruit). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features, such as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study.

overall_design: Small RNA libraries were derived from leaves, flowers and fruit of Capsicum annuum. Total RNA was isolated using the TriReagent (Molecular Research Center) for leaves and flowers and the Plant RNA Purification Reagent (Invitrogen) for fruit, and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Barbara Baker for providing the plant material, as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.

first: Monica

last: Green

first: Blake

last: Meyers


title: Mycorrhizal Metabolome: L. bicolor :: P. trichocapra

status: Public on Mar 25 2011

city: Argonne National Laboratory

first: Peter

last: Larsen

address: 970 S. Cass Ave

country: USA

institute: Argonne National Labs

type: Expression profiling by high throughput sequencing

summary: Mycorrhizae, symbiotic interactions between soil fungi and tree roots, are ubiquitous in terrestrial ecosystems.  The fungi contribute phosphorous, nitrogen and mobilized nutrients from organic matter in the soil and in return the fungus receives photosynthetically-derived carbohydrates.  This union of plant and fungal metabolisms is the mycorrhizal metabolome.  Understanding this symbiotic relationship at a molecular level provides important contributions to the understanding of forest ecosystems and global carbon cycling.  We generated next generation short-read transcriptomic sequencing data from fully-formed ectomycorrhizae between Laccaria bicolor and aspen (Populus tremuloides) roots.  The transcriptomic data was used to identify statistically significantly expressed gene models using a bootstrap-style approach, and these expressed genes were mapped to specific metabolic pathways.  Integration of expressed genes that code for metabolic enzymes and the set of expressed membrane transporters generates a predictive model of the ectomycorrhizal metabolome.  Results indicate the specific compounds glycine, glutamate, and allantoin are synthesized by L. bicolor and that these compounds or their metabolites may be used for the benefit of aspen in exchange for the photosynthetically-derived sugars fructose and glucose.The analysis illustrates an approach to generate testable biological hypotheses to investigate the complex molecular interactions that drive ectomycorrhizal symbiosis. These models are consistent with experimental environmental data and provide insight into the molecular exchange processes for organisms in this complex ecosystem.  The method used here for predicting metabolomic models of mycorrhizal systems from deep RNA sequencing data can be generalized and is broadly applicable to transcriptomic data derived from complex systems.

sample_organism:
Populus tremuloides
Laccaria bicolor

platform_organism:
Populus tremuloides
Laccaria bicolor

overall_design: Fully formed L.bicolor::P.trichocapra mycorrhizae in duplicate

middle: E

first: Avinash

last: Sreedasyam

first: Geetika

last: Trivedi

first: Leland

middle: J

last: Cseke

first: Frank

middle: R

last: Collart


title: RNA-Seq of rRNA depleted circulating endothelial progenitor cells (cEPCs) in trisomy 21 and euploid samples

status: Public on May 05 2011

city: Naples

first: Valerio

last: Costa

address: Via P. Castellino 111

country: Italy

institute: Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR, Naples, Italy

type: Non-coding RNA profiling by high throughput sequencing

summary: Massive-scale RNA-seq on rRNA-depleted samples allowed us to survey in a specific cell type, affected in Down syndrome, the complete set of coding and non-coding RNA species - emerging as novel contributors to pathogenic mechanisms - to an unprecedented level of resolution and sensitivity. In particular, we demonstrated the deregulation of crucial gene pathways by quantitatively measuring the expression of known RefSeq genes. Moreover, we identified novel regions of active transcription falling outside already annotated loci, and investigated also the expression of non-polyadenilated long, as well as short, non coding RNAs. In addition, we found novel splice isoforms for a large subset of genes, and novel extended UTRs, both 5' and 3', for known genes - possibly representing novel miRNA targets or regulatory sites for gene transcription, respectively.

overall_design: 2 RNA samples examined: 1 euploid control and 1 trisomic sample.

first: Alfredo

last: Ciccodicola

first: Claudia

last: Angelini


title: Evolutionary conservation of phototransduction and clock gene expression in the cave beetle Ptomaphagus hirtus (Coleoptera, Leiodidae)

status: Public on Mar 19 2011

city: Houston

first: Bryce

last: Daines

state: TX

address: One Baylor Plaza

department: Molecular and Human Genetics

institute: Baylor College of Medicine

summary: Caves are populated with a diverse fauna of highly adapted species that tend to exhibit a consistent suite of both regressive and constructive trait modifications. Because molecular studies of cave adaptation have largely concentrated on vertebrate models, our ability to recognize universalities in the genetic trajectories underlying cave adaptation remains limited. We have initiated efforts to elucidate the molecular evolution of the flightless small carrion beetle Ptomaphagus hirtus (Ptomaphagus hirtus), which represents one of the highly endemic signature inhabitants of the Mammoth Cave system of Kentucky. Ptomaphagus hirtus has been considered blind despite the presence of lateral eye rudiments. However, analysis of the Ptomaphagus hirtus adult head transcriptome by deep RNA sequencing reveals the conservation and expression of all essential insect phototransduction genes including a single long wavelength-sensitive opsin. Consistent with the preservation of visual ability, Ptomaphagus hirtus expresses all core members of the clock gene network and exhibits a similar degree of negative phototaxis as does a closely related flight-active species in light-dark choice assays. The structural reduction of the peripheral Ptomaphagus hirtus visual system is reflected by the lack of five eye pigmentation specific genes in the head transcriptome. Taken together our data suggest that wavelength contingent and probably also spatial vision have been lost in Ptomaphagus hirtus, while irradiance vision and contingent behavioral modules have remained preserved. We predict that the adaptive state of Ptomaphagus hirtus is representative for a large number of microphthalmic species adapted to the twilight zone of caves and other subterranean habitats

overall_design: Poly(A)+ transcripts were isolated from a pooled sample of 25 adult Ptomaphagus hirtus heads, reverse transcribed and sequenced on the Illumina GAII

first: Rui

last: Chen

first: Markus

last: Friedrich


title: Differential expressions of microRNAs involve the development of superior and inferior spikelets in rice (Oryza sativa)

city: Zhengzhou

first: Quan-Zhi

last: Zhao

state: Henan Province

address: NO 63 Nongye Road

department: Agricultural College

country: China

institute: Henan Agricultural University

summary: We aimed to identify differential expression of microRNAs between superior and inferior spikelets by using a deep sequencing approach developed by Solexa (Illumina). Two small RNA libraries were constructed from superior and inferior spikelets at 18 days after fertilization, and more than nine million small RNA sequence reads were generated for each library. Totals of 351 and 312 known miRNAs were obtained from the superior and inferior spikelets, respectively. Analysis of the expression profiles of these miRNAs showed that 189 miRNAs were differentially expressed between superior spikelets and inferior spikelets. In addition, 43 novel miRNAs were identified mostly by the accumulations of miRNA*s were also expressed differentially. Further analysis shows that these miRNAs may individually participate in regulating hormone metabolism, carbohydrate metabolic pathways, and cell division during rice grain development. These results indicate that slow grain filling and low grain weight of rice inferior spikelets probably relation to the expression and function differences between superior and inferior spikelet miRNAs.

overall_design: Examination of 2 different small RNA expression profilings in superior and inferior spikelets at 18 days after fertilization.

first: Quan

middle: Z

first: Ting

last: Peng

first: Jing

last: Zhang

first: Jun

last: Li

first: Yan

middle: X

last: Du


title: High-throughput sequencing of small RNAs in Lactuca sativa

status: Public on Apr 30 2011

city: Udine

first: Emanuele

last: De Paoli

state: UDINE

address: via delle Scienze 206

department: Agricultural, Food, Environmental and Animal Sciences

institute: University of Udine

summary: Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different Lactuca sativa tissues (including leaves, flowers, and fungus-infected leaves). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features such as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study.

overall_design: Small RNA libraries were derived from leaves and flowers of Lactuca sativa, cultivar Salinas, including leaves infected with the fungus Bremia lactucae, strain CA-III. Total RNA was isolated using TriReagentÂ® (Molecular Research Center) and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Richard Michelmore and Oswaldo Ochoa for providing the plant material as well as Kan Nobuta for assistance with the computational methods.

first: Monica

last: Accerbi

first: Pamela

last: Green

first: Blake

middle: C

last: Meyers


title: miRNA profiling of Adenosine treated Endothelial Progenitor Cells (EPCs)

status: Public on Mar 15 2011

city: Luxembourg

first: Yvan

last: Devaux

address: 120 route d'Arlon

country: Luxembourg

institute: LIH

summary: Studies of miRNA profiling in early and late endothelial progenitor cells treated or not by cardioprotective nucleoside adenosine.

overall_design: Early outgrowth endothelial progenitor cells were obtained by adhesion of peripheral blood mononuclear cells of healthy volunteers. Late endothelial progenitor cells were obtained by purification of CD34+ peripheral blood cells and were cultured and amplified in endothelial-specific medium containing growth factors. Both cell types were treated by adenosine (10micromol/L) for 6 hours. Total RNA was extracted using mirVana Kit and quantified by Nanodrop. RNA was labeled and hybridized using Agilent miRNA Complete Labeling and Hyb Kit. 3 to 4 arrays per sample were hybridized and scanned with the Genepix 4000B Scanner (Molecular Devices). Six independent experiments were performed.


