title: A Probabilistic Model of Transcription Dynamics applied to Estrogen Signalling in Breast Cancer Cells

city: Nijmegen

last: Stunnenberg

address: Geert Grooteplein 28

department: Department of Molecular Biology (274)

institute: Radboud University

summary: Gene  transcription mediated   by  RNA  polymerase  II  (pol-II)  is a  key  step  in  gene  expression. The dynamics  of pol-II moving along the transcribed region influences the rate  and timing  of gene expression. In this work we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data  measured  using ChIP-Seq.  The model can be used to estimate  transcription speed and to  infer  the  temporal  pol-II  activity  profile at  the  gene promoter.  Model  parameters are  determined using either  maximum  likelihood estimation or via Bayesian inference using Markov chain  Monte  Carlo sampling.   The  Bayesian approach  provides  confidence intervals  for parameter estimates  and  allows the use of priors  that capture domain  knowledge, e.g.  the  expected  range  of transcription speeds, based  on previous experiments.  The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes.  By clustering  the inferred promoter activity  time  profiles, we are able to determine which genes respond quickly to stimuli and  group genes that share  activity  profiles and  may therefore  be co-regulated.  We apply  our methodology  to biological data  obtained using  ChIP-seq  to  measure  pol-II  occupancy  genome-wide  when  MCF-7  human  breast cancer cells are treated with estradiol  (E2).  The transcription speeds we obtain  agree with those obtained previously  for smaller  numbers  of genes with  the  advantage  that our approach  can be applied  genome- wide.   We  validate  the  biological  significance  of the  pol-II  promoter  activity  clusters  by  investigating cluster-specific  transcription factor  binding  patterns and determining  canonical  pathway  enrichment.

overall_design: MCF-7 cells were mock treated or with 10nM 17b-E2 to seven time points (5', 10', 20', 40', 80', 160', 320')  Genome-wide identification of RNA polymerase II (RNAPII) occupancy at different time points following E2 induction of MCF-7 cells

first: Hendrik

first: Ciirawa

last: Maina

first: Korbinian

last: Grote

first: George

last: Reid

first: Antti

last: Honkela

first: Neil

last: Lawrence

first: Magnus

last: Rattray

first: Filomena

last: Matarese


title: Analysis of the Epigenomic and Transcriptional Landscapes during mammalian spermatogenesis [Bisulfite-Seq]

status: Public on May 19 2014

city: Singapore

first: Diana

middle: HP

last: Low

address: #03-06, 61 Biopolis Drive, Proteos

country: Singapore

institute: Institute of Molecular and Cell Biology

type: Methylation profiling by high throughput sequencing

summary: Adult germline stem cells (AGSCs) are multifunctional - they must self renew, maintain genome pluripotency, and prepare for gametogenesis – which involves meiotic and chromatin repackaging phases. To better understand AGSCs and gametogenesis, we derived high-resolution profiles of transcription, DNA methylation, 5hmC, and multiple histone modifications at key stages. First, AGSCs display chromatin ‘poising’ of enhancers and promoters of genes utilized in embryo development. Second, the pluripotency network in AGSCs is remarkably distinct from ESCs - lacking Nanog, Sox2, or Prdm14 expression.  Third, spermatogenesis involves stage-specific transcription and distinctive chromatin dynamics, but virtually no changes in DNAme.  Surprisingly, we observe co-incidence of RNA polymerase II, high H3K4me3, and DNA methylation at 20-35% of genes transcribed during gametogenesis - including piRNA clusters - but often observe attendant promoter 5hmC.  Our work reveals key differences between AGSCs and other germ/stem cells, and reveals both logical and unexpected chromatin-transcription relationships accompanying germline developmental transitions.

overall_design: Methylation profiles of 8-week old wild type (WT) mice were generated by bisulfite sequencing using Illumina HiSeq 2000.

middle: H

first: Sue

middle: S

last: Hammoud


title: Developmental Dynamics of Kranz Cell Transcriptional Specificity in Maize Leaf Reveals Early Onset of C4-related Processes

status: Public on May 18 2014

city: New Haven

first: Timothy

last: Nelson

state: CT

department: Department of Molecular, Cellular and Developmental Biology

country: USA

institute: Yale University

type: Expression profiling by high throughput sequencing

summary: The comparison of the cell-specific transcriptomes of bundle sheath (BS) and mesophyll (M) cells from successive developmental stages of maize leafs reveals that the number of genes preferentially transcribed in one cell type or the other varies considerably from the sink-source transition to mature photosynthetic stages. The number of differentially expressed (DE) genes is maximal at a stage well prior to full maturity, including those that encode key functions for C4 photosynthesis. The developmental dynamics of BS/M differential expression can be used to identify candidates for other C4-related functions and to simplify the identification of specific pathways members from otherwise complex gene families. The candidates for C4-related transcription factors identified with this developmental DE strategy overlap with those identified in studies using alternative strategies.

overall_design: Nine day old third leaves of maize sections, located at -1 cm, +4 cm and +9 cm (leaf tip), relative to the sink-source transition, were collected. BS and M cells were captured from each section. There are two duplications for each section and each cell types. A total of 12 libraries were constructed for RNA-seq.

first: S

middle: L

last: Tausta


title: Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

city: Charlottesville

first: Mazhar

last: Adli

state: VA

address: 1340 Jefferson Park Ave

department: Mol.Bio and Genetics

institute: Uni

type: Genome binding/occupancy profiling by high throughput sequencing

summary: RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ~10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide

overall_design:
1.sgRNA1-6 binding sites were identified  with ChipSeq by using HA antibody (there are 2 replicates for sgRNA1-3, one sample for sgRNA4-6,one control without sgRNA)
2.PCR products which amplifies " off-target genomic sites" were deep sequenced in the presence of WT Cas9+sgRNA or WT Cas9 alone( unique adaptor was used for each sgRNA and mixed for multiplex run)

first: Adli

last: Mazhar

first: Kuscu

last: Cem


title: Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

status: Public on May 17 2014

city: Seattle

first: Michael

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

type: Non-coding RNA profiling by high throughput sequencing

summary: HIV infection of CD4  T cells induces a range of host transcriptional changes in mRNAs as well as microRNAs that may coordinate changes in mRNAs.

overall_design: We applied next-generation sequencing, analyzing the small RNA fraction of HIV-infected cells at 5, 12, and 24 h postinfection (RNA-Seq).

first: Stewart

middle: T

last: Chang

first: Matthew

middle: J

last: Thomas

first: Pavel

last: Sova

first: Richard

middle: R

last: Green

first: Robert

middle: E

last: Palermo

middle: G


title: Condensin association with chromosomes depends on a DNA binding domain formed by its HEAT-repeat subunits

status: Public on May 16 2014

city: Heidelberg

first: Ilaria

last: Piazza

address: 1, Meyerhofstrasse

country: Germany

institute: EMBL

summary: Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensin’s HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain.

overall_design: Analysis of condensin binding genomewide in a wild type and a condensin mutant

first: Anna

last: Rutkowska

first: Alessandro

last: Ori

first: Marta

last: Walczak

first: Vicent

last: Pelechano

first: Martin

last: Beck

first: Christian

middle: C

last: Haering


title: Genome-wide map of GATA6 DNA binding in human CRC cells

status: Public on May 20 2014

city: Barcelona

first: Camille

last: Stephan Otto Attolini

state: Select a State or Province

address: Baldiri Reixac 10

department: Biostatistics and bioinformatics Unit

country: Spain

institute: IRB Barcelona

summary: We describe the genome-wide DNA-binding of GATA6 in a human CRC cell line (LS174T). GATA6 is found to bind the promoter of genes involved in the maintenance of intestinal stem cells, including genes of the Wnt and TGFbeta/BMP pathways. With this we describe a novel GATA6-dependent mechanism of stem cell maintenance in colorectal tumors.

overall_design: Examination of GATA6 binding and H3K4me1, H3K4me3 and H3K27ac levels in a human CRC cell line by Chromatin immunoprecipitation followed by deep sequencing.

first: P

last: Martinelli

first: G

last: Whissel

first: E

last: Carrillo de Santa Pau

middle: P

last: Vicent

first: C

last: Stephan-Otto Attolini

first: F

middle: X

last: Real

last: Batlle


title: INO80 complex in the core regulatory network governing ESC self-renewal

city: Durham

first: Li

last: Wang

address: 111 T.W. Alexander Dr

institute: NIH

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

overall_design: Refer to individual Series


title: RNA-Seq Profiling of a Defective Seed Coat Mutation in Glycine max Reveals Differential Expression of Proline Rich and Other Cell Wall