title: RNA Capture-Seq resolves the deep complexity of the human transcriptome (454)

summary: Transcriptomic analyses have revealed an unexpected complexity of the human transcriptome, whose breadth and depth exceeds current RNA sequencing capacity 1-3. Here we combine tiling arrays and RNA sequencing technologies, in an approach we term RNA Capture-Seq, to enable the assembly and characterisation of transcripts whose expression level is below the detection limits of conventional sequencing approaches. By this technique we identify novel exons and splicing patterns to even well-studied genes, such as the p53 and Hox loci, and expose widespread, regulated and remarkably complex noncoding transcription in intergenic regions. We show that unassigned tags observed in conventional RNA sequencing datasets are not noise but can derive from rare transcripts fully assembled by RNA Capture-Seq. We expect RNA Capture-Seq to prove an invaluable technique for future research into gene expression and its relationship to phenotypic variation.

overall_design:
Application of RNA Capture sequencing from human fibroblasts for identifying rare novel transcripts.
Hybridization enhancing oligos are designed to cover the full sequencing adaptor and MID sequences during hybridisation.
Enhancing Oligo A 5   CCATCTCATCCCTGCGTGTCTCCGACTCAG/3ddc/
Enhancing Oligo B 5'   CCTATCCCCTGTGTGCCTTGGCAGTCTCAG/3ddc/

first: John

last: Rinn


title: RNA Capture-Seq resolves the deep complexity of the human transcriptome (Illumina)

status: Public on Sep 12 2011

city: Sydney

first: Tim

middle: R.

last: Mercer

state: New South Wales

address: 384 Victoria Street Darlinghurst NSW 2010

country: Australia

institute: Garvan

type: Expression profiling by high throughput sequencing

summary: Transcriptomic analyses have revealed an unexpected complexity of the human transcriptome, whose breadth and depth exceeds current RNA sequencing capacity 1-3. Here we combine tiling arrays and RNA sequencing technologies, in an approach we term RNA Capture-Seq, to enable the assembly and characterisation of transcripts whose expression level is below the detection limits of conventional sequencing approaches. By this technique we identify novel exons and splicing patterns to even well-studied genes, such as the p53 and Hox loci, and expose widespread, regulated and remarkably complex noncoding transcription in intergenic gene deserts. We show that unassigned tags observed in conventional RNA sequencing datasets are not noise but can derive from rare transcripts fully assembled by RNA Capture-Seq. We expect RNA Capture-Seq to prove an invaluable technique for future research into gene expression and its relationship to phenotypic variation.

middle: R

middle: L


title: Chromatin occupancy of TCF7L2 in hepatocytes

city: San Antonio

first: Luke

last: Norton

address: 7703 Floyd Curl Dr

country: USA

institute: UTHSCSA

type: Genome binding/occupancy profiling by high throughput sequencing

summary: TCF7L2 is one of the strongest type 2 diabetes (T2DM) candidate genes to emerge from GWAS studies, but the mechanisms by which it regulates the pathways which are important in the pathogenesis of type 2 diabetes are unknown. Previous in vitro and in vivo studies have focused on the link between TCF7L2 and insulin secretion as an explanation for the association between TCF7L2 and T2DM. However, TCF7L2 and the Wnt/β-catenin pathway are important for metabolic zonation in the liver. This raises the interesting possibility that TCF7L2 may influence glucose homeostasis by regulating hepatic glucose production (HGP). To examine this question, we utilized the H4IIE cell as a model of HGP.  Inhibition of HGP in H4IIE cells from lactate and pyruvate was highly sensitive to physiological concentrations of insulin and metformin. Silencing of TCF7L2 protein expression induced a 5-fold increase in basal HGP (P<0.0001), and this was accompanied by marked increase in the expression of several key gluconeogenic genes. FBPase, PEPCK and G6Pase mRNA were up-regulated 2.5-fold (P<0.0001), 1.4-fold (P<0.01) and 2.3-fold (P<0.0001), respectively, compared to scramble siRNA. Compared to their respective baseline values, insulin and metformin suppressed HGP equally in the scramble and TCF7L2 siRNA cells, but HGP remained elevated in TCF7L2 silenced cells due to the increased baseline HGP. Using chromatin immunoprecipitation sequencing (ChIP-Seq), we investigated the direct transcriptional targets of TCF7L2 in hepatocytes. A total of 2119 ChIP peaks were detected, of which 36% were located inside gene boundaries and, overall, a total of 65% of all binding events were within 50 Kb of a gene. De novo motif analysis revealed remarkable conservation of the long and short TCF7L2 consensus binding sites in the rat hepatocytes. Pathway analysis showed that the top two disease categories over-represented in our dataset were “non-insulin dependent diabetes” (155 genes; P = 1.63 x 10-10) and “diabetes mellitus” (245 genes; P = 7.4 x 10-12). Inspection of genes in these categories revealed that TCF7L2 directly binds to multiple genes important in the regulation of glucose metabolism in the liver, including PEPCK, FBP1, IRS1, IRS2, AKT2 ADIPOR1, PDK4 and CPT1A. Our findings suggest a novel mechanism for the regulation of HGP by TCF7L2, and provide a possible explanation for the association of TCF7L2 polymorphisms with the incidence of T2DM.

overall_design: two samples: TCF7L2 ChIP-Seq and Input DNA


title: Transcription profile analysis of S. cerevisiae strains

status: Public on Sep 10 2011

city: Hangzhou

first: Daoqiong

last: Zheng

state: Zhejiang

address: 866 Yuhangtang Road

department: Institute of Microbiology

country: China

institute: College of Life Sciences, Zhejiang University

summary: We report the application of single-molecule-based sequencing technology for transcription profile analysis of S. cerevisiae strains with different genetic background. By combining the whole genome sequence of these strains, we sought to explore the effects of genome mutations on the transcription diversities.

overall_design: Comparsion of transcription profiles in S. cerevisiae strains

first: Xuechang

last: Wu


title: Phyloepigenetic comparison of apes

status: Public on Sep 09 2011

city: Los Angeles

first: Dario

last: Boffelli

state: CA

address: 700 N San Vicente Blvd

department: Pediatrics

institute: Cedars Sinai

type: Methylation profiling by high throughput sequencing

summary: We have determined methylation state differences in the epigenomes of neutrophils purified from human and chimpanzee. We used deep sequencing of ends generated by digestion with a methylation-sensitive restriction enzyme, followed by analysis with the MetMap computational pipeline to infer methylation states from the sequencing data. Using the orangutan as an outgroup, analysis of DNA sequence substitutions in CG-dense regions that are either methylated or unmethylated in all three species indicates that methylation states in the neutrophil reflect methylation states in the germline. Differences in methylation states were not correlated with differences in the local genomic sequences, indicating that they can be determined independently of local DNA sequence. Methylation differences were not distributed randomly among the individuals we analyzed, but recapitulated the known phylogenetic relationships of the three species in a pattern consistent with their stable inheritance. This data provide the first comprehensive dataset indicating that epigenetic states are maintained as independent characters that are predictably transmitted within species. Heritable epigenetic differences such as those we have identified could readily have functional and adaptive consequences, and contribute to the phenotypic divergence of human and chimpanzee.

sample_organism:
Pan troglodytes
Pongo pygmaeus
Homo sapiens

overall_design: Comparison of methylation states in a single, uncultered cell type from human and chimpanzee


title: Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function [ChIP-Seq]

status: Public on Sep 08 2011

city: Brookline

first: Harrison

middle: Wren

last: Gabel

state: MA

address: 7 Linden St Apt 3

department: Neurobiology

institute: Harvard Medical School

summary:
Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes; rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitateÂ a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.
To examine MeCP2 binding across the neuronal genome and where on the genome MeCP2 is phosphorylated at Serine 421 in response to neuronal activity we performed anti-total MeCP2 and anti-phospho-Serine 421 specific Chromatin immunoprecipitation from cultured cortical neurons that were either left unstimulated or membrane depolarized for 2 hours by addition of 55mM KCl to the media. ChIP DNA was verified for successful IP by qPCR then cloned and sequenced using ABI SOLiD system 4.

overall_design: ChIP was performed from E16 +7DIV  disociated cortical cultures from one or two independent dissections using an anti-c-terminal antiserum recognizing MeCP2 independent of its phosphorylation state or with an anti-pS421 antiserum that specifically immunoprecipitates MeCP2 phosphorylated at serine 421. Samples were qPCR validated and sequenced using ABI SOLiD system 4 library preparation and sequencing.

first: Sonia

last: Cohen

first: Martin

last: Hemberg

first: Ashley

middle: N

last: Hutchinson

first: L

middle: A

last: Sadacca

first: Daniel

middle: H

last: Ebert

first: David

last: Harmin

first: Rachel

middle: S

last: Greenberg

first: Zhaolan

last: Zhou

first: William

middle: C

last: Wetsel

first: Anne

middle: E

last: West

middle: W

first: Michael


title: piRNA production requires heterochromatin formation in Drosophila

city: New York

first: Prashanth

last: Rangan

state: New York

address: 540 1st Aveune

department: Developmental genetics

institute: NYU/HHMI

summary: Here, we analyzed H3K9me3 derived from wild type ovarian tissue. Here we show that deposition of histone 3 lysine 9 by the methyltransferase dSETDB1 (egg) is required for piRNA cluster transcription. In the absence of dSETDB1, cluster precursor transcription collapses in germline and somatic gonadal cells and TEs are activated, resulting in germline loss and a block in germline stem cell differentiation. We propose that heterochromatin protects the germline by activating the piRNA pathway.

overall_design: Examination of H3K9me3 marks in the wild type gonad

first: Ruth

last: Lehmann


title: SAGE-Seq gene expression profiles of Hs578T, MCF7, and SUM159PT cells treated with STAT3 or non-targeting siRNAs, DMSO, or CXCR2, PTGIS, HAS1, PFKFB3, JAK, or NQO1 inhibitor

city: Boston

first: Kornelia

last: Polyak

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: To investigate potential links between Stat3 transcriptional activity and other signaling pathways in breast cancer, we determined the gene expression profiles of three breast cancer cell lines treated with JAK, PTGIS, PFKFB3, CXCR2, HAS1, or NQO1 inhibitor (all of which decreased Stat3 transcriptional activity in Hs 578T cells except for the NQO1 inhibitor), inhibitor treatment vehicle alone (DMSO), STAT3 siRNAs, or non-targeting siRNAs. Using the resulting data, we identified a gene signature that was significantly regulated by STAT3 siRNAs and similarly affected by the JAK and at least 3 other inhibitors (or by 4 other inhibitors) but not by the NQO1 inhibitor in Hs 578T cells that was enriched in genes involved in development and correlated with shorter distant metastasis-free survival in primary lymph-node-negative invasive breast tumors. These results emphasize the central importance of Stat3 in CD44+/CD24- stem-cell-like breast cancer cells.

overall_design: This study includes SAGE-Seq libraries obtained using 27 samples that each underwent individual protocols. There are nine samples of each of three cell lines - Hs 578T, MCF7, and SUM159PT. The samples in each group were treated with either one of the six inhibitors, DMSO only (the background control for inhibitor-treated cells), STAT3 siRNAs, or non-targeting siRNAs (the background control for STAT3 siRNAs).

first: Lauren

last: Marotta

first: Sibgat

last: Choudhury

first: SungJin

last: Huh


title: Transcriptome-Wide Binding Sites for Components of the Saccharomyces cerevisiae Non-poly(A) termination Pathway: Nrd1, Nab3 and Sen1

city: Baltimore

first: Jeff

last: Corden

state: MD

address: 725 N Wolfe Street

department: Molecular Bio and Gen

institute: JHU

summary: We report here the transcriptome-wide distribution of yeast Rpb2, Sen1, Nrd1 and Nab3 binding sites. These data sets provide highresolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3'-antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link to many expected ncRNAs but surprisingly binds to pre-mRNA transcripts suggesting a role in 3' end formation and/or termination.

overall_design: Six samples by adaptation of PAR-ClIP procedure

first: Nuttara

last: Jamonnak

first: Tyler

last: Creamer

first: Miranda

last: Darby

first: Paul

last: Schaughency

first: Haiping

last: Hao

first: Sarah

last: Wheelan

first: Jeffry


title: Effect of splicing on histone H3 K36 methylation

status: Public on Sep 05 2011

city: Aurora

first: David

last: Bentley

state: CO

institute: UCHSC

summary: Histone H3 K36me3 and RNA pol II were localized by ChIP before and after inhibition of splicing by spliceostatin for 12 hr. Total matched reads for the control and SSA treated Hela samples were 11.6 and 8.2 million for anti-H3K36me3 and 6.6 and 5.0 million for anti-pol II.

overall_design: ChIP-seq of histone H3 K36me3 and RNA pol II in Hela cells before and after spliceosotatin treatment

first: Hyunmin

last: Kim


title: Transcriptome analysis and molecular marker discovery in red pepper, Capsicum annuum L. TF68

city: Yesan

first: Fu-Hao

last: Lu

address: 314, Plant Resources Department

country: South Korea

institute: Kongju National University

summary: Transcriptome from high throughput sequencing-by-synthesis is a good resource of molecular markers. In this study, we present utility of massively parallel sequencing by synthesis for profiling the transcriptome of red pepper (Capsicum annuum L. TF68) by 454 GS-FLX pyrosequencing. Through the generation of approximately 30.63 megabases (Mb) of Expressed Sequence Tags (ESTs) data with the average length of 375 base pairs (bp), 9,818 contigs and 23,712 singletons were obtained by assembly. Using BLAST alignment against NCBI non-redundant and a UniProt protein database, 30% of the tentative consensus sequences were assigned to specific function annotation, while 24% returned alignments of unknown function, leaving up to 46% with no alignment. Functional classification using FunCat revealed that sequences with putative known function were distributed cross 18 categories. Furthermore, over 200 high quality single nucleotide discrepancies were discovered using the Bukang cDNA collection as a reference database. Moreover, 758 simple sequence repeat (SSR) motif loci were mined from over 600 contigs, from which 572 primer sets were designed. The SSR motifs corresponded to di- and tri- nucleotide motifs (27.03 and 61.92%, respectively). These molecular markers may be of great value for application in linkage mapping and association mapping research.

overall_design: 1 sample TF68 accession examined

first: Myeong-Cheoul

last: Cho

first: Yong-Jin

last: Park


title: Effect of wheel running exercise on gene expression in skeletal muscles of mice

status: Public on Sep 03 2011

city: Rochester

first: Stephen

last: Welle

state: NY

address: 601 Elmwood Avenue

institute: University of Rochester

summary: This analysis was done to determine which muscle group has the most skeletal muscle transcriptional reponses to wheel running activity. Seven male mice with C57BL/6 genetic background were placed individually in cages with running wheels and allowed free access to the wheels 24 hr per day. Access to running wheels began when the mice were 6 months old, and continued for 12 weeks. The morning after the night of running, the mice were euthanized and several muscle groups were snap froze in liquid nitrogen to preserve RNA. The same muscle groups were collected from three age-matched "sedentary" mice that were housed in ordinary cages that did not permit sustained running. For each muscle group, separate pools of polyadenylated RNA from the sedentary and wheel-running mice were prepared for deep sequencing of cDNA with the SOLiD 3 platform.

overall_design: Four muscle groups were examined (quadriceps femoris; gastrocnemius/plantaris; tibialis anterior; triceps brachii). For each muscle, RNA was pooled from 3 sedentary or 7 wheel-running mice (8 pools total).


title: Cellular reprogramming by the conjoint action of ERalpha, FOXA1 and GATA3 to a ligand-inducible growth state

city: Singapore

first: Guoliang

last: Li

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

department: Computational and Systems Biology

country: Singapore

institute: Genome Institute of Singapore

summary: Despite the role of the estrogen receptor alpha (ERalpha) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERalpha into ERalpha-negative cells paradoxically has been growth inhibition. We map the binding profiles of ERalpha and its interacting transcription factors (TFs), FOXA1 and GATA3, in MCF-7 breast carcinoma cells. We observe that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERalpha alone. We demonstrate that these enhanceosome-occupied sites are associated with optimal enhancer characteristics with highest p300 coactivator recruitment, RNA Pol II occupancy, and chromatin opening. The enhancesome binding sites appear to regulate the genes driving core ERalpha function. Most importantly, we show that transfection of all three TFs was necessary to reprogram the ERalpha-negative MDA-MB-231 and BT-459 cells to restore the estrogen responsive growth and to transcriptionally resemble the estrogen-treated ERalpha-positive MCF-7 cells. Cumulatively, these results suggest that all of the enhanceosome components comprising ERalpha, FOXA1 and GATA3 are necessary for the full repertoire of the cancer-associated effects of the ERalpha.

overall_design: The analysis of ERalpha, FOXA1, and GATA3 in MCF-7 cancer cells was done by ChIP-seq data obtained either with estradiol (E2) stimulation or without stimulation using vehicle as a control. Using the ERalpha bindings defined by ChIP-seq (GSE23893), FOXA1 bindings (GSE26831), and GATA3 bindings (this Series), we analyzed the enhanceosome effect of the overlapped binding sites from ERalpha, FOXA1 and GATA3.

first: Say

last: Kong

first: Edison

middle: T

last: Liu


title: Genome-wide maps of TCF7L1 binding during early adipogenesis

status: Public on Sep 02 2011

city: Philadelphia

first: Jonathan

last: Schug

state: Pennsylvania

address: 12-156 Smilow Ctr Trans Research

department: School of Medicine

institute: University of Pennsylvania

summary: Here we show through genome-wide binding studies that transcription factor 7-like 1 (TCF7L1) represses structure-related genes during adipogenesis. Intriguingly, TCF7L1 is induced in a cell contact-dependent manner by confluency in preadipocytes and is required for adipocyte differentiation by repressing transcription of cell structure genes. TCF7L1 is also sufficient to bestow adipogenic potential upon non-adipogenic cells.  These results implicate TCF7L1 as a novel adipogenic competency factor that uniquely determines adipogenic fate through cell structure organization required for adipocyte gene activation.

overall_design: Examination of TCF7L1 binding in preadipocytes treated for 24 hours with adipogenic stimuli.

first: Ana

middle: G

last: Cristancho

first: Mitchell

last: Lazar


title: Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function

first: Harrison

last: Gabel

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

overall_design: Refer to individual Series


title: Effect of wheel running exercise and myostatin depletion on gene expression in triceps brachii muscles of mice

summary: The purpose of this study was to determine whether postdevelopmental myostatin depletion influenced the changes in skeletal muscle gene expression profiles induced by a long-term increase in physical activity. Myostatin levels in muscles of adult male mice with floxed myostatin genes were reduced ~85% by activating Cre recombinase. Control mice with normal myostatin genes had the same Cre-activating treatment. Some of the mice were housed in ordinary cages throughout the study, limiting their physical activity. Other mice were given free access to running wheels for the final 12 weeks of the study. At the end of the study, comprehensive gene expression profiles of triceps brachii muscles were determined by RNA sequencing (RNA-Seq), with muscles from mice selected for similarity of running behavior throughout the period of wheel access. Wheel running increased expression of hundreds of mRNAs encoding proteins involved in oxidative energy metabolism, and this response was not affected by myostatin deficiency. The running-induced increase in the ratio of Myh1 mRNA (which encodes myosin heavy chain type 2x) to Myh4 mRNA (which encodes myosin heavy chain type 2b) also was not affected by myostatin depletion. At every threshold of P (computed by analysis of variance), the number of transcripts with interactions between activity level and myostatin level was fewer than the number expected by chance. These data suggest that myostatin is not required for transcriptional adaptations to moderate-intensity exercise.

overall_design: 12 samples, 6 from sedentary mice and 6 from active (wheel running) mice. 3 control and 3 myostatin-deficient mice within each activity level.


title: Control of Embryonic Stem Cell Lineage Commitment by Core Promoter Factor, TAF3 (RNA-Seq data)

city: Berkeley

first: Devin

last: Scannell

address: 378 Stanley Hall #3220

institute: UC Berkeley

summary: We report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to chromosomal regions bound by CTCF and cohesin. Enrichment for TAF3/CTCF/cohesin bound regions distinguishes TAF3-activated from TAF3-repressed genes. Our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions to safeguard the finely-balanced transcriptional programs that give rise to pluripotency.

overall_design: Comparison of genome-wide expression patterns between TAF3-knockdown and WT embryonic stem cells using mRNA-Seq. Significantly differentially expressed protein-coding genes were identified by comparing control and knock-down samples at each timepoint (ES, embryoid body day 3 (EB3), EB6). Single and paired-end samples were combined at each timepoint, resulting in 3 tests for each gene (based on 8, 4, 4 independent measurements at ES ,EB3, EB6, respectively).

first: Robert

last: Tjian


title: Effects of wheel running exercise and myostatin depletion on skeletal muscle gene expression in mice


title: Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage [ChIP-Seq]

first: Wen-Hui

last: Lien

address: 1230 York Ave Box#300

department: Mammalian cell biology and development

institute: The Rockefeller Unoversity

summary: Mouse hair follicles undergo synchronized cycles. Cyclical regeneration and hair growth is fueled by stem cells (SCs). During the rest phase, the HF-SCs remain quiescent due to extrinsic inhibitory signals within the niche. As activating cues accumulate, HF-SCs become activated, proliferate, and grows downward to form transient-amplifying matrix progenitor cells. We used ChIP-seq to reveal the genome-wide maps of histone modifications underlying the states of hair follicle stem cells and their transient-amplifying progeny before differentiation.

overall_design: Quiescent hair follicle stem cells (qHF-SCs), activated hair follicle stem cells (aHF-SCs) and transient-amplifying matrix cells (HF-TACs) were FACS-purified for ChIP-sequcencing.

first: Elaine

last: Fuchs


title: Control of Embryonic Stem Cell Lineage Commitment by Core Promoter Factor, TAF3


title: Control of Embryonic Stem Cell Lineage Commitment by Core Promoter Factor, TAF3 (ChIP-Seq data)

overall_design: Genome-wide binding of TAF3, TAF1, TBP and PolII assayed by ChIP-Seq.


title: Targets of Ty3 transposition in Saccharomyces cerevisiae

city: Irvine

first: Suzanne

last: Sandmeyer

address: University

department: Biological Chemistry

institute: University of CA, Irvine

type: Genome variation profiling by high throughput sequencing

summary: The goal of these experiments was to define the targets of Ty3 transposition in Saccharomyces cerevisiae.  Ty3 is a retroviruslike element that is found at the transcription initiation site of chromosomal tRNA genes.

overall_design: A Ty3 that can be induced by growth in galactose-containing medium and which was marked by an insertion of HIS3 downstream of the second open reading frame of the element (POL3) was induced to undergo transposition by plating cells onto galactose containing medium and replica-plating onto medium selective for cells that had undergone transposition.  These cells were collected, DNA was extracted, and inverse PCR was performed using primers inside the Ty3 element in order to generate a library of insertion sites flanked by Illumina sequence-compatible primers.

first: Xiaojie

last: Qi

first: Kenneth

last: Daily

first: Kim

last: Nguyen

first: Haoyi

last: Wang

first: Sholeh

last: Forouzan

first: Paul

last: Rigor

first: Mark

last: Johnston

first: Rob

last: Mitra

first: Pierre

last: Baldi


title: ChimeraScan: A tool for identifying chimeric transcription in sequencing data

status: Public on Sep 01 2011

city: Ann Arbor

first: Matthew

last: Iyer

state: MI

address: 5316 CCGC 0940 1400 E. Medical Center Drive

department: Michigan Center for Translational Pathology

institute: University of Michigan

summary: Next Generation Sequencing technologies have enabled de novo gene fusion discovery that could reveal candidates with therapeutic significance in cancer. Here we present an open-source software package, ChimeraScan, for the discovery of chimeric transcription between two independent transcripts.

overall_design: Three cancer cell lines with known gene fusions

first: Arul

middle: M

last: Chinnaiyan

first: Christopher

last: Maher


title: Oryza glaberrima small RNA discovery and expression profiling

city: St. Louis

first: Blake

middle: C.

last: Meyers

state: MO

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

type: Non-coding RNA profiling by high throughput sequencing

summary: Examination of 3 tissue types in Oryza glabberima (accession CG14) by high throughput sequencing for small RNA discovery and expression profiling

overall_design: sRNA and mRNA from leaves, roots, and panicles

first: Stacey

last: Simon


title: Identification and analysis of microRNAs associated with drought tolerance in cowpea

city: Shanghai

first: Renyi

last: Liu

state: Shanghai

address: 3888 Chenhua Road

department: Bioinformatics

institute: Shanghai Center for Plant Stress Biology

summary: To identify novel microRNAs that are associated with drought tolerance in two different cowpea genotypes, we generated small RNA sequences from adult cowpea plants under control and dought stress treatments. Over 79 million raw reads were generated and numerous novel microRNAs are identified, including some associated with drought tolerance.

overall_design: Sequencing of small RNAs in two cowpea genotypes under control and drought stress conditions.

first: Blanca

last: Barrera-Figueroa

first: Jian-kang

last: Zhu


title: Digital gene expression profiling of the Phytophthora sojae transcriptome

city: Nanjing

first: Wenwu

last: Ye

address: Department of Plant Pathology, Nanjing Agricultural University

institute: Nanjing Agricultral University, China

summary: The transcriptome of the oomycete plant pathogen Phytophthora sojae was profiled at 10 different developmental and infection stages based on a 3'-tag digital gene expression (DGE) protocol. More than 90 million clean sequence tags were generated and compared to the P. sojae genome and its 19,027 predicted genes. A total of 14,969 genes were detected, of which 10,044 were deemed reliable because they mapped to unambiguous tags. A comparison of the whole-library genes expression patterns suggested four groups: 1) mycelia and zoosporangia (MY and SP); 2) zoospores and cysts (ZO and CY); 3) germinating cysts (GC); 4) five infection site libraries (IF1.5 to IF24h). The libraries from the different groups showed major transitional shifts in gene expression. From the ten libraries, 722 gene expression pattern clusters were obtained and the top 16 ones, containing more than half of the genes, comprised enriched genes with different functions including protein localization, triphosphate metabolism, signaling process, and non-coding RNA metabolism. An evaluation of the average expression level of 30 pathogenesis related gene families revealed that most were infection induced, but with diverse expression patterns and levels. A web-based server named the Phytophthora Transcriptional Database (PTD) has been established.

sample_organism:
Glycine max
Phytophthora sojae

platform_organism:
Glycine max
Phytophthora sojae

overall_design: The five axenically grown stages were mycelia (MY), zoosporangia (SP), zoospores (ZO), cysts (CY), and germinating cysts (GC). The five infection stages, 1.5, 3, 6, 12 and 24 h after inoculation onto susceptible soybean leaf tissues (IF1.5h to IF24h).

first: Xiaoli

first: Kai

last: Tao

first: Yuping

last: Lu

first: Tingting

last: Dai

first: Suomeng

last: Dong

first: Daolong

last: Dou

first: Gijzen

last: Mark

first: Yuanchao


title: Histone Modifications by ChIP-seq from ENCODE/Stanford/Yale/Davis/Harvard

status: Public on Aug 31 2011

city: Stanford

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Peggy Farnham mailto:pfarnham@usc.edu for questions concerning data collection and usage and Philip Cayting mailto:pcayting@stanford.edu for data scoring and submission inquiries). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).

This track, produced as part of the ENCODE Project, displays maps of histone modifications genome-wide using ChIP-seq in different cell lines. The ChIP-seq method involves first using formaldehyde to cross-link histones and other DNA-associated proteins to genomic DNA within cells. The cross-linked chromatin is subsequently extracted, sheared, and immunoprecipitated using specific antibodies. After reversal of cross-links, the immunoprecipitated DNA is sequenced and mapped to the human reference genome. The relative enrichment of each antibody-target (epitope) across the genome is inferred from the density of mapped fragments.
Chemical modifications (e.g. methylation or acetylation) of the histone proteins present in chromatin influence gene expression by changing how accessible the chromatin is to transcription factors. Shown for each experiment (defined as a particular antibody and a particular cell type) is a track of enrichment for the specifically modified histone (Signal), along with sites that have the greatest enrichment (Peaks). Also included for each cell type is the input signal, which represents the control condition where no antibody targeting was performed. In general the following chemical modifications have associated genetic phenotypes:
H3K4me3 and H3K9Ac are considered to be marks of active or potentially active promoter regions. H3K4me1 and H3K27Ac are considered to be marks of active or potentially active enhancer regions. H3K36me3 and H3K79me2 are considered to be marks of transcriptional elongation. H3K27me3 and H3K9me3 are considered to be marks of inactive regions.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design:
Cells were grown according to the approved ENCODE cell culture protocols. Briefly, cells were crosslinked, chromatin was extracted and sonicated using a Bioruptor sonicator (Diagenode) to an average size of 300-500bp, and individual ChIP assays were performed using antibodies to modified histones. For the K562 and Ntera2 histone ChIP-seq samples, immunoprecipitates were collected using protein G-coupled magnetic beads; a detailed ChIP and library protocol can be found at http://www.roadmapepigenomics.org/protocols. For the U2OS histone ChIP-seq samples, immunoprecipitates were collected using StaphA cells; a detailed protocol can be found at http://expression.genomecenter.ucdavis.edu/chip.html. Library DNA was quantitated using either a Nanodrop or a BioAnalyzer and sequenced on an Illumina GA2.
The sequencing reads were mapped to the genome using the Eland alignment program. ChIP-seq data was scored based on sequence reads (length ~30 bps) that align uniquely to the human genome. From the mapped tags, a signal map of ChIP DNA fragments (average fragment length ~ 200 bp) was constructed where the signal height is the number of overlapping fragments at each nucleotide position in the genome.
For each 1 Mb segment of each chromosome, a peak height threshold was determined by requiring a false discovery rate <= 0.05 when comparing the number of peaks above threshold as compared to the number obtained from multiple simulations of a random null background with the same number of mapped reads (also accounting for the fraction of mapable bases for sequence tags in that 1 Mb segment). The number of mapped tags in a putative binding region is compared to the normalized (normalized by correlating tag counts in genomic 10 kb windows) number of mapped tags in the same region from an input DNA control. Using a binomial test, only regions that have a p-value <= 0.05 are considered to be significantly enriched compared to the input DNA control.

first: Peggy

last: Farnham

first: Michael

last: Snyder

first: Philip

last: Cayting


title: Tdrd1 acts as a molecular scaffold for Piwi proteins and piRNA targets in zebrafish.

city: Groningen

first: Eugene

last: Berezikov

address: Antonius Deusinglaan 1

department: European Research Institute for the Biology of Ageing

country: Netherlands

institute: University Medical Center Groningen

summary: RNA libraries from immunoprecipitates of Tdrd1, Ziwi and Zili, total testis RNA, total RNA from 3 week old wild-type and tdrd1 mutant gonads.

overall_design: Both size selected and non-size selected libraries were made. Sequencing was performed using Illumina platform.

first: Hsin-Yi

last: Huang

first: Rene

middle: F

last: Ketting


title: SRA of transcriptome of Hevea brasiliensis

city: Beijing

first: lili

last: hao

address: Beichen west road

institute: BIG

summary: We first report the use of next-generation massively parallel sequencing technologies and de novo transcriptome assembly to gain insight into the wide range of transcriptome of Hevea brasiliensis. The output of sequenced data showed that more than 12 million sequence reads with average length of 90nt were generated. Totally 48,768 unigenes (mean size = 488 bp) were assembled through transcriptome de novo assembly, which represent more than 3-fold of all the sequences of Hevea brasiliensis deposited in the GenBank. Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. Total 37,373 unigenes were successfully annotated and more than 10% of unigenes were aligned to known proteins of Euphorbiaceae. The unigenes contain nearly complete collection of known rubber-synthesis-related genes. Our data provides the most comprehensive sequence resource available for study rubber tree and demonstrates the availability of Illumina sequencing and de novo transcriptome assembly in a species lacking genome information.

overall_design: The transcriptome of latex and leaf in Hevea brasiliensis

first: Zhihui

last: Xia

first: Xi

first: Chaozu

last: He

first: Huimin

last: Xu

first: Dejun

last: Li

first: Jinling

last: Zhai

first: Hongli

last: Luo


title: Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression

status: Public on Aug 29 2011

first: Matthew

last: Iyer


title: Multiple tissue RNA sequencing of two Arabidopsis thaliana MAGIC founder accessions

city: Salt Lake City

first: Richard

last: Clark

state: Utah

department: Department of Biology

institute: University of Utah

summary: We sequenced messenger RNA isolated from seeding, root, and floral bud tissue for 2 MAGIC founder accessions of Arabidopsis thaliana. The resulting data was used for validation of gene models, and provides a resource to assess tissue-specific expression.

overall_design: Examination of RNA expression across tissues (seedling, root, floral bud) for 2 accessions (Col-0, Can-0).


title: Integrative Annotation of Human Large Intergenic Non-Coding RNAs Reveals Global Properties and Specific Subclasses

status: Public on Aug 28 2011

city: Cambridge

first: Nataly

middle: Moran

last: Cabili

address: 7  Cambridge Center

institute: Broad Institute ;  Harvard University

summary: Large intergenic non-coding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes. Determining the function of individual lincRNAs remains a challenge. Recent advances in RNA sequencing (RNA-Seq) and computational methods allow for an unprecedented analysis of such transcripts. Here, we present an integrative approach to define a reference catalogue of over 8,000 human lincRNAs. Our catalogue unifies previously existing annotation sources with transcripts we assembled from RNA-Seq data collected from ~4 billion RNA-Seq reads across 24 tissues and cell types.  We characterize each lincRNA by a panorama of more than 30 properties, including sequence, structural, transcriptional, and orthology features. We find that lincRNA expression is strikingly tissue specific compared to coding genes, and that they are typically co-expressed with their neighboring genes, albeit to a similar extent to that of pairs of neighboring protein-coding genes. We distinguish an additional sub-set of transcripts that have high evolutionary conservation but may include short open reading frames, and may serve either as lincRNAs or as small peptides. Our integrated, comprehensive, yet conservative reference catalogue of human lincRNAs reveals the global properties of lincRNAs and will facilitate experimental studies and further functional classification of these genes.

overall_design: We extracted profiled the transcriptome expression polyadenylated mRNA-Seq.  We then used these to reconstruct the transcriptome using de-novo assemblers and identify long non coding RNAs and their expression.

first: Moran

first: Cole

last: Trapnell

first: Loyal

last: Goff

first: Barbara

last: Tazon-Vega

first: Magdalena

last: Koziol

first: Aviv

last: Regev

first: John

last: Rinn


title: Multiple reference genomes and transcriptomes for Arabidopsis thaliana


title: The tumor suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements

city: Vienna

first: Jonas

last: Steinmann

address: Dr. Bohr Gasse 3

country: Austria

institute: IMBA

summary: In Drosophila, defects in asymmetric cell division can result in the formation of stem cell derived tumors. Here, we reveal a different mechanism that can result in the formation of very similar terminal brain tumor phenotypes. We demonstrate that brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. We use ChIP-seq to identify L(3)mbt-binding sites and show that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting the insulator protein mod(mdg4) results in upregulation of SWH pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Our data reveal that very different primary defects can result in the formation of brain tumors, which behave quite similarly in their advanced stages.

overall_design: Examination of l(3)mbt binding in third instar larval brain and wing/haltere/third leg imaginal discs tissue.

first: Constance

last: Richter

first: Katarzyna

last: Oktaba

first: Juergen

last: Knoblich


title: RNA-Seq of Gonads and Carcasses in D. simulans and D. pseudoobscura

status: Public on Aug 26 2011

city: Bethesda

first: Brian

last: Oliver

address: 50 South Drive

department: LBG

institute: NIDDK, NIH

summary: We sequenced dissected ovaries and testes (with reproductive tracts) as well as female and male carcasses in two species of Drosophila in order to validate gene predictions from the ModENCODE project.

sample_organism:
Drosophila pseudoobscura
Drosophila simulans

overall_design: Comparison of dissected reproductive tracts and remaining carcasses between D. simulans and D. pseudoobscura

first: Carlo

last: Artieri

first: Nicolas

last: Mattiuzzo

last: Malone


title: Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology

institute: Harvard University

summary: New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2â²-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.

overall_design: We sequenced in-vitro transcribed RNaseP in wild type and barcoded variants and probed them with SHAPE-Seq.  Comparison to existing crystalography data and previous SHAPE experiments verified the accuracy of the technique and the absence of bias due to our multiplexiing strategy.

first: Julius

last: Lucks

first: Stefanie

last: Mortimer

first: Shujun

last: Luo

first: Sharon

last: Aviran

first: Gary

last: Schroth

first: Lior

last: Pachter

first: Jennifer

last: Doudna

first: Adam

last: Arkin


title: Genomic maps of lincRNA occupancy reveal principles of RNA-chromatin interactions.

status: Public on Sep 29 2011

first: Kun

last: Qu

address: 269 Campus Dr. CCSR 2150

department: Dermatology

institute: Stanford University

summary: Long intergenic noncoding RNAs (lincRNAs) are key regulators of chromatin state, yet the nature and sites of RNA-chromatin interaction are mostly unknown. Here we introduce Chromatin Isolation by RNA Purification (ChIRP), where tiling oligonucleotides retrieve specific lincRNAs and bound protein and DNA sequences, which are enumerated by deep sequencing. ChIRP-seq of two lincRNAs reveal that RNA binding sites in the genome are focal, sequence-specific, and numerous. Human telomerase RNA TERC occupies telomeres and Wnt pathway genes. HOTAIR lincRNA preferentially binds a GA-rich homopurine DNA motif to nucleate broad domains of Polycomb occupancy and histone H3 lysine 27 trimethylation. HOTAIR occupancy occurs independently of EZH2, defining the order of RNA guidance of Polycomb occupancy. ChIRP-seq is readily applicable to numerous RNAs in different cell types and biological states, thus enabling the study of RNA regulation of chromatin and gene expression at a genomic scale.

sample_organism:
Drosophila melanogaster
Homo sapiens

platform_organism:
Drosophila melanogaster
Homo sapiens

overall_design: Examination of 3 lincRNAs in 5 cell types

first: Ci

last: Chu

first: Howard

last: Chang


title: Multilineage Priming of Enhancer Repertoires Precedes Commitment to the B and Myeloid Cell Lineages in Hematopoietic Progenitors

city: La Jolla

first: Yin

middle: Chun

last: Lin

address: 9500 Gilman Drive

department: Division of Biological Sciences

institute: UCSD

summary:
Recent studies have documented genome-wide binding patterns of transcriptional regulators and their associated epigenetic marks in hematopoietic cell lineages. In order to determine how epigenetic marks are established and maintained during developmental progression, we have generated long-term cultures of hematopoietic progenitors by enforcing the expression of the E-protein antagonist Id2. Hematopoietic progenitors that express Id2 are multipotent and readily differentiate upon withdrawal of Id2 expression into committed B lineage cells, thus indicating a causative role for E2A (Tcf3) in promoting the B cell fate. Genome-wide analyses revealed that a substantial fraction of lymphoid and myeloid enhancers are premarked by the poised or active enhancer mark H3K4me1 in multipotent progenitors. Thus, in hematopoietic progenitors, multilineage priming of enhancer elements precedes commitment to the lymphoid or myeloid cell lineages.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Genome-wide maps of chromatin state in early erythroid precursors versus later, more differentiated erythroblasts.

status: Public on Aug 24 2011

first: Bill

last: Wong

address: 9 Cambridge Center

institute: Whitehead Institute

summary: Here we globally  analyzed mRNA and epigenetic changes in both early erythroid progenitors and late erythroblasts. Concomitant with gene induction there was an increase in RNA Pol II binding and activation marks near the transcriptional start site (TSS) and the elongation mark H3K79me2 (but not H3K36me3),both near the TSS and along the full gene length. In contrast, most repressed genes became depleted of elongation marks. We also found that relative changes in histone modification levels--in particular, H3K79me2 and H4K16ac-- most predictive of gene expression patterns. Our data suggests that in terminal erythropoiesis both transcriptional initiation and elongation contribute to induction of erythroid genes; in contrast, gene repression is mainly mediated by inhibition of Pol II elongation. Our data provides a comprehensive map of the epigenome during terminal erythroid differentiation.

overall_design: Examination of 7 different histone modifications and Pol II binding in 2 cell types.

first: Shilpa

last: Hattangadi


title: Genome-wide Analyses of Transcription Factor GATA3-Mediated Gene Regulation in Distinct T Cell Types

city: Memphis

first: Brian

middle: J

last: Abraham

state: TN

address: 262 Danny Thomas Blvd

department: Computational Biology

institute: St. Jude Children's Research Hospital

summary: We report genome-wide characterization of GATA3 binding sites in eleven well-defined developmental and effector cell types of the T lymphocyte lineage.  By utilizing a conditional allele of GATA3, we investigated the impact of GATA3 expression on the mRNA expression patterns in several of these cell types.  Correlation of GATA3 binding with gene expression changes indicates that GATA3 regulates a large number of stage- and cell-specific genes involved in multiple signaling and transcriptional pathways critical for T cell differentiation and immune responses.

overall_design:
1) RNA-Seq: Examination of RNA transcript levels in 6 cell types in wild-type and Gata3-knockout cells.
2) ChIP-Seq: Examination of GATA3 binding sites in 11 cell types.
3) Methyl-Seq: Examination of histone modification levels in DP cells from wild-type and Gata3-knockout mice.

first: Gang

last: Wei

first: Ryoji

last: Yagi

first: Raja

last: Jothi

first: Kairong

last: Cui

first: Suvenna

last: Sharma

first: Leelavati

last: Narlikar

first: Daniel

last: Northrup

first: Qingsong

last: Tang

first: William

last: Paul

first: Jinfang

last: Zhu

first: Keji

last: Zhao


title: A Transcriptomic Atlas of Mouse Neocortical Layers

city: Oxford

first: T

middle: Grant

last: Belgard

address: Le Gros Clark Building, South Parks Road

department: Department of Physiology, Anatomy and Genetics

country: United Kingdom

institute: University of Oxford

summary: In the mammalian cortex, neurons and glia form a patterned structure across six layers whose complex cytoarchitectonic arrangement likely contributes greatly to cognitive abilities.  We sequenced transcriptomes from layers 1-6b of the adult (P56) mouse primary somatosensory cortex, along with dorsal cortex and lateral cortex, to understand the transcriptional levels and functional repertoires of coding and noncoding loci for cells that constitute these layers.  5,835 protein-coding genes and 66 noncoding RNA loci are differentially expressed (patterned) across the layers, based on a machine-learning model (naive Bayes) approach. Layers 2-6b are each associated with specific functional and disease annotations that provide insights into their biological roles.  This new resource greatly extends currently available resources, such as the Allen Mouse Brain Atlas and microarray data sets, by providing quantitative expression levels, by being genome-wide, by including novel loci, and by identifying alternatively spliced transcripts that are differentially expressed across layers.

overall_design: Eight adult male mice (56 days old; C57BL/6J strain) were killed by cervical dislocation according to approved schedule one UK Home Office guidelines (Scientific Procedures Act, 1986).  The eight were comprised of two groups of four littermates each.  The mice were decapitated, the skull opened down the midline and the brain removed.  Newly dissected brains were rinsed in RNAse free PBS and stored at -20°C in RNAlater (Ambion).   Whole brains were embedded in 5% agarose and sectioned using a vibrating microtome into 200 micrometer coronal sections using a 1:1 mixture of RNAlater and PBS.  Six sections corresponding approximately to cortical layers I-III, IV, upper V, lower V, VI, and VIb (henceforth referred to as samples A-F, respectively) were dissected out under visual guidance, using transillumination on a dissecting microscope (MZFLIII, Leica) and stored separately in RNAlater at -80°C until all microdissection was complete.  For RNA extraction samples from individual zones from the eight mice were combined and all tissue samples were processed at the same time.  To provide a biological replicate sample, the RNA for sample B was extracted and pooled by litter, providing two samples each representing 4 mice (heretofore known as samples B1 and B2).  In the same way, layers of dorsal cortex and lateral cortex were dissected from eight additional adult male mice.

first: Ana

last: Marques

first: Peter

middle: L

last: Oliver

first: Hatice

last: Ozel Abaan

first: Tamara

last: Sirey

first: Fernando

last: Garcia-Moreno

first: Zoltan

last: Molnar

first: Elliott

last: Margulies

first: Chris

middle: P

last: Ponting


title: HEB and E2A function as SMAD/FOXH1 cofactors

status: Public on Aug 23 2011

first: Se-Jin

last: Yoon

address: 300 Pasteur Drive, Alway M311

department: Genetics

summary: Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We have identified a new motif, termed SMAD Complex Associated (SCA) that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two bHLH proteins - HEB and E2A - bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Further, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E2A is a novel Nodal signaling cofactor that associates with SMAD2/3 and FOXH1 and is necessary for mesendoderm differentiation.

overall_design: ChIP-seq of Smad2/3 and Input in X.tropicalis, stage 10.5 embryo.

first: Andrea

last: Wills

first: Edward

last: Chuong

first: Rakhi

last: Gupta

first: Julie

last: Baker


title: Transcriptional programs in transient embryonic zones of the cerebral cortex defined by high-resolution mRNA-sequencing

status: Public on Aug 22 2011

city: New Haven

first: Albert

middle: E.

last: Ayoub

state: CT

address: 333 Cedar St. SHM C317

department: Neurobiology

institute: Yale University

summary: Here, we apply differential transcriptome analysis on microscopically isolated cell populations, to define five transcriptional programs that represent each transient embryonic zone and the progression between these zones. The five transcriptional programs contain largely uncharacterized genes in addition to transcripts necessary for stem cell maintenance, neurogenesis, migration, and differentiation. Additionally, we found intergenic transcriptionally active regions that possibly encode novel zone-specific transcripts. Finally, we present a high-resolution transcriptome map of transient zones in the embryonic mouse forebrain.

overall_design: mRNAseq performed after laser microdissection of cells from transient embryonic zones in the mouse cortex

first: Sunghee

last: Oh

first: Yanhua

last: Xie

first: Jing

last: Leng

first: Justin

last: Cotney

first: Martin

last: Dominguez

first: James

last: Noonan

first: Pasko

last: Rakic


title: Characterization of small interfering RNAs derived from the geminivirus/betasatellite complex using deep sequencing

status: Public on Aug 21 2011

city: Hangzhou

first: Xiuling

last: Yang

state: Zhejiang Province

address: 268# Kaixuan Road

department: Institute of Biotechnology

institute: Zhejiang University

summary: Small RNA (sRNA)-guided RNA silencing is a critical antiviral defense mechanism employed by a variety of eukaryotic organisms. Although the induction of RNA silencing by bipartite and monopartite begomoviruses has been described in plants, the nature of begomovirus/betasatellite complexes remains undefined. We profiled Tomato yellow leaf curl China virus (TYLCCNV) and its associated betasatellite (TYLCCNB)-derived small RNAs (V-sRNAs and S-sRNAs) using Solexa-based deep sequencing to evaluate the role of betasatellites in V-sRNA modulation. Both sense and anti-sense V-sRNAs and S-sRNAs accumulated preferentially as 22 nucleotide species in infected Solanum lycopersicum and Nicotiana benthamiana plants, indicating that secondary siRNAs were triggered. High resolution mapping of V-sRNA and S-sRNA revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV and betaC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5’ terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3’ terminal of AC1. betaC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form. In addition, the betasatellite affected the amount of V-sRNAs detected in S. lycopersicum and N. benthamiana plants.

overall_design: characterization of Tomato yellow leaf curl China virus and Tomato yellow leaf curl China betasatellite-derived small interfering RNAs from five cDNA libraries of two plant species

first: Xiu

first: Yu

last: Wang

first: Wei

last: Guo

first: Yan

first: Qi

first: Long

last: Fan

first: Xue

last: Zhou


title: Sperm methylation profiles reveal features of epigenetic inheritance and evolution in primates

city: Los Angeles

first: Andrew

middle: D

last: Smith

address: 1050 Childs Way

department: Molecular and Computational Biology

institute: University of Southern California

summary: During germ cell and preimplantation development, mammalian cells undergo nearly complete reprogramming of DNA methylation patterns. We profiled the methylomes of human and chimp sperm as a basis for comparison to methylation patterns of ES cells. While the majority of promoters escape methylation in both ES cells and sperm, the corresponding hypomethylated regions show substantial structural differences. Repeat elements are heavily methylated in both germ and somatic cells; however, retrotransposons from several subfamilies evade methylation more effectively during male germ cell development, while other subfamilies show the opposite trend. Comparing methylomes of human and chimp sperm revealed a subset of differentially methylated promoters and strikingly divergent methylation in retrotransposon subfamilies, with an evolutionary impact that is apparent in the underlying genomic sequence. Thus, the features that determine DNA methylation patterns differ between male germ cells and somatic cells, and elements of these features have diverged between humans and chimpanzees.

sample_organism:
Pan troglodytes
Homo sapiens

platform_organism:
Pan troglodytes
Homo sapiens

overall_design: Genomic DNA form Human and Chimp (2 human individuals; 2 chimp individuals; individuals sequenced separately) mature sperm was isolated, bisulfite converted and sequenced using the Illumina G2 platform

first: Antoine

last: Molaro

first: Emily

last: Hodges

first: Fang

last: Fang

first: Qiang

last: Song

first: Richard

last: McCombie

first: Gregory

last: Hannon


title: Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability

status: Public on Aug 19 2011

city: Durham

first: Neelanjan

last: Mukherjee

state: NC

address: 101 Science Drive

department: Institute for Genome Sciences & Policy

institute: Duke University

type:
Expression profiling by array
Other

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


title: Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation

city: Kansas City

first: Chris

last: Seidel

department: Genomics

institute: Stowers Institute


title: Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability [sequence data]

summary: RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.

overall_design: HuR (ELAVL1) PAR-CLIP

first: Jack

last: Keene


title: seq-AB9050_H3K36me3_N2_L3

status: Public on Aug 17 2011

city: Chapel Hill

first: Tristan

last: De Buysscher

address: CB# 3280, Coker Hall

department: Biology

institute: University of North Carolina at Chapel Hill

summary:
ChIP-seq analysis of elements that specify nucleosome positioning and occupancy, control domains of gene expression, induce repression of the X chromosome, guide mitotic segregation and genome duplication, govern homolog pairing and recombination during meiosis, and organize chromosome positioning within the nucleus.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: ChIP-seq against H3K36me3 in L3 stage N2 worms.

first: Julie

last: Ahringer

first: Paulina

last: Kolasinska-Zwierz

first: Isabel

last: Latorre


title: seq-JL00001_DPY27_N2_L3

overall_design: ChIP-seq against DPY-27 in L3 stage N2 worms.

first: Anne

last: Canonge


title: Genome-wide map of vitamin D receptor (VDR) binding in THP-1 cells

city: Kuopio

first: Sami

last: Heikkinen

address: Yliopistonranta 1E

department: Institute of Biomedicine

country: Finland

institute: University of Eastern Finland

summary: Analysis of acute effects of ligand-treatment on vitamin D receptor binding genome-wide using ChIP-seq. THP-1 monocytic leucemia cells were treated with 1?,25(OH)2D3 (1,25D) or left unstimulated to investigate the acute effects of VDR chromatin occupancy. We identified in total 2340 VDR binding sites with and without the ligand. Without the ligand, there is a considerable presence of VDR already on the chromatin. However, upon a short (40 min) ligand treatment VDR shifts from sites that rarely contain a DR3 type element to sites that frequently contain one or more DR3-type element.

overall_design: Genome-wide identification of VDR binding in THP-1 cells at the unstimulated state and after 40 min ligand (10 nM 1?,25(OH)2D3 (1,25D, calcitriol)) treatment.

last: Väisänen

first: Petri

last: Pehkonen

first: Carsten

last: Carlberg


title: dRNA-Seq of Chlamydia pneumoniae

status: Public on Aug 18 2011

city: Würzburg

first: Marco

last: Albrecht

address: Am Hubland

department: Department of Microbiology

country: Germany

institute: University of Würzburg

summary: The obligate intracellular human pathogen Chlamydia pneumoniae was subjected to dRNA-Seq to gain insights into the transcriptome. The two distinct life cycle forms elementary bodies (EB) and reticulate bodies (RB) were isolated from human Hep2 cell line by differential gradient centrifugation.

overall_design: Total RNA was isolated and partially treated with Terminator Exonuclease to digest RNA without 5'-PPP and thereby enrich for native 5' ends.

first: Cynthia

last: Sharma

last: Reinhardt

first: Jörg

last: Vogel

first: Thomas

last: Rudel


title: Msx1 ChIP-Seq from C2C12 murine myoblast cells expressing Flag-Msx1 protein

first: Richard

middle: A

last: Young

institute: Whitehead Institute for Biomedical Research

summary: The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. We used genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1 in C2C12 murine myoblast cells.

overall_design: C2C12 myoblast cells were seeded at a density of 2.5 x 10^5 cells / 10-cm plate one day prior to infection, and infected with a retrovirus containing a Flag-Msx1 construct for two consecutive days. The resulting cells were then crosslinked with formaldehyde, and DNA was enriched by chromatin immunoprecipitation (ChIP) with an anti-Flag antibody and analyzed by Solexa sequencing. Enriched regions were identified using a Poissonian background model, and were further compared to an additional background of sequences from a sample of Flag-immunoprecipitated DNA from C2C12 cells infected with an empty vector to determine enrichment. ChIP was performed using an antibody against the Flag epitope (Sigma M2, F3165).

first: Roshan

last: Kumar


title: Seedling transcriptome sequencing of the Arabidopsis thaliana MAGIC founder accessions

last: Clark

institute: University of Utah

summary: We have sequenced messenger RNA isolated from seedling tissue for 19 accessions of Arabidopsis thaliana (with biological replication).  The 19 accessions for which RNA-Seq reads were collected have served as the founders for the MAGIC lines, a high-resolution recombinant inbred line mapping resource.  RNA sequencing data was used to examine differential gene expression among the accessions.

overall_design: Examination of RNA expression in seedling tissue carried out using biological replicates.


title: Regulation of Mammalian Cell Growth and Survival By an RNA Polymerase II-Pausing Factor

city: Boston

first: Jianlong

last: Sun

address: 1 Blackfan Street

department: Hematology/Oncology

institute: Boston Children's Hospital


title: Regulation of Mammalian Cell Growth and Survival By an RNA Polymerase II-Pausing Factor (ChIP-seq)

summary: Many mammalian genes are occupied by paused RNA polymerase II (pol II) at promoter-proximal regions on both sides of transcription start sites (TSSs).  However, the consequences of pol II pausing on gene expression and cell biology are not fully understood.  Here we report that genetic ablation of the b subunit of mouse negative elongation factor (Nelf-b), a key pol II-pausing factor, results in slower progression at multiple cell cycle stages and increased apoptosis.  Consistently, a whole-genome analysis indicates that growth and cell death-related genes are highly enriched among the direct target genes of Nelf-b.  In particular, Nelf-b deletion increases pol II density in the promoter-distal region of stress response genes and their overall expression levels in the absence of any external stress signals.  In addition, our work also reveals a previously unappreciated role of Nelf-b role in curbing TSS-upstream transcription of many mammalian genes.  We suggest that Nelf-mediated pol II pausing balances the cellular needs for growth/survival and stress response by preventing excessive basal transcription of stress-induced genes.

overall_design: Examination of Nelf-b bindings sites on chromosomes in mouse embryonic fibroblasts

first: Rong

last: Li


title: Small RNA sequencing in normal and psoriatic skin

city: Saint Louis

first: Xiang

last: Zhou

address: Campus Box 1045, One Brookings Dr.

department: Computer Science and Engineering

institute: Washington University in St. Louis

summary: We report the application of Illumina small RNA sequencing to normal human skin, as well as uninvolved and involved psoriatic skin. By obtaining over 600 million qualified reads from 20 healthy controls and 47 psoriasis biopsies (uninvolved/involved), we have generated a complete small RNA profile in normal and diseased human skin, with particular emphasis on miRNAs. We report the discovery of 284 putative novel miRNAs as well as 98 differentially expressed miRNAs in psoriatic skin.

overall_design: miRNA discovery and expression profiling in 67 normal and psoriatic human skin biopsies

last: Bowcock

first: Weixiong

last: Zhang


title: Novel piRNA Pathway Components Identified Among the Class of TUDOR Domain Containing Proteins

status: Public on Aug 16 2011

first: Julius

last: Brennecke

address: Dr. Bohrgasse 3

institute: IMBA

summary: PIWI proteins and their bound piRNAs form the core of a gonad specific small RNA silencing pathway in animals that protects the genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology, where TUDOR domains of various proteins recognize and bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analyzed the Drosophila TUDOR protein family and identified three previously not characterized TUDOR domain-containing genes (CG4771, CG14303 and CG11133) as essential piRNA pathway members. We characterized CG4771 (Avocado) in detail and demonstrate a critical role for this protein during primary piRNA biogenesis in somatic and germline cells of the ovary. Avocado physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Avocado also interacts with the Tdrd12 orthologs CG11133 and CG31755, which are essential for primary piRNA biogenesis in the germline and probably functionally replace the related and soma specific factor Yb.

overall_design: small RNA libraries were prepared from total RNA isolation of 8 different genotypes

first: Dominik

last: Handler

first: Daniel

last: Olivieri


title: The Msx1 homeoprotein recruits Polycomb to the nuclear periphery during development

overall_design: Refer to individual Series


title: Transcriptional activity regulates alternative cleavage and polyadenylation

status: Public on Aug 15 2011

city: Chicago

first: Zhe

last: Ji

state: ILLINOIS

address: 303 E Superior St, Lurie 6-107

institute: Northwestern University

summary: Genes containing multiple polyadenylation (polyA) sites express mRNA isoforms with variable 3’ untranslated regions (3’UTRs). We found that short and long 3’UTR isoforms were relatively more abundant when genes were highly and lowly expressed, respectively, in human and mouse cells. Consistently, upregulated and downregulated genes were more likely to have shortened and lengthened 3’UTRs, respectively, when genes changed expression under different cell conditions or in response to extracellular stimuli. Using nuclear run-on assays, we found that RNA polymerase II (Pol II) was more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, in line with the difference in polyA site usage, highly expressed genes tend to have higher H3K4me3 and H3K36me3 levels but a lower density of nucleosome around promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that the efficiency of 3’ end processing is generally coupled to transcriptional activity, leading to modulation of polyA site choice by transcription and thus connecting transcriptional control with post-transcriptional regulation via pre-mRNA processing.

overall_design: Nascent RNA sequencing of C2C12 cell

first: Wenting

last: Luo

first: Wencheng

first: Mainul

last: Hoque

first: Zhenhua

last: Pan

first: Yun

last: Zhao

first: Bin

last: Tian


title: piRNA production requires heterochromatin formation in Drosophila

city: New York

first: Colin

middle: Dean

last: Malone

address: NYUMC, Skirball Institute, 540 1st Ave, 4-10

department: Skirball Institute - Developmental Genetics

institute: NYU Medical Center

summary:
Here, we analyzed two small RNA libraries derived from ovarian tissue mutant for either the Drosophila SETDB1 gene, or the Bam gene. Here we show that deposition of histone 3 lysine 9 by the methyltransferase dSETDB1 (egg) is required for piRNA cluster transcription. In the absence of dSETDB1, cluster precursor transcription collapses in germline and somatic gonadal cells and TEs are activated, resulting in germline loss and a block in germline stem cell differentiation. We propose that heterochromatin protects the germline by activating the piRNA pathway.
Keyword : Epigenetics

overall_design: 2 libraries were analyzed, with 1 being a developmental control (Bam Mutant).

first: Prashanth

last: Rangan

first: Ruth

last: Lehmann


title: RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

first: Ryan

last: Dale

address: Rm 10D39, 10 Center Drive

department: National Institute of Child Health and Human Development

institute: National Institutes of Health

summary: Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression.  In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown.  Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator.  AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production.  Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions.  Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.

overall_design: ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)

first: Elissa

last: Lei

first: Nellie

last: Moshkovich

middle: K


title: Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila.

summary:
Here, we analyzed small RNA libraries derived from ovarian tissues heterozygous or mutant for the Tudor gene, Vreteno. In the absence of vret, Piwi-bound piRNAs are lost without changes in piRNA precursor transcript production, supporting a role for Vret in primary piRNA biogenesis. In the germline, piRNAs can engage in an Aub/Argonaute 3 (AGO3)-dependent amplification in the absence of Vret, suggesting that Vret function can distinguish between primary piRNAs loaded into Piwi/Aub complexes and piRNAs engaged in the amplification cycle. We propose that Vret acts at an early step in primary piRNA processing where it plays an essential role in transposon regulation.
Keyword : Epigenetics

overall_design: 2 libraries were analyzed, with 1 being a control (heterozygote).

first: Andrea

last: Zamparini


title: Post-transcription initiation function of the ubiquitous SAGA complex in tissue-specific gene activation (ChIP-seq)

first: Chris

middle: W

last: Seidel

summary: SAGA is a highly conserved transcriptional co-activator complex involved in multiple steps of transcription with activities that function both pre and post initiation. Loss of individual subunits results in developmental defects, suggesting a role in development. To better understand the roles of SAGA functions in developmental gene expression and it's relationship with RNA polymerase II, we examined its composition, binding profile, and the effects of subunit loss on gene expression in two distinct cell types in late stage Drosophila embryos: muscle and neurons.

overall_design: Chromatin IP of FLAG-tagged SAGA subunit Ada2b, and RNA polymerase II (antibody 4H8), was performed in neuronal (elav+) or muscle (mef2+) cells isolated by FACS from late stage embryos, and compared to whole cell extracts (input). The control constists of an IP performed in neuronal cells from a non-tagged strain.

first: Vikki

last: Weake

first: Allison

last: Peak

first: Jerry

last: Workman


title: Transcription Factor Binding Sites by Epitope-Tag ChIP-seq from ENCODE/University of Chicago

status: Public on Aug 12 2011

city: Stanford

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Kevin White mailto:kpwhite@uchicago.edu (Principal Investigator), Subhradip Karmakar mailto:subhradip@uchicago.edu (Project Lead), Nick Bild mailto:nbild@bsd.uchicago.edu (Data Analyst), Alina Choudhury mailto:achoudhury@uchicago.edu (Laboratory Technician), Marc Domanus mailto:mdomanus@anl.gov (Sequencing Technician at Argonne National Lab)). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).

This ENCODE track maps human transcription factor binding sites, genome-wide using second generation massively parallel sequencing. This mapping uses expressed transcription factors as GFP tagged fusion proteins after BAC (Bacterial artificial chromosomes) recombineering.
The U. of Chicago and Max Planck Institute (Dresden) pipeline generates recombineered (recombination-mediated genetic engineering) BACs for the production of cell lines or animals that express fusion proteins from epitope tagged transgenes.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design:
Cells were grown according to the approved ENCODE cell culture protocols. (http://hgwdev/ENCODE/protocols/cell)
Recombineering strategy: To facilitate high-throughput production of the transgenic constructs, the program BACFinder (Crowe, Rana et al. 2002) automatically selects the most suitable BAC clone for any given human gene and generates the sets of PCR primers required for tagging and verification (Poser, Sarov et al. 2008). Recombineering is used for tagging cassettes at either the N or C terminus of the protein. The N-terminal cassette has a dual eukaryotic-prokaryotic promoter (PGK-gb2) driving a neomycin-kanamycin resistance gene within an artificial intron inside the tag coding sequence. The selection cassette is flanked by two loxP sites and can be permanently removed by Cre recombinase-mediated excision. The C-terminal cassette contains the sequence encoding the tag followed by an internal ribosome entry site (IRES) in front of the neomycin resistance gene. In addition, a short bacterial promoter (Gb3) drives the expression of the neomycin-kanamycin resistance gene in E. coli. The tagging cassettes, containing 50 nucleotides of PCR-introduced homology arms are inserted into the BAC by recombineering, either behind the start codon (for the N-terminal tag) or in front of the stop codon (for the C-terminal tag) of the gene. E. coli cells that have successfully recombined the cassette are selected for kanamycin resistance in liquid culture. Each saturated culture from a specific recombineering reaction derived 10-200 independent recombination events. Checking two independent clones for each PCR through the tag insertion point, 97% (85/88) yielded a PCR product of the expected size. Most of the clones that failed to grow were missing the targeted genomic region. An estimated 10% of the BACs used are chimeric, rearranged or wrongly mapped. Thus, initial results indicate that the necessary recombineering steps can be carried out with high fidelity. The White lab produced all epitope tagged transcription and chromatin factor BACs, as well as the genome wide ChIP data and analysis. An application of this approach to the analysis of closely related paralogs (RARa and RARg) yielded transcription factors, chromatin factors, cell lines, ChIP chip data and ChIP-seq data (Hua, Kittler et al. Cell 2009). Such paralogous transcription factors often can not otherwise be distinguished by antibodies.
Sample Preparation: ChIP DNA from samples are sheared to ~800bp using a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of 150-200bp are isolated by gel extraction and amplified using limited cycles of PCR.
Sequencing System: Illumina GAIIx and HySeq next-generation sequencing produced all ChIP-seq data.
Processing and Analysis Software: Raw sequencing reads are aligned using Bowtie version 0.12.5 (Langmead et al. 2009). The "-m 1" parameter is applied to suppress alignments mapping more than once in the genome. Reads are aligned to the UCSC hg19 assembly. Wiggle format signal files are generated with SPP 2.7.1 for R 2.7.1. Macs 1.3.7 is used to call peaks. The Macs parameters used vary by experiment. The White lab used goat anti-GFP antibody to perform ChIP in untagged K562 cells as a background control. The test IP was performed in the same way as the background control. Results are expressed as values of the test normalized to the background

first: Kevin

last: White

first: Subhradip

last: Karmakar

first: Nick

last: Bild

first: Alina

last: Choudhury

first: Marc

last: Domanus


title: Whole genome bisulfite sequencing of Entamoeba histolytica

status: Public on Aug 10 2011

city: Heidelberg

first: Guenter

last: Raddatz

address: Im Neuenheimer Feld 580

institute: German Center for Cancer Research

summary: Up until now, the existence of Dnmt2-mediated DNA methylation has mostly been supported by focal analyses in organisms that contain Dnmt2, but no Dnmt1 or Dnmt3 DNA methyltransferase. In these organisms, several independent studies have also provided support for a biologically important function of Dnmt2-dependent DNA methylation. For example, Dnmt2-dependent methylation in Entamoeba histolytica, the causative agent of amebic dysentery, has been connected to the parasite s virulence. However, global DNA methylation levels in Entamoeba have been found to be very low. In addition, no specific features, such as CpG-specificity and specificity for certain genetic subcompartments have been described. This distinguishes Dnmt2-dependent methylation patterns from all other known methylomes and has raised questions about the validity of the underlying results. We have used whole-genome bisulfite sequencing for an unbiased characterization of the Entamoeba histolytica methylome at single-base resolution in a E.histolytica strain HM-1:IMSS devoid of significant level of EhDnmt2 (Ehmeth) expression.

overall_design: Paired-end BS-sequencing was performed on an Illumina Genome Analyzer with read lengths of 105 base pairs and an average insert size of 200 bp.

first: F

last: Lyko

first: G

first: S

last: Ankri

first: A

last: Tovy


title: Compatibility with Killer explains the Rise of RNAi-deficient Fungi

city: Cambridge

first: Ines

middle: Anna

last: Drinnenberg

address: 9 Cambridge Center

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


title: Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids and poly-beta-1,6-N-acetylglucosamine

status: Public on Aug 11 2011

city: Clayton

first: Paul

middle: Francis

last: Harrison

state: VIC

address: Building 76, Monash University, Wellington Rd

department: Microbiology

institute: Victorian Bioinformatics Consortium

summary: We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis. Consequently, strains harboring these mutations are unable to produce the major Gram negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC19606, to that of an isogenic, LPS-deficient, lpxA mutant strain. Analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, up-regulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-beta-1,6-N-acetylglucosamine (PNAG) were also up-regulated and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein, AssC, in culture supernatants of the A. baumannii wild-type strain, but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

overall_design: Comparison of a gene expression in biological duplicate samples derived from parent bacterial strain to an isogenic mutant strain.

first: Rebekah

last: Henry

first: Nuwan

last: Vithanage

first: Torsten

last: Seeman

first: Scott

last: Coutts

first: Jennifer

middle: H

last: Moffatt

first: Roger

last: Nation

first: Jian

last: Li

first: Marina

last: Harper

first: Ben

last: Adler

first: John

last: Boyce


title: Nuclear hormone 1alpha,25-dihydroxyvitamin D3 elicits a genome-wide shift in the locations of VDR chromatin occupancy

first: Sami

last: Heikkinen


title: The impact of RNAi on the Saccharomyces cerevisiae transcriptome

summary: The RNA interference (RNAi) pathway is found in most eukaryotic lineages but curiously is absent in others, including that of Saccharomyces cerevisiae. Here, we show that reconstituting RNAi in S. cerevisiae causes loss of a beneficial dsRNA virus, known as killer virus. Incompatibility between RNAi and killer viruses extends to other fungal species, in that RNAi is absent in all species known to possess dsRNA killer viruses, whereas killer viruses are absent in closely related species that retained RNAi. Thus, the advantage imparted by acquiring and retaining killer viruses explains the persistence of RNAi-deficient species during fungal evolution.

overall_design: Examine mRNA abundance of S. cerevisiae wild-type (DPB249), +AGO1 (DPB252), +DCR1 (DPB255) and +AGO1, DCR1 (DPB258).

first: I

middle: R

last: Fink

first: D

last: Bartel


title: Identification and Characterization of MicroRNAs from Phaeodactylum tricornutum by High-throughput Sequencing and Bioinformatics Analysis

status: Public on Aug 08 2011

city: Qingdao

first: Guangce

last: Wang

address: Nanhai Road 7

institute: Institute of Oceanology, the Chinese Academy of Sciences (IOCAS)

summary: Diatoms, which are important planktons widespread in various aquatic environments, are believed to play a vital role in primary production as well as silica cycling. The genomes of the pennate diatom Phaeodactylum tricornutum and the centric diatom Thalassiosira pseudonana have been sequenced, revealing some characteristics of the diatomsâ mosaic genome as well as some features of their fatty acid metabolism and urea cycle, and indicating their unusual properties. To identify microRNAs (miRNAs) from P. tricornutum and to study their probable roles in nitrogen and silicon metabolism, we constructed and sequenced small RNA (sRNA) libraries from P. tricornutum under normal (PT1), nitrogen-limited (PT2) and silicon-limited (PT3) conditions. A total of 13 miRNAs were identified. They were probable P. tricornutum-specific novel miRNAs. These miRNAs were differentially expressed in PT1, PT2 and PT3, and their potential targets were involved in various processes. Our results indicated that P. tricornutum contained novel miRNAs that differed from miRNAs of other organisms and that they might play important regulator roles in P. tricornutum metabolism.

overall_design: Constructing and sequencing small RNA (sRNA) libraries from P. tricornutum under normal (PT1), nitrogen-limited (PT2) and silicon-limited (PT3) conditions

first: Aiyou

last: Huang

first: Linwen

last: He


title: Genetic Framework for GATA Factor Function in Vascular Biology

city: Palo Alto

first: Philip

last: Cayting

state: CA

address: 1501 S California Ave

institute: Stanford University

summary:
Vascular endothelial dysfunction underlies the genesis and progression of numerous diseases. Whereas the GATA transcription factor GATA-2 is expressed in endothelial cells and is implicated in coronary heart disease, it has been studied predominantly as a master regulator of hematopoiesis. As many questions remain unanswered regarding GATA-2 function in the vascular biology realm, we used ChIP-seq and loss-of-function strategies to define the GATA-2-instigated genetic network in human endothelial cells. By contrast to erythroid cells, GATA-2 occupied a unique target gene ensemble, consisting of genes encoding key determinants of endothelial cell identity and inflammation. GATA-2-occupied sites characteristically contained motifs that bind Activator Protein-1 (AP-1), a pivotal regulator of inflammatory genes. GATA-2 frequently occupied the same chromatin sites as c-JUN and c-FOS, heterodimeric components of AP-1. Though all three components were required for maximal AP-1 target gene expression, GATA-2 was not required for AP-1 chromatin occupancy. GATA-2 conferred maximal phosphorylation of chromatin-bound c-JUN at Ser 73, which stimulates AP-1-dependent transactivation, in a chromosomal context-dependent manner. This work establishes a link between a GATA factor and inflammation, mechanistic insights underlying GATA-2-AP-1 cooperativity, and a rigorous genetic framework for understanding GATA-2 function in normal and pathophysiological vascular states.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: Examination of GATA2 ChIP-seq in HUVEC cells.

first: Amelia

last: Linnemann

first: Henriette

last: O'Geen

first: Sunduz

last: Keles

first: Peggy

last: Farnham

first: Emery

last: Bresnick


title: Deep sequencing and functional annotation reveal miRNAs implicated in the cell senescence phenotype

status: Public on Aug 09 2011

first: Dario

last: Boffelli

address: 700 N San Vicente Blvd

department: Pediatrics

institute: Cedars Sinai

summary: In cell senescence, cultured cells cease proliferating and acquire aberrant gene expression patterns. MicroRNAs (miRNAs) modulate gene expression through translational repression or mRNA degradation, and have been implicated in senescence. We have used deep sequencing to carry out a comprehensive survey of miRNA expression and its involvement in cell senescence. Informatic analysis of small RNA sequence datasets from young and senescent IMR90 human fibroblasts identifies many known miRNAs, and a small number of novel miRNAs, that are regulated (either up or down) with cell senescence. Comparison with mRNA expression profiles revealed potential mRNA targets of the senescence-regulated miRNAs. The target mRNAs are enriched for genes involved in biological processes associated with cell senescence. This result greatly extends existing information on the role of miRNAs in cell senescence, and is consistent with miRNAs having a causal role in the process.

overall_design: Comprehensive survey of miRNA from young and senescent IMR90 fibroblasts using deep sequencing

first: Joseph

last: Dhahbi


title: Integrated epigenome profiling of repressive histone modifications, DNA methylation and gene expression in normal and malignant urothelial cells [ChIP-Seq data]

status: Public on Aug 03 2011

city: Gießen

first: Andreas

last: Gogol-Döring

address: Wiesenstraße 14

department: MNI

institute: Technische Hochschule MIttelhessen

summary: We profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq in normal urothelial cells and cell lines representing non-invasive and invasive tumors.

overall_design: Two bladder cancer cell lines (RT112 and EJ/T24 from ATCC) and normal human urothelial (NHU) cells were treated according to standard ChIP-Seq protocols using antibodies against the histone modifications H3K9m3 and H3K27m3 (from Millipore). The sequencing reads were mapped against the human genome (GRCh37/hg19).

first: Ewa

last: Dudziec

first: Victoria

last: Guy

first: Wei

last: Chen

first: James

last: Catto


title: Gene expression of polyoma middle T antigen induced mammary tumors [RNA_Seq : MOLF x PyMT]

status: Public on Aug 06 2011

city: Rockville

first: Ying

last: Hu

email: yhu@mail.nih.gov, hunterk@mail.nih.gov

address: 9609 Medical Center Dr.

department: NCI/CCR

institute: NIH

summary: Mouse genetic crosses were established between the PyMT model of metastatic breast cancer and MOLF strain.  Tumors were harvested from the animals for gene expression analysis to identify genes and network modules associated with progression to distant metastatic disease.

overall_design: 30 samples from the MOLF cross were assayed using Illumina next generation sequencers

first: Kent

last: Hunter


title: Alternative splicing of the TRPV1 gene underlies infrared sensation in vampire bats

status: Public on Aug 04 2011

city: Berkeley

first: Nicholas

middle: T

last: Ingolia

email: ingolia@berkeley.edu, nick@ingolia.org

address: 1 Barker Hall # 3202

department: Molecular and Cell Biology

institute: University of California, Berkeley

summary: Vampire bats and snakes have taken thermosensation to the extreme by developing specialized systems for detecting infrared radiation. As such, these creatures provide a window into the molecular and genetic mechanisms underlying evolutionary tuning of thermoreceptors in a species or cell type specific manner. In each case, robust thermal sensitivity likely reflects specialized anatomical features of infrared sensing pit organs, as well as intrinsic heat sensitivity of trigeminal nerve fibers that innervate these structures. Here we show that vampire bats use a molecular strategy involving alternative splicing of the TRPV1 gene to generate a channel specifically within trigeminal ganglia that has a reduced thermal activation threshold. Selective expression of splicing factors in trigeminal, but not dorsal root ganglia, together with unique organization of the vampire bat TRPV1 gene underlies this mechanism of sensory adaptation. Comparative genomic analysis of the TRPV1 locus supports phylogenetic relationships within the proposed Pegasoferae clade of mammals.

sample_organism:
Desmodus rotundus
Carollia brevicauda

platform_organism:
Desmodus rotundus
Carollia brevicauda

overall_design: Gene expression measurements implicate a TRPV1 splice isoform as the heat-sensitive channel in vampire bats

first: Elena

middle: O

last: Gracheva

first: Julio

middle: F

last: Cordero-Morales

first: Jose

last: Gonzalez

first: Carla

middle: I

last: Aranguren

first: Carlo

last: Manno

first: Jonathan

middle: S

last: Weissman

first: David

last: Julius


title: Comparative Analysis of RNA-Seq Alignment Algorithms and the RNA-Seq Unified Mapper (RUM).

first: Eric

last: Pierce

state: MA

address: 243 Charles Street

department: Ophthalmology

institute: Massachusetts Eye and Ear Infirmary

summary:
A critical task in high throughput sequencing is aligning millions of short reads to a reference genome. Alignment is especially complicated for RNA sequencing (RNA-Seq) because of RNA splicing. A number of RNA-Seq algorithms are available, and claim to align reads with high accuracy and efficiency while detecting splice junctions. RNA-Seq data is discrete in nature; therefore with reasonable gene models and comparative metrics RNA-Seq data can be simulated to sufficient accuracy to enable meaningful benchmarking of alignment algorithms. The exercise to rigorously compare all viable published RNA-Seq algorithms has not previously been performed.
RESULTS:

We developed an RNA-Seq simulator that models the main impediments to RNA alignment, including alternative splicing, insertions, deletions, substitutions, sequencing errors, and intron signal. We used this simulator to measure the accuracy and robustness of available algorithms at the base and junction levels. Additionally, we used RT-PCR and Sanger sequencing to validate the ability of the algorithms to detect novel transcript features such as novel exons and alternative splicing in RNA-Seq data from mouse retina. A pipeline based on BLAT was developed to explore the performance of established tools for this problem, and to compare it to the recently developed methods. This pipeline, the RNA-Seq Unified Mapper (RUM) performs comparably to the best current aligners and provides an advantageous combination of accuracy, speed and usability.

overall_design: RNA-Seq of mouse retinal RNA, as described.

first: Michael

last: Farkas

first: Gregory

last: Grant