title: High-throughput sequencing of small RNAs in Cycas rumphii

status: Public on May 10 2011

summary: Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different Cycas rumphii tissues  (leaves, ovules, and microsporangia). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study.

overall_design: Small RNA libraries were derived from leaves, ovules and microsporangia of Cycas rumphii. Each tissue represented a mixture of developmental stages. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen) and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Eric Brenner for providing the plant material and Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods..


title: Genome-wide CTCF binding maps in chicken red blood cells

status: Public on Apr 15 2011

city: Barcelona

first: David

last: Martin

address: C/ Dr. Aiguader, 88

department: Bioinformatics and Genomics

country: Spain

institute: CRG

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Chromatin immunoprecipitation against CTCF followed by Illumina High-throughput sequencing.

overall_design: Examination of CTCF binding in red blood cells at 2 stages of development

first: Christian

last: ValdÃ©s-Quezada

first: Mayra

last: Furlan-Magaril

first: FÃ©lix

last: Recillas-Targa


title: High-throughput sequencing of small RNAs in Aristolochia fimbriata

summary: Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from different Aristolochia fimbriata tissues (including leaves and flower buds). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features such as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study.

overall_design: Small RNA libraries were derived from mature leaves and flower buds of Aristolochia fimbriata. Each tissue represented a mixture of developmental stages, with a bias towards those from early, actively differentiating cells.  Total RNA was isolated using the TriReagent (Molecular Research Center) and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Claude dePamphilis and Paula Ralph for providing the plant material, as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.


title: DNA copy number alterations in endobronchial squamous metaplastic lesions predict lung cancer

status: Public on Aug 02 2011

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

country: Netherlands

institute: Vrije Universiteit Medical Center

summary:
BACKGROUND
Autofluorescence bronchoscopy (AFB) is a valid strategy for detecting premalignant endobronchial lesions. However, no biomarker can reliably predict lung cancer risk of subjects with AFB-visualized premalignant lesions. Our present study was set out to identify AFB-visualized squamous metaplastic lesions with malignant potential by DNA copy number profiling.

METHODS
Regular AFB-examinations in 474 subjects at risk of lung cancer identified 6 subjects with SqM lesions at baseline and carcinoma (in situ) at the initial SqM site at follow-up bronchoscopy. These progressive SqM lesions were compared for immunostaining pattern and arrayCGH-based chromosomal profiles to 23 SqM of subjects who remained cancer-free. Specific copy number alterations (CNAs) linked to cancer risk were identified and accuracy of CNAs to predict endobronchial cancer in this series was determined.

RESULTS
At baseline, p53, p63 and Ki-67 immunostaining were not predictive for a differential clinical outcome of SqM lesions. The mean number of CNAs in baseline SqM of cases was significantly higher compared to controls (p<0.01). Chromosomal regions significantly more frequently altered in SqM of cases were 3p26.3-p11.1, 3q26.2-q29, 9p13.3-p13.2, and 17p13.3-p11.2 (FWER<0.10). CNAs were specifically detected at the site of future cancer. In cases, baseline-detected CNAs persisted in subsequent biopsies taken from the initial site, and levels increased towards cancer progression. CNAs at 3p26.3-p11.1, 3q26.2-29, and 6p25.3-24.3, predicted cancer risk for AFB-visualized SqM with 97% accuracy.

CONCLUSION
Our data strongly suggest that the presence of specific DNA copy number alterations in endobronchial SqM lesions predict endobronchial cancer.

overall_design: Fresh frozen specimens of 29 squamous metaplastic AFB-visualized baseline biopsies, 23 location-matched follow-up biopsies, 6 biopsies of anatomically distinct sites of 4 cases. Test samples were compared to an external pool of normal male/female reference DNA.

first: Robert

middle: A

last: van Boerdonk

first: Thomas

middle: G

last: Sutedja

first: Peter

last: Snijders

first: Emilie

last: Reinen

first: Saskia

middle: M

last: Wilting

first: Mark

last: van de Wiel

first: Frederik

middle: B

last: Thunnissen

first: Sylvia

last: Duin

first: Clarissa

last: Kooi

first: Bauke

last: Ylstra

first: Chris

last: Meijer

first: Gerrit

first: Johannes

last: Daniëls

first: Pieter

last: Postmus

first: Egbert

middle: F

last: Smit

first: Daniëlle

last: Heideman


title: Small RNA sequences from Schizosaccharomyces japonicus

status: Public on Mar 09 2011

city: Austin

first: Matthew

middle: Wayne

last: Vaughn

address: 10100 Burnet Rd

department: Texas Advanced Computing Center

institute: University of Texas at Austin

summary: Small interfering RNAs (siRNAs) are known to be involved in both transposon silencing and centromere function, leading us to investigate the interplay between these two roles in the Schizosaccharomyces lineage. In S. pombe, the centromeric repeats produce dicer-dependent siRNAs that are required for maintenance of centromeric structure, function and transcriptional silencing via Argonaute-dependent heterochromatin formation13. However, transposons are silenced in S. pombe by RNAi-independent mechanisms and do not produce abundant siRNAs. To investigate whether centromere-directed siRNA production is conserved within the transposon-rich centromeres of S. japonicus, we isolated and sequenced small RNAs from log-phase S. japonicus cultures. The small RNAs have a modal size of 23 nucleotides and 94% map to transposons, both telomeric and centromeric.

overall_design: Isolation and computational analysis of small RNAs from wild-type S. japonicus

last: Martienssen

first: Klavs

last: Hansen


title: Gene expression in wild type and Cagal102 knockout Candida albicans strains.

city: Bangalore

first: Genotypic

last: technology

state: Karnataka

address: 259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE

country: India

institute: Genotypic Technology

summary:
CaGAL102 is a sequence homolog of Rmlb. In Candida knock out of this gene causes abnormal hyphal morphogenesis and increased sensitivity to cell wall damaging agents. The knock out strain is also avirulent in mouse model of systemic infection. To get a larger insight into the function of the protein product of this gene we carried out global transcription analysis through micro array experiment. The gene is expressed under normal growth conditions and the knock out causes the cells to become hyphal under these conditions. Many of the cell wall proteins were upregulated recapitulating the cell morphology.
Keywords: Candida albicans, Gene knockout, genome wide transcription profiling study

overall_design: Wild type Sc5314 cells and the mutant CAS12 were grown in YPD till (OD 600nm) 1 following which total RNA was extracted using hot phenol method. The RNA was checked using Bioanalyser (Agilent). Single dye experiment was carried out using Cy3 labelled pooled wild type samples and two independent mutant RNA samples.

first: Parag

middle: P

last: Sadhale


title: Gene expression throughout vertebrate embryogenesis

status: Public on Apr 19 2011

city: Miami

first: Marjorie

middle: F.

last: Oleksiak

state: FL

address: 4600 Rickenbacker Causeway

department: Marine Biology and Fisheries

institute: University of Miami

summary: This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage.  Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing.  Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660.  Five adjacent stages have disproportionately more significant changes in gene expression (>200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre- and post-hatch, and during complete yolk absorption.  The fewest differences among adjacent stages occur during gastrulation.  Yet, at stage 16 (pre-mid-gastrulation), the largest number of genes has peak expression.  This stage has an over-representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases).  Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur.  Similar correlated patterns of expression occur among all significant genes.  These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development.

overall_design: A double-loop design was used for the microarray hybridizations, where each sample is hybridized to 2 arrays using both Cy3- and Cy5-labelled fluorophores (Kerr and Churchill 2001a; Kerr and Churchill 2001b).  The loop consisted of Cy3- and Cy5-labelled embryo aRNAs from 4 biological pools for each of 40 stages (S).  In total, 160 biological pools were hybridized to 80 microarrays.  Each array had different combinations of biological pools (Altman and Hua 2006).  The double loop formed was S1→ S2 → S3→ … S40→ S1 and S40→ S39→ S38→ … S2→ S1 → S40, where each arrow represents a separate hybridization (array) with the biological pool at the base of the arrow labeled with Cy3 and the biological pool at the head of the arrow labelled with Cy5.

first: Goran

last: Bozinovic


title: Genome-wide discovery and analysis of microRNAs and other small RNAs from rice embryogenic callus

status: Public on Mar 07 2011

city: Orsay

first: Chong-Jian

last: CHEN

address: IGM, Bat 400, Université Paris-Sud

country: France

institute: Institut de Génétique et Microbiologie

summary: Small RNAs constitute a new and unanticipated layer of gene regulation present in the three domains of life. In plants, all organs are ultimately derived from a few pluripotent stem cells localized in specialized structures called apical meristems. The development of meristems involves a coordinated balance between undifferentiated growth and differentiation, a phenomenon requiring a tight regulation of gene expression. We used in vitro cultured embryogenic calli as a model to investigate the roles of meristem-associated small RNAs. Using high-throughput sequencing, we sequenced 20 million short reads with size of 18-30nt from rice undifferentiated and differentiated calli. We confirmed 50 known microRNA families, representing one third of annotated rice microRNAs. Using a specific computational pipeline for plant microRNA identification, we identified 24 novel microRNA families. Among them, 53 microRNA or microRNA* sequences appear to vary in expression between differentiated and undifferentiated calli, suggesting a role in meristem development. Our analysis also revealed a new class of plant small RNAs derived from 5' or 3' ends of mature tRNA analogous to the tRFs in human cancer cell. We independently verified the expression of these small RNAs from 5' end of mature tRNA using qRT-PCR.

overall_design: Examination of 2 different small RNA expression profilings in 2 developmental stages of meristems.

last: Chen

first: Qing

last: liu

first: Yu-Chan

last: Zhang

first: Liang-Hu

last: Qu

first: Yue-Qin

first: Daniel

last: Gautheret


title: Evaluation of differential expression in the vascular transcriptome among Picea species

status: Public on Feb 18 2012

city: Québec

first: John

last: MacKay

state: Québec

address: 1030, avenue de la Médecine, Pavillon C.-E. Marchand

department: Centre d'étude de la forêt

country: Canada

institute: Université Laval

summary: The goal of this experiment is to assess tissue preferential transcript accumulation and fold difference between two tissues that support secondary vascular growth in three spruces: Picea glauca, Picea sitchensis and Picea mariana. Tissues compared are secondary xylem (wood forming tissue located on the internal side of the cambial meristem) and phelloderm (composite sample of the phloem and phelloderm tissues located on the outer side of the cambial meristem).

sample_organism:
Picea glauca
Picea sitchensis
Picea mariana

platform_organism:
Picea glauca
Picea sitchensis

overall_design: One-color comparison of 3 spruce species in 2 tissue types: xylem and phelloderm. 20 biological repetitions per tissue for Picea glauca and 15 for Picea sitchensis and Picea mariana, for a total of 100 slides.

first: Elie

last: Raherison

first: Sébastien

last: Caron

first: Isabelle

last: Giguère


title: r2d2 Ago1 Immunoprecipitation ovary

status: Public on Oct 18 2010

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

institute: Ontario Institute for Cancer Research

summary:
modENCODE_submission_2749
This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Keywords: RIP-seq

overall_design: EXPERIMENT TYPE: RIP-seq. BIOLOGICAL SOURCE: Strain: r2d2 1; Developmental Stage: Adult Female; Genotype: w; r2d2[1]; Sb/TM6B; Sex: Female;  EXPERIMENTAL FACTORS: Adult_ovaries

first: Eric

last: Lai

first: Gregory

last: Hannon


title: S. aureus gene expression following AFN-1252 treatment

status: Public on Jul 02 2010

city: Memphis

first: Matthew

middle: Wilmer

last: Frank

state: TN

address: 262 Danny Thomas Place

department: Infectious Diseases

institute: St Jude Children's Research Hospital

summary: AFN-1252 is an inhibitor of fatty acid biosynthesis. Gene expression profiles were generated by microarray analysis of S. aureus cells following treatment with AFN-1252, an inhibitor of fatty acid synthesis.

overall_design: Fatty acid biosynthesis in S. aureus was inhibited by AFN-1252 for 15 minutes, and RNA was extracted to look at the gobal gene expression.

middle: W

first: Charles

middle: O

last: Rock


title: High-throughput sequencing reveals a simple model of nucleosome energetics

status: Public on Aug 20 2010

city: Piscataway

first: George

last: Locke

state: NJ

address: 136 Frelinghuysen Rd

department: Physics

institute: Rutgers University

summary: We use nucleosome maps obtained by high-throughput sequencing to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a secondary role. Furthermore, similar sequence signatures are exhibited by control experiments in which genomic DNA is either sonicated or digested with micrococcal nuclease in the absence of nucleosomes, making it possible that current predictions based on highthroughput nucleosome positioning maps are biased by experimental artifacts.

sample_organism:
Escherichia coli
Saccharomyces cerevisiae

overall_design:
Included are raw (eland) and mapped (wig) reads.  The mapped reads are provided in eland and wiggle formats, and the raw reads are included in the eland file.

This series includes only Mnase control data. The sonicated control is part of this already published accession, as is a in vitro nucleosome map:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15188

We also studied data (in vitro and in vivo maps as well as a model) from
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13622

and from:
http://www.ncbi.nlm.nih.gov/sra/?term=SRA001023

first: Denis

last: Tolkunov

first: Zarmik

last: Moqtaderi

first: Kevin

last: Struhl

first: Alexandre

middle: V

last: Morozov


title: Gene expression analysis of FXR null, SHP null, and double FXR/SHP null mice

status: Public on Dec 10 2010

first: Scott

middle: Andrew

last: Ochsner

department: Molecular and Cellular Biology

summary: Bile acid (BA) homeostasis is maintained through a feedback loop operated by the nuclear hormone receptors FXR and SHP. Here we show that contrary to the current models placing FXR upstream of SHP in a linear regulatory pathway, the phenotypic consequence of the combined loss of both receptors is much more severe than the relatively modest impact of the loss of either Fxr or Shp alone. This is highlighted by the dramatic elevation of hepatic and serum BA levels in the double knockout (DKO) mice as early as three weeks of age coupled with a commensurate increase in Cyp7A1 expression and alterations in BA homeostatic genes. In addition, we find several genes necessary for C21 steroid biosynthetic pathway as novel targets for FXR and SHP. The elevated BAs result in severe hepato-pathology but the DKO mice surprisingly do not develop complete liver failure and live for over a year. Their survival is accompanied by an adaptive proliferation of the resident liver progenitor cell population, known as oval cells. Overall, these data demonstrate that FXR and SHP function coordinately to maintain BA homeostasis, and identify DKO mice as a novel genetic model for juvenile cholestatic disorders and for oval cell activation.

overall_design: Liver samples collected from FXR-/-, SHP-/-, and FXR-/-/SHP-/- animals at 3 or 5 weeks were hybridized to Illumina mouse REF-8 v1.1 arrays in duplicate.

first: Sayeepriyadarshini

last: Anakk

first: David

middle: D

last: Moore


title: miRNA* loading on Ago2

status: Public on Aug 03 2010

city: Worcester

first: Hervé

last: Seitz

state: MA

address: 364 Plantation Street

department: Biochemistry and Molecular Pharmacology

institute: University of Massachusetts Medical School

summary: In flies, microRNAs (miRNAs) are synthesized as a short imperfect duplex, where the miRNA is paired to another small RNA, dubbed the "miRNA*". Usually, miRNA*s are degraded in the last steps of miRNA maturation, but we found evidence that some of them can be loaded on the Ago2 protein.

overall_design: Small RNAs (18-29 nt) from fly heads (WT, ago2 mutants, dcr-2 homozygous and heterozygous mutants) were sequenced using a Solexa Genome Analyzer instrument. The ago2 cDNA library was loaded twice on the instrument, yielding two technical replicates.

first: Megha

last: Ghildiyal

first: Phillip

last: Zamore


title: Expression profiling of Moraxella catarrhalis RH4 during in vitro growth in brain heart infusion (BHI) medium

status: Public on May 07 2010

city: Nijmegen

first: Hester

last: Bootsma

address: Kapittelweg 29

department: Pediatrics

institute: Radboud University Medical Centre

type: Expression profiling by genome tiling array

summary:
Transcriptome analysis of M. catarrhalis RH4 predicted coding sequences and clustered regularly interspaced short palindromic repeats (CRISPRs) regions.
The results described in this study are further discussed in De Vries, S.P.W., van Hijum, S.A.F.T., Schueler, W., Riesbeck, K., Hays, J.P., Hermans, P.W.M., Bootsma, H.J.; Genome analysis of Moraxella catarrhalis strain RH4: a human respiratory tract pathogen; Journal of Bacteriology

overall_design: Total RNA from lag phase (OD620 = 0.2-0.3), exponential phase (OD620 = 1.2-1.4), or stationary phase (OD620 = 2.0-2.2) bacteria grown in triplicate in BHI medium, and hybridized in duplicate on 4x72K custom design Nimblegen arrays

first: Stefan

last: de Vries

first: Sacha

last: van Hijum

first: Wolfgang

last: Schueler

first: Kristian

last: Riesbeck

first: John

last: Hays

first: Peter

last: Hermans

middle: J


title: MIRNA Gene Evolution in Arabidopsis lyrata and Arabidopsis thaliana: 454 sequencing

status: Public on May 14 2010

city: Saint Louis

first: James

last: Carrington

state: MO

address: 975 North Warson Road

institute: Donald Danforth Plant Science Center

summary: Deep sequencing of small RNA from three closely related brassicaceae A. thaliana, A. lyrata and Capsella rubella was done to systematically analyze the evolution of MIRNA genes and their targets.

sample_organism:
Arabidopsis lyrata
Capsella rubella

platform_organism:
Arabidopsis lyrata
Capsella rubella

overall_design: Small RNA were extracted from A. lyrata and C. rubella total RNA by size fractionation and converted to DNA amplicons by serial adaptor ligation to both ends followed by RT-PCR.  DNA amplicons were sequenced using an Ilumina Genome Analyzer.  Resulting sequences were computationally trimmed to remove 3' adaptor sequences.

first: Noah

last: Fahlgren

first: Sanjuro

last: Jogdeo

first: Kristin

last: Kasschau

first: Christopher

last: Sullivan

first: Elisabeth

last: Chapman

first: Sascha

last: Laubinger

first: Lisa

last: Smith

first: Mark

last: Dasenko

last: Givan

first: Detlef

last: Weigel


title: Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas

status: Public on Feb 09 2010

city: Newark

first: Linda

last: Rymarquis

state: DE

address: 15 Innovation way rm 241

institute: University of Delaware

summary:
Regulation of gene expression by small RNAs (∼20–30 nucleotides in length) plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) commonly direct the inactivation of cognate sequences through a variety of mechanisms, including RNA degradation, translation inhibition, and transcriptional repression. Recent studies have provided considerable insight into the biogenesis and the mode of action of miRNAs and siRNAs. However, relatively little is known about mechanisms of quality control and small RNA decay in RNA interference (RNAi) pathways. Here we show that deletion of MUT68, encoding a terminal nucleotidyltransferase in the alga Chlamydomonas reinhardtii, results in elevated miRNA and siRNA levels. We found that MUT68 plays a role in the untemplated uridylation of the 3′ ends of small RNAs in vivo and stimulates their degradation by the RRP6 exosome subunit in vitro. Moreover, RRP6 depletion also leads to accumulation of small RNAs in vivo. We propose that MUT68 and RRP6 cooperate in the degradation of mature miRNAs and siRNAs, as a quality control mechanism to eliminate dysfunctional or damaged small RNA molecules.

Keywords: small RNA

overall_design: Four small RNA libraries were created: two from Mut68 and two from the Maa7-IR44 control using unique barcodes at the 3' end of the 5' adapter.  Libraries were pooled and sequenced.

first: Fadia

last: Ibrahim

first: Eun-Jeong

last: Kim

last: Becker

first: Eniko

last: Balassa

first: Pamela

last: Green

first: Heriberto

last: Cerutti


title: Pilot Experiment for Cell-specific Translatome: AP1 Domain vs. Whole Flower

status: Public on Aug 25 2010

city: Pasadena

first: Yuling

last: Jiao

state: CA

address: 1200 E California Blvd

department: Biology

institute: California Institute of Technology

summary: A pilot experiment for an RNA-seq dataset (SRP003234).  This experiment compares AP1 domain-specific translating RNA with total flower translating mRNA.

overall_design: Two-condition experiment, AP1 flower domains vs. whole flower at flower stage 4.

last: Jia

first: Eliiot

last: Meyerowitz


title: Meyerowitz Lab Arabidopsis Operon Array v4 - 4200A scanner - AP1-GR ap1cal inflorescences - time series (hours)

status: Public on Apr 02 2010

city: Bellaterra

first: Jose Luis

last: Riechmann

state: Barcelona

address: Campus UAB - Edifici CRAG

institute: Center for Research in Agricultural Genomics

summary: This experiment describes gene expression after the activation of APETALA1-GR, to study and identify AP1 target genes. We used a 35S:AP1-GR ap1 cal line to induce a synchronized response activating the AP1-GR fusion protein in ap1 cal inflorescence-like meristems through dexamethasone treatment. Tissue samples were collected immediately after the treatment, as well as subsequent timepoints. The expression profiles of the individual samples were then analyzed by gene expression profiling using whole-genome oligonucleotide arrays (non-commercial; Meyerowitz Lab Arabidopsis Operon Array v4).  Keywords: time course

overall_design:
Four sets of biologically independent tissue samples were collect at 0, 2, 4 ,8, and 12 hours after the application of dexamethasone (Dex-; activation of the AP1-GR fusion protein) or a mock solution (Mock-; control). In each of the biological replicates of the time course experiments, all the samples derived from dexamethasone (Dex)-treated plants were labeled with one dye (i.e., Cy3), and all the samples derived from the corresponding Mock-treated plants were labeled with the alternative dye (i.e., Cy5). The dyes used for labeling RNA from a given treatment type (Dex and Mock) were switched for two of the replicate experiments, to reduce dye-related artifacts. Dex- and Mock-derived samples for each timepoint and biological replicate were co-hybridized. This experimental setup resulted in a total of 5 hybridizations per set (0h, 2h, 4, 8h, and 12h; Dex vs. Mock at each timepoint), and two biological replicate sets labeled with each dye polarity (Mock-Cy3/Dex-Cy5, and vice versa).

The combined ratio data results are available as a supplementary file on the Series record.

first: Jose

middle: L

first: Vijaya

middle: S

last: Kumar

first: Thilia

last: Ferrier

first: Frank

last: Wellmer


title: The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE

status: Public on Feb 01 2011

city: Dijon

first: Carlos

middle: Mauricio

last: Molina

state: Bourgogne

address: 17 Rue Sully

department: UMRLEG

institute: INRA

summary:
Background
The combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche’s 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level.
Results
We used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress.
Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways.
From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available.
Conclusions
This report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms.
As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea.
Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26bp tags by SuperSAGE.

overall_design:
SuperSAGE libraries from chickpea roots and nodules were constructed starting from hydro aeroponics-generated material.
Control and stressed plants were grown in parallel

Sequencing Instrument:
First gerenration 454-Machine
Place of sequencing:
454 Life Sciences, Branford, CT, USA

first: Mainassara

last: Zaman

first: Faheema

last: Kahn

first: Nadia

last: Fatnassi

first: Ralf

last: Horres

first: Bjorn

last: Rotter

first: Diana

last: Steinhauer

first: Laurie

last: Amenc

first: Jean-Jacques

last: Drevon

first: Peter

last: Winter

first: Guenter

last: Kahl


title: MicroRNA discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries

city: Seattle

first: Muneesh

last: Tewari

state: WA

address: 1100 Fairview Ave, N

department: Human Biology

institute: Fred Hutchinson Cancer Research Center

summary: We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESCs). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high-quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 36 novel miRNAs highly conserved across vertebrates, and 291 novel candidate miRNAs. A set of nine known and seven novel miRNAs were expressed in undifferentiated hESC and then strongly down-regulated with differentiation. Predicted mRNA targets of these miRNAs exhibited statistically significant enrichment for Gene Ontology functional categories relevant to ESC biology. Our study reveals that hESC express a larger complement of miRNAs than previously appreciated and it provides a resource for future studies of miRNA-mediated regulation in human embryonic stem cells.

overall_design: Two samples were 454 sequenced: undifferentiated human embryonic stem cells and differentiated human embryonic stem cells.

first: Merav

last: Bar

first: Stacia

middle: K

last: Wyman

first: Brian

middle: R

last: Fritz

first: Rachael

last: Parkin

first: Evan

last: Kroh

first: Angelique

last: Nelson

first: Carol

last: Ware

first: Jerald

last: Radich

first: Robert

last: Gentleman


title: Deep sequencing of small RNAs from C. elegans during aging

status: Public on Jan 01 2011

city: NEW HAVEN

first: ALEXANDRE

last: DE LENCASTRE

address: 219 PROSPECT ST KBT 924

institute: YALE UNIVERSITY

summary: Aging is under genetic control in C. elegans but the mechanisms of lifespan regulation are not completely known. MicroRNAs (miRNAs) regulate various aspects of development and metabolism and one miRNA has been previously implicated in lifespan. Here we show that multiple miRNAs change expression in C. elegans aging, including novel miRNAs, and that mutations in several of the most up-regulated miRNAs lead to lifespan defects. Some act to promote normal lifespan and stress resistance while others inhibit these phenomena. We find that these miRNAs genetically interact with genes in the DNA damage checkpoint response pathway and in the insulin signaling pathway. Our findings reveal that miRNAs both positively and negatively influence lifespan. Since several miRNAs up-regulated during aging regulate genes in conserved pathways of aging and thereby influence lifespan in C. elegans, we propose that miRNAs may play important roles in stress response and aging of more complex organisms.

overall_design: 4 sample examined: Wild-type (N2) and long-lived daf-2(e1379) animals at days 0 and 10 of adulthood

first: Alexandre

last: de Lencastre

first: Frank

last: Slack


status: Public on Nov 13 2010

city: Seoul

first: Chang Ohk

last: Sung

address: Song-Pa gu,Pung-nap dong

country: South Korea

institute: Asan Medical Center

type: Genome variation profiling by genome tiling array

summary: Genomic aberrations in primary central nervous system lymphoma (PCNSL).

overall_design: Two-condition experiment, PCNSLs vs. healthy men. Biological replicates: 12 tumors replicates, 13 peripheral blood replicates.

first: Young-Hyeh

last: Ko

first: Chang-Ohk

first: Sang-Cheol

last: Kim

first: Sivasundaram

last: Karnan

first: Kennosuke

last: Karube

first: Hyung-Jin

last: Shin

first: Do-Hyun

last: Nam

first: Yeon-Lim

last: Suh

first: Seok-Hyung

first: Ji-Yeon

first: Soek-Jin

first: Won-Seog

first: Masao

last: Seto


title: Deletion of chromosome 4q predicts outcome in Stage II colon cancer

status: Public on Jul 13 2010

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

institute: Vrije Universiteit Medical Center

summary: Background: Around 30% of all stage II colon cancer patients will relapse and die of their disease. At present no objective parameters for identification of high-risk stage II colon cancer patients, who will benefit from adjuvant chemotherapy, are established. With traditional histopathological features definition of high-risk stage II colon cancer patients is inaccurate. Therefore more objective and robust markers for prediction of relapse are needed. DNA copy number aberrations have proven to be robust prognostic markers, but have not been investigated for this specific group of patients. The aim of the present study is to identify chromosomal aberrations that can predict relapse of tumor in patients with stage II colon cancer. Materials and Methods: DNA was isolated from 40 formaldehyde fixed paraffin embedded stage II colon cancer samples with extensive clinicopathological data. Samples where hybridized using Comparative Genomic Hybridization (CGH) arrays to determine DNA copy number changes and microsatellite stability was determined by PCR. To analyze differences between stage II colon cancer patients with and without relapse of tumor a Wilcoxon rank-sum test was implemented with multiple testing correction Results: Patients with stage II colon cancer who had relapse of disease showed significant more losses on chromosome 4, 5, 15q, 17q and 18q. When microsatellite stable (MSS) patients were analyzed separately, only losses on chromosome 4q22.1-4q35.2 predicted worse outcome in stage II colon cancer patients. No differences in clinicopathological characteristics between patients with and without relapse were observed. Conclusion: Losses on 4q22.1-4q35.2 predict worse outcome in MSS stage II colon cancer patients and may aid in the selection of patients for adjuvant therapy.

overall_design: 40 Stage II colorectal cancer (CRC) tissue samples (FFPE), 16 with and 24 without relapse of tumor

first: Rebecca

last: Brosens

first: Eric

last: Belt

first: Josien

middle: C

last: Haan

first: Tineke

middle: E

last: Buffart

first: Nicole

last: T van Grieken

first: Heike

last: Grabsch

first: Miquel

middle: A

last: Cuesta

first: Alexander

last: Engel

first: Bauke

last: Ylstra

first: Gerrit

last: Meijer


title: SuperSAGE analysis of the Nicotiana attenuata transcriptome after wounding

status: Public on Oct 06 2010

city: Jena

first: Klaus

last: Gase

address: Hans-Knoell-Strasse 8

department: Molecular Ecology

country: Germany

institute: Max-Planck-Institute for Chemical Ecology

summary: Fatty acid-amino acid conjugates (FACs) in the oral secretion (OS) of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) in N. attenuata. The second fully expanded leaf of rosette stage plants were wounded by rolling a fabric pattern wheel three times on each side of the midvein and the wounds were immediately supplied with either 20 uL of 0.02% (v/v) Tween-20/water (solvent control) or 10 uL of synthetic N-linolenoyl-glutamic acid (18:3-Glu; 0.03 nmol/µL in 0.02% (v/v) Tween-20/water). Tissue was collected after 30 min of the treatments and frozen in liquid nitrogen. Total RNA was extracted by the phenol/chloroform-LiCl method and poly(A)+-RNA was purified from total RNA. Subsequent steps for the construction of the SuperSAGE libraries and 454 sequencing were performed as previously described using double NlaIII digestions (Molina et al., 2008, BMC Genomics, 9:553).

overall_design:
Two SuperSAGE libraries were generated after wounding and FAC elicitation of Nicotiana attenuata leaves.
Leaves were harvested after 30 min of the treatments, total RNA extracted, polyA+ mRNA isolated and used for cDNA synthesis and SuperSAGE analysis as described my Matsumura et al (2003).

first: Gustavo

last: Bonaventure

first: Paola

last: Gilardoni

first: Ian

middle: T

last: Baldwin


title: Ath_Agilent_v1_Riechmann array expression profile apetala1

first: Jose Luis

last: Riechmann

summary:
Comparion of gene expression profiles of wild-type and apetala1 inflorescences
The gene expression profile of inflorescences of the floral homeotic mutant apetala1 was compared to wild-type inflorescences (Ler) using a whole-genome oligonucleotide array (Agilent, custom-commercial). Experiment was performed in triplicate.

overall_design: This series contains the data for three replicate experiments performed with three sets of independent samples.  The combined ratio data results are available as a supplementary file on the Series record.

first: Jose

first: Viaya

last: Kumar

first: Tara

last: Mastro

first: Thilia

last: Ferrier

first: Frank

last: Wellmer


title: Tracing the derivation of embryonic stem cells from the inner cell mass by single cell RNA-seq analysis

status: Public on May 10 2010

city: Foster City

first: Catalin

last: Barbacioru

address: 850 Lincoln Centre Dr

institute: Applied Biosystems

summary:
Here we modified a single cell whole transcriptome amplification method to make it capable of amplifying cDNAs as long as 3kb efficiently and unbiasedly. We combined this modified single cell cDNA amplification method with Applied Biosystems next generation sequencing SOLiD™ System to set up a single cell whole transcriptome assay.  The modified amplification strategy allows us to amplify full-length cDNAs for most of the expressed genes. We show that it is feasible to get digital gene expression profiles at single cell resolution. This allows us to ask fundamental biological questions that could not be addressed previously, especially in the embyronic stem cell studues , and to understand transcriptome complexity at the resolution of a single cell.
Four-cell stage embryos were recovered from MF1 females mated with MF1 male mice (Nagy et al. 2003). The zona pellucida was removed by treatment with acidic tyrode solution. The individual blastomeres were separated by gentle pipeting using a glass capillary. Mature oocytes were isolated from Dicer knockout [Dicer-/Flox, Zp3-Cre] and Ago2 knockout [Ago2-/Flox, Zp3-Cre] female mice (14,17). Cumulus cells were removed from oocytes by treatment with hyaluronidase. All the ‘mature oocytes’ mentioned in the text are ovulated mature oocytes.

overall_design: RNA-Seq from 13 single mouse ES cells, 9 single mouse E3.5 ICM, 3 Day3-Oct4+ outgrowth, 3 Day5-Oct4+ outgrowth, 3 Day5-Oct4- outgrowth and 3 mouse E.4.5 Epiblast

first: Fuchou

last: Tang

first: Kaiqin

last: Lao

first: M

last: Surani


title: Ath_Agilent_v2_Riechmann array expression profile AP1-GR ap1cal inflorescences - chx experiment

summary: This experiment describes gene expression after the activation of APETALA1-GR, to study and identify AP1 target genes. We used a 35S:AP1-GR ap1 cal line to induce a synchronized response activating the AP1-GR fusion protein in ap1 cal inflorescence-like meristems through dexamethasone or dexamethasone+cycloheximide treatment. Tissue samples were collected at 3hrs after the treatment. The expression profiles of the individual samples were then analyzed by gene expression profiling using whole-genome oligonucleotide arrays (Agilent, custom-commercial).

overall_design: We treated inflorescences of 35S:AP1-GR ap1-1 cal-1 plants with a dexamethasone-containing or a mock solution, or with identical solutions that contained in addition 10 μM cycloheximide. Tissue was collected 3 hours after the treatment. Samples from each of the four biological replicates resulted in a set of four hybridization pairs: Mock vs. Dex, Mock vs. Chx, Mock vs. Dex+Chx, and Chx vs. Dex+Chx. Dye polarities were switched between biological replicates.

first: Vijaya


title: Meyerowitz Lab Arabidopsis Operon Array v4 - 4000B scanner - AP1-GR ap1cal inflorescences - time series (hours)


title: Global gene expression analysis of green spotted puffer fish gills in response to environmental calcium changes

status: Public on Jan 03 2011

city: Gambelas

first: Patricia

middle: I. S.

last: Pinto

address: Ed.7, University of Algarve, Campus de Gambelas

country: Portugal

institute: Centro de Ciências do Mar (CCMAR), CIMAR-Laboratório Associado

summary: This study aims to understand the molecular adaptation mechanisms of fish gills to environmental Ca2+ changes. Using SuperSAGE, we compared the gene expression profiles of T. nigroviridis gills transferred from natural brackish water (10 ppt salinity,2.9 mM Ca2+)  to artificial brackish water with control (2.9 mM), low (0.01 mM) or high (10 mM) Ca2+ concentrations for 2 or 12h.

overall_design: To obtain SuperSAGE tags, we followed the original protocol described by Matsumura and colleagues (Matsumura et al. 2003) with some modifications. The SuperSAGE ditags were directly sequenced with by high throughput pyrosequencing (454 LifeSciences).

middle: I

first: Hideo

last: Matsumura

first: Ryohei

last: Terauchi

first: Adelino

last: Canario


title: Accumulation of TuMV-derived siRNAs in aerial parts of Arabidopsis thaliana plants at 7 and 10 dpi

status: Public on Apr 09 2010

first: James

last: Carrington

summary: At 7 and 10 dpi with wild type TuMV or  buffer (mock) whole plants (root not included) were collected and total RNA extracted for sRNA sequencing using the Illumina plataform.

sample_organism:
Arabidopsis thaliana
Turnip mosaic virus

overall_design: Three plant genotypes were analyzed: Col-0, dcl1-7 single mutant and dcl2-1 dcl3-1 dcl4-2 triple mutants. Three replicate libraries were made per treatment. For each library, hits to TuMV and to Arabidopsis are inclued in separate files.

first: Hernan

last: Garcia-Ruiz


title: Expression profiling of yeast kinase and phosphatase knockouts

status: Public on Dec 09 2010

city: Utrecht

first: Patrick

last: Kemmeren

state: Utrecht

address: Universiteitsweg 100

department: Department of Molecular Cancer Research

institute: UMC Utrecht

summary: To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene-sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multi-process control.

overall_design: RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Up to five deletion strains were grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.

first: Sake

last: van Wageningen

first: Philip

last: Lijnzaad

first: Thanasis

last: Margaritis

first: Joris

last: Benschop

first: Ines

last: de Castro

first: Dik

last: van Leenen

first: Marian

last: Groot Koerkamp

first: Cheuk

last: Ko

first: Tony

last: Miles

first: Nathalie

last: Brabers

first: Mariel

last: Brok

first: Tineke

last: Lenstra

first: Dorothea

last: Fiedler

first: Like

last: Fokkens

first: Rodrigo

last: Aldecoa

first: Eva

last: Apweiler

first: Katrin

last: Sameith

first: Loes

last: van de Pasch

first: Sander

last: van Hooff

first: Linda

last: Bakker

first: Nevan

last: Krogan

first: Berend

last: Snel

last: Holstege


title: Digital gene expression profiling for maize development - 2mm ear libraries, B73 vs. ramosa3 mutant

status: Public on Oct 20 2010

city: St. Louis

first: Andrea

last: Eveland

address: 975 N. Warson Road

summary: Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks.  In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait.  In this work, we developed and tested a framework for analysis of tag-based, digital gene expression (DGE) profiles using Illumina’s high-throughput sequencing technology and the newly-assembled B73 maize reference genome.  We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in determinacy of axillary meristems.  The RA3 gene encodes a trehalose-6-phosphate-phosphatase and may act at the interface between developmental and metabolic processes.  Deep sequencing of DGE libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20-21nt reads with frequencies spanning four orders of magnitude.  Unique sequence tags were anchored to 3´-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing.  We mapped 86% of non-redundant signature tags to the maize genome, which associated with 37,117 working gene models and un-annotated regions of expression.  66% of maize genes were detected at ≥ nine reads in immature maize ears.  We used comparative genomics to leverage existing information from Arabidopsis and rice in functional analyses of differentially expressed maize genes.  Results from this study provide a basis for analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.

overall_design: 3' tag-based DGE libraries were generated using Illumina 1G technology.  Libraries were prepared from 2mm ear samples and represent: 3 biological replicates of wild-type B73, 3 biological replicates of ramosa3 (ra3) mutants, and 1 technical replicate of a ra3 sample.  Each sample was sequenced in a single lane of a flow cell.  Each lane includes library preparations from both DpnII and NlaIII digests for that sample.  Sequences from DpnII digests start with GATC and those from NlaIII digests start with ATG.

first: Namiko

last: Satoh-Nagasawa

first: Alexander

last: Goldshmidt

first: Sandra

last: Meyer

first: Mary

last: Beatty

first: Hajime

last: Sakai

first: Doreen

last: Ware

first: David

last: Jackson


title: Maize gene expression during infection with Ustilago maydis strain SG200∆fox1

status: Public on Dec 31 2010

city: Karlsruhe

first: Jörg

middle: Thomas

last: Kämper

address: Hertzstrasse 16, Geb. 06.40

department: Department for Applied Biosciences

institute: Karlsruhe Institute of Technology

summary: Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host Zea mays. The biotrophic interaction is initiated upon host penetration, and involves expansion of the host plasma membrane around hyphae, which is thought to facilitate the exchange of nutrients and virulence factors. Transcriptional regulators involved in the establishment of an infectious dikaryon and penetration into the host have been identified, however, regulators involved in the post-penetration stages remained to be elucidated. In the study we report the identification of an Ustilago maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumour development in planta. Δfox1 hyphae induce plant defences including the overproduction and accumulation of H2O2 in and around infected cells. This oxidative burst acts as an intercellular signal, which elicits a specific host defence response phenotypically represented by the encasement of proliferating hyphae in extensions of the plant cell wall. Maize microarrays experiments were performed to identify genes involved in the observed plant defence responses on leaf tissue infected with U. maydis strain SG200∆fox1 4 dpi.

overall_design: In three independent experiments plants were infected with the U. maydis strain SG200 and SG200∆fox1, respectively. Samples from infected leaves were taken at 4 days post infection. Samples from uninfected control plants were taken at the same time points.

last: Zahiri

first: Ramon

last: Wahl

first: Gunther

last: Döhlemann

first: Regine

last: Kahmann


title: Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection

status: Public on Dec 03 2009

city: Hoersholm

first: Andreas

last: Petri

address: Kogle Alle 6

department: microRNA

country: Denmark

institute: Santaris Pharma

summary:
The aim of this deep sequencing project is to determine whether treatment of chronically Hepatitis C infected chimpanzees with an antiviral compound leads to accumulation of viral mutants.

The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. Here, we show that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia with no evidence for viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes (IRGs) and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.

overall_design: Animals 4x0513 and 4x0514 were chosen for analysis as the high SPC3659 concentration could induce adaptive mutations in these animals in the 5’ NCR encompassing the miR-122 seed sites. Four time points were selected, i.e. baseline (-4 weeks), end of treatment (11 weeks), first rise in viremia after treatment (17 or 23 weeks, respectively), and end of the follow-up period (end of study at 27 weeks).

first: Morten

last: Lindow


title: Genome-wide Ste12-binding site mapping in MATa segregants of YJM789 x S96 cross

status: Public on Feb 18 2010

city: New Haven

first: Wei

last: Zheng

state: CT

address: Suite 503, 300 George Street

institute: Yale University

summary: In this study, we mapped for the first time differences in transcription binding among individuals and elucidated the genetic basis of such variation.  Whole-genome Ste12 binding profiles were determined using ChIP-Seq in pheromone-treated cells of 43 segregants of a cross between two highly diverged yeast strains, YJM789 and S288c as well as the parental lines. We identified extensive Ste12 binding variation among individuals and mapped underlying cis- and trans- acting loci responsible for such variation. We showed that the majority of TF binding variation is cis-linked and that many variations are associated with polymorphisms residing in the binding motifs of Ste12 as well as those of several known and proposed Ste12 cofactors. We also identified two trans factors, AMN1 and FLO8, that modulate Ste12 binding to promoters of more than 10 genes under α-factor treatment.  Neither of these two genes was known to regulate Ste12 previously, and we suggest that they may be key mediators of gene activity and phenotypic diversity.  Ste12 binding strongly correlates with gene expression for more than 200 genes indicating that binding variation is functional.  Many of the variable bound genes are involved in cell wall organization and biogenesis.  Overall, we identified key regulators of molecular diversity among individuals and provide novel insights into mechanisms of gene regulation.

overall_design: Two ChIP-Seq experiments and one Input DNA-Seq experiment for the yeast strains S96, HS959 and 43 MATa segregants were performed under alpha factor treatment conditions; an additional replicate was also performed for some of the strains. One ChIP-Seq experiment for each parental strain was performed without alpha factor treatment, and one ChIP-Seq experiment for each of the 24 deletion strains was performed under alpha factor treatment.

first: Hongyu

last: Zhao

first: Eugenio

last: Mancera

first: Lars

last: Steinmetz

first: Michael

last: Snyder


title: Arabidopsis regeneration from multiple tissues occurs via a root development pathway

status: Public on Feb 01 2010

first: Yuling

last: Jiao

summary: Unlike most animal cells, plant cells can easily regenerate new tissues from a wide variety of organs when properly cultured. The common elements that provide varied plant cells with their remarkable regeneration ability are still largely unknown. Here we describe the initial process of Arabidopsis in vitro regeneration, where a pluripotent cell mass termed callus is induced. We demonstrate that callus resembles the tip of a root meristem, even if it is derived from aerial organs such as petals, which clearly shows that callus formation is not a simple reprogramming process backwards to an undifferentiated state as widely believed. Furthermore, callus formation in roots, cotyledons and petals is blocked in mutant plants incapable of lateral root initiation. It thus appears that the ectopic activation of a lateral root development program is a common mechanism in callus formation from multiple organs.

overall_design: Four sets of biologically independent tissue samples were collect for root, cotyledon and petal explants just after being excised from plants (d0) or after ten days on callus-inducing medium (CIM, d10). Samples derived from the same organ were co-hybridized in the array experiments. Dyes used for labeling the RNA populations derived from the individual samples were switched in the replicate experiments to reduce dye-related artefacts.

first: Kaoru

last: Sugimoto

first: Elliot

last: Meyerowitz


title: Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing

status: Public on Nov 21 2009

city: Boston

first: Deborah

middle: R.

last: Yoder-Himes

address: 77 Louis Pasteur Ave, 1038 NRB

department: Microbiology and Molecular Genetics

institute: Harvard Medical School

summary: Transcriptional profiling of RNA-seq data from two Burkholderia species grown under conditions mimicking the cystic fibrosis lung and the soil environment

overall_design: Two condition, two strain experiment. Biological replicates: 2 replicates

first: D

first: P

last: Chain

first: Y

last: Zhu

first: O

last: Wurtzel

first: E

middle: M

last: Rubin

first: J

last: Tiedje

first: R

last: Sorek


title: Characterization of Drosophila mitochondrial transcripts through high throughput EST data

status: Public on Dec 09 2009

city: Campinas

first: Tatiana

middle: Teixeira

last: Torres

state: SP

address: Rua Candido Rondon, 400

department: CBMEG

country: Brazil

institute: Unicamp

summary:
We used high throughput EST sequences to characterize and generate expression profiles for all transcripts encoded in the mitochondrial genome of D. melanogaster and D. pseudoobscura. Mapping of ESTs confirmed previously predicted transcripts and revealed non-canonical transcript structures. Expression of mitochondrial transcripts was not different between the two species, but it was significantly different for males and females. We also detected difference in expression levels among mRNAs from genes located in the same transcriptional unit and among genes interacting in the same protein complexes, NADH dehydrogenase and cytochrome c oxidase complexes, suggesting a complex post-transcriptional regulation to maintain mitochondrial function.

Keywords: Transcriptome Analysis

sample_organism:
Drosophila melanogaster
Drosophila pseudoobscura

overall_design:
Four samples analysed: D. melanogaster males, D. melanogaster females, D. pseuoobscura males, D. pseudoobscura females
The number of reads that mapped to each transcript is proportional to the transcript abundance, so it can be used as a measure of the absolute level of expression of that transcript. The gene expression measure as the absolute EST counts and the expression relative to the library size for each mitochondrial gene in each library is displayed in the supplementary file linked below.

first: Christian

last: Schlotterer


title: Sequence and expression analysis of microRNAs during ovule and fiber development in cotton

status: Public on Nov 12 2009

city: South San Francisco

first: Vikram

last: Agarwal

address: 1130 Veterans Blvd

institute: Calico Life Sciences

summary: Cotton fibers are seed trichomes, and their development undergoes a series of rapid and dynamic changes from fiber cell initiation, elongation to primary and secondary wall biosynthesis and fiber maturation. Previous studies showed that cotton homologues encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development.  Many of these factors are targets of microRNAs (miRNAs). miRNAs are ~21 nucleotide (nt) RNA molecules derived from non-coding endogenous genes and mediate target regulation by mRNA degradation or translational repression. Here we show that among ~4-million reads of small RNAs derived from the fiber and non-fiber tissues, the 24-nt small RNAs were most abundant and were highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 28 putative miRNAs families, including 25 conserved and 3 novel miRNAs were identified in at least one of the cotton tissues examined. Thirty-two pre-miRNA hairpins representing 19 unique families were detected in Cotton Gene Indices version 9 (CGI9) using mirCheck. Sequencing, miRNA microarray, and small RNA blot analyses showed that many of these miRNAs differentially accumulated during ovule and fiber development.  The cotton miRNAs examined triggered target cleavage in the same predicted sites of the cotton targets in ovules and fibers as that of the orthologous target genes in Arabidopsis.  Targets of the potential new cotton miRNAs matched the previously characterized ESTs derived from cotton ovules and fibers.  The miRNA targets including those encoding auxin response factors were differentially expressed during fiber development.  We suggest that both conserved and new miRNAs play an important role in the rapid and dynamic process of fiber and ovule development in cotton.

overall_design: Clone and sequence small RNAs from immature ovules fiber-bearing ovules and leaves.

first: Mingxiong

last: Pang

first: Andrew

middle: W

last: Woodward

first: Xueying

last: Guan

first: Misook

last: Ha

first: Vanitharani

last: Ramachandran

first: Xuemei

last: Chen

first: Barbara

last: Triplett

first: David

last: Stelly

first: Z


title: Gene expression effects of morpholino directed spRunt-1 knockdown on hatched blastula stage embryos

status: Public on Jan 07 2010

city: Salisbury Cove

first: Anthony

middle: James

last: Robertson

state: ME

address: Box 35 Old Bar Harbor Road

department: Functional Genomics

institute: Mount Desert Island Biolabs

summary: Runt domain (Runx) transcription factors constitute a metazoan family of sequence specific DNA binding proteins that are essential for animal development.  The sea urchin Runx gene SpRunt-1, which is expressed globally during early embryogenesis, is required for blastula stage cell proliferation and subsequently for cell survival.   To obtain a comprehensive list of SpRunt-1 regulatory targets we screened a  custom microarray representing the blastula stage transcriptome of Strongylocentrotus purpuratus, comparing relative hybridization signals  generated by labeled RNA from 18 hr control versus SpRunt-1 morphant  embryos.

overall_design:
For an 8x15k chip the 8 arrays were 4 biological replicates
and a dye swap of the same 4.

Biological Replicates -

Four separate embryo cultures, each from a different mating
pair were initiated and divided into a control group and
a treatment group.  All of the embryos in the treatment group
were micoinjected with a splice-blocking morpholino
antisense oligonucleotide (MASO) targeted to SpRunt-1.

All control and sample embryos were developed to hatched blastula
stage (18 hours post-fertilization) in filtered seawater at 15°C.
Total RNA was extracted from each of the four separate samples
(3,600 embryos in each sample) with an RNA yield near 5 micro grams
per sample.  Total RNA was also extracted from the untreated embryos
of each separate culture pooled together (about 14,400) with an RNA
yield near 20 micro grams.

Technical (dye swap) Replicates -

Control derived Cy3 (green) labeled aRNA was mixed with sample derived
Cy5 (red) labeled aRNA in one biological replicate set of 4 samples and
using half of the extracted RNA amount.  The other half of the RNA was
reciprocally labeled with control derived aRNA Cy5 labeled and sample
derived aRNA Cy3 labeled.

middle: J

first: Benjamin

last: King

first: Alison

last: Coluccio

first: James

last: Coffman


title: High throughput sequencing of RNAPII-Ser5P ChIP DNA

status: Public on Nov 04 2009

city: Singapore

first: Chia-Lin

last: Wei

address: 60 Biopolis Street #02-01

department: Genome Technology & Biology

country: Singapore

institute: Genome Institute of Singapore

summary: The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, suggesting that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hotspots optimized for efficient and coordinated transcriptional control.

overall_design: Wild type Mus musculus RNAPII-Ser5P ChIP DNA

first: Jennifer

last: Mitchell

first: Peter

last: Fraser


title: Limitations and possibilities of small RNA digital gene expression profiling: spleen and liver comparison (SOLiD)

status: Public on Jul 29 2009

first: Elzo

last: de Wit

address: Uppsalalaan 8

institute: Hubrecht Institute

summary:
Analysis of RNA samples by massive parallel sequencing holds the promise to assay gene expression in both a quantitative and qualitative fashion and therefore allows for digital gene expression (DGE) profiling. We assessed the effect of different experimental approaches by generating small RNA libraries from a biological sample as well as an equimolar pool of synthetic miRNAs and analyzed the results using capillary dideoxy sequencing and next-generation sequencing platforms (Roche/454, AB/SOLiD and Illumina/Solexa). Whereas different sequencing platforms provided highly similar results, large differences in DGE profiles were observed depending on the library preparation method used. Nevertheless, our results indicate that the preferential nature of the library preparation methods is systematic and highly reproducible and we show that DGE is well suited for the quantification of relative expression differences between samples.

Keywords: Transcriptome analysis

overall_design: Examination of miRNA expression in BN-Lx rat spleen and liver

first: Sam

last: Linsen

first: Edwin

last: Cuppen


title: Structural and Functional Analysis of Viral siRNAs using 454 sequencing

status: Public on Apr 12 2010

city: Norwich

first: Simon

last: Moxon

state: Norfolk

address: University of East Anglia

country: United Kingdom

institute: University of East Anglia

summary:
We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation-based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs were the same in different plant species and in the absence of RDR6. We used the TerminatorTM 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were not complementary to highly abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs.

Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double-stranded RNA and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double-stranded RNA or by RNA-dependent RNA polymerase.

sample_organism:
Nicotiana benthamiana
Cymbidium ringspot virus

overall_design: Size-fractionated (19-24 nt) small RNA from early systemic leaves of CymRSV-infected Nicotiana benthamiana total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification, the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.

first: Gyorgy

last: Szittya

first: Vitantonio

last: Pantaleo

first: Gabor

last: Toth

first: Rachel

last: Rusholme-Pilcher

first: Jozsef

last: Burgyan

first: Tamas

last: Dalmay


title: Limitations and possibilities of small RNA digital gene expression profiling: library preparation comparison (SOLiD)

overall_design: Examination of three different library preparation methods for small RNAs, two replicates per library method


title: Limitations and possibilities of small RNA digital gene expression profiling: library preparation comparison (454)


title: Ath_Agilent_v1_Riechmann array expression profile AP1-GR ap1cal inflorescences - time series (hours)

status: Public on Apr 02 2010

city: Bellaterra

first: Jose Luis

last: Riechmann

address: Campus UAB - Edifici CRAG

institute: Center for Research in Agricultural Genomics

summary:
This experiment describes gene expression after the activation of APETALA1-GR, to study and identify AP1 target genes. We used a 35S:AP1-GR ap1 cal line to induce a synchronized response activating the AP1-GR fusion protein in ap1 cal inflorescence-like meristems through dexamethasone treatment. Tissue samples were collected immediately after the treatment, as well as subsequent timepoints. The expression profiles of the individual samples were then analyzed by gene expression profiling using whole-genome oligonucleotide arrays (Agilent, custom-commercial).
Keywords: time course

overall_design:
Four sets of biologically independent tissue samples were collected at 0, 2, 4, 8, and 12 hours after the application of dexamethasone (Dex-; activation of the AP1-GR fusion protein) or a mock solution (Mock-; control). Dex- and Mock-derived samples for each timepoint and biological replicate were co-hybridized; one sample was labeled with Cy3 and the other with Cy5. The dyes used for labeling RNA from a given treatment type (Dex and Mock, at a given timepoint) were switched for two of the replicate experiments, to reduce dye-related artifacts. This experimental setup resulted in a total of 5 hybridizations per set (0h, 2h, 4, 8h, and 12h; Dex vs. Mock at each timepoint), and an even number (2) of hybridizations of each dye polarity per timepoint.

The combined ratio data results are available as a supplementary file on the Series record.

first: Jose

middle: L

first: Vijaya

middle: S

last: Kumar

first: Tara

last: Mastro

first: Thilia

last: Ferrier

first: Frank

last: Wellmer


title: Orchestration of floral initiation by APETALA1

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

overall_design: Refer to individual Series


title: Drosophila Ecdysone Receptor Study

status: Public on May 01 2008

first: Zareen

last: Gauhar

address: 333 Cedar St, 386 NSB

department: Genetics

institute: Yale

type: Genome binding/occupancy profiling by genome tiling array

summary:
We used the DamID method to systematically identify the binding sites of Ecdysone Receptor and its heterodimeric partner USP across the whole genome in Drosophila Kc cells. We find that the EcR sites are a subset of the USP sites and that only a proportion are ecdysone regulated from an accompanying ecdysone profiling study. The role of EcR/USP in the ecdysone network appears to be coordinated by the recruitment of many transcription factors as well as signaling molecules.
Keywords: DamID, chromatin profiling, DNA microarray

overall_design:
In this study we mapped the genomic binding sites of steroid hormone nuclear receptor EcR and USP in Kc167 cells using the DamID method.
DamID involves the low level expression of a fusion protein consisting of DNA adenine methyltransferase (Dam) and a chromatin protein of interest. This fusion protein is targeted to the native binding sites of the chromatin protein, where Dam methylates adenines in the surrounding DNA. The methylated DNA fragments were isolated and amplified by selective PCR, labeled with a fluorescent dye and hybridized to whole genome tiling microarrays.
In this study,experiments were done with samples obtained from independent experiments and include dye swaps.

first: Ling

middle: V

last: Sun

first: Sujun

last: Hua

first: Christopher

last: Mason

first: Victor

last: Stolc

first: Kevin

middle: P

last: White


title: mRNA-Seq Whole Transcriptome Analysis of a Single Cell

status: Public on Feb 23 2009

first: Catalin

last: Barbacioru

summary: Here we modified a single cell whole transcriptome amplification method to make it capable of amplifying cDNAs as long as 3kb efficiently and unbiasedly. We combined this modified single cell cDNA amplification method with Applied Biosystems next generation sequencing SOLiD™ System to set up a single cell whole transcriptome assay.  The modified amplification strategy allows us to amplify full-length cDNAs for most of the expressed genes. We show that it is feasible to get digital gene expression profiles at single cell resolution. This allows us to ask fundamental biological questions that could not be addressed previously, especially in the early embryonic development field, and to understand transcriptome complexity at the resolution of a single cell.

overall_design: gene expression profiling from two single wild-type oocytes, two single Dicer knockout oocyte, and one single Ago2 knockout oocyte

first: Fuchou

last: Tang

first: Kainqin

last: Lao

first: M

last: Surani


title: Relapse versus diagnostic acute myeloid leukaemia samples analysed using Affymetrix 10K SNP array

city: London

first: Manoj

last: Raghavan

address: Charterhouse Square

department: Centre for Medical Oncology, Institute of Cancer

institute: Barts and the London, Queen Mary's School of Medicine and Dentistry

type:
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary:
Relapse is the commonest cause of death in acute myeloid leukaemia (AML), but the mechanisms leading to relapse are unclear. Recently, acquisition of segmental uniparental disomy (UPD) by mitotic recombination (MR) has been reported in 15-20% of AML samples at diagnosis using whole genome single nucleotide polymorphism (SNP) arrays. These cytogenetically invisible abnormalities are associated with homozygous mutations in several types of malignancy. Clonal evolution of heterozygous to homozygous mutations by MR could provide a mechanism for relapse.
Keywords: DNA copy number, loss of heterozygosity

overall_design: DNA from 27 pairs of diagnostic and relapsed AML samples were analysed using Affymetrix 10K SNP arrays. The genotype data of relapsed AML were compared with the data from the corresponding presentation AML.

first: Lan-Lan

last: Smith

first: Debra

last: Lillington

first: Tracy

last: Chaplin

first: Ioannis

last: Kakkas

first: Gael

last: Molloy

first: Claude

last: Chelala

first: Jude

last: Fitzgibbon

first: T

last: Lister

first: Bryan

middle: D

last: Young

first: James

last: Cavenagh

first: Jean-Baptiste

last: Cazier


title: Maize gene expression during infection with Ustilago maydis

status: Public on Mar 01 2008

first: Jörg

last: Kämper

summary:
The fungal pathogen Ustilago maydis establishes a biotrophic relationship with its host plant maize. Hallmarks of the disease are large plant tumors in which fungal proliferation occurs. Plants have developed various defense pathways to cope with pathogens. We used microarrays to detail the global programme of gene expression during the infection process of Ustilago maydis in its host plant to get insights into the defense programs and the metabolic reprogramming needed to supply the fungus with nutrients.
Keywords: time course

overall_design: In three independent experiments plants were infected with the solopathogenic U. maydis  strain SG200. Samples from infected leaves were taken at 12 and 24 hours post infection, as well as 2, 4 and 8 days post infection. Samples from uninfected control plants were taken at the same time points.

first: Joerg

last: Kaemper

first: Ramon

last: Wahl

first: Gunther

last: Doehlemann


status: Public on Sep 01 2009

city: Waltham

first: Nelson

middle: C

last: Lau

address: 415 South St MS029

institute: Brandeis University

summary: Various small RNA libraries from purified microtubules or Xiwi immunoprecipitates or total extract from X.tropicalis or X.laevis egg extract.   Various small RNA libraries from single X.tropicalis eggs. Analysis of this series of files is described in the manuscript "Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi".

sample_organism:
Xenopus laevis
Xenopus tropicalis

platform_organism:
Xenopus laevis
Xenopus tropicalis

overall_design: Small RNAs were ligated with linkers and converted to cDNA by reverse transcription.  cDNA library was amplified by PCR and was sequenced with either the 454 Genome Sequencer FLX platform or the Illumina GA-II platform.

first: Toshihiro

last: Ohsumi

first: Mark

last: Borowsky

first: Robert

middle: E

last: Kingston

first: Michael

last: Blower


title: Limitations and possibilities of small RNA digital gene expression profiling: synthetic miRNAs replicates (SOLiD)

first: Elzo

last: de Wit

overall_design: Examination library method on preparation of equimolar pool of miRNAs, three replicates

first: Sam

last: Linsen

first: Edwin

last: Cuppen


title: Late-term turkey embryo liver gene expression profiling

status: Public on Jan 31 2008

city: Raleigh

first: Jean

middle: Eduardo

last: de Oliveira

state: NC

address: 2711 Founders Dr

department: Poultry Science

institute: North Carolina State University

summary:
Turkey embryos are very sensitive to perturbations in energy metabolism because they have a wider hatching window than chicken embryos. Mortality of turkey embryos during late-term incubation is high relative to chickens, and many surviving hatchlings have compromised vitality. Intestinal maturation at hatch is also crucial to survival and post-hatch performance. The study of poultry embryo metabolism during the last stages of incubation is difficult due to many shifts and changes that occur in preparation for hatching. Microarray technology is suitable to study complex biological systems like avian late-term embryonic development. Therefore, the objectives of this study were to create a customized focused oligonucleotide microarray based on chicken genome sequences that could be used to study late-term avian metabolism and intestinal maturation, and use this array to survey turkey embryos gene expression from 20 days of incubation until hatch. The key features of this microarray are that all genes present have been annotated and gene spot replication (4) within each array chip. Microarray analysis was performed on liver, pectoral muscle, hatching muscle, and duodenum
Keywords: time course, embryo development

overall_design: Pooled samples from 6 embryos were arraged on a complete interwoven loop design where each treatment (embryonic ages 20, 22, 24, 26 and 28) was replicated 4 times (2 dye-swaps).

first: Chistopher

last: Ashwell

first: Peter

middle: R

last: Ferket

first: Zehava

last: Uni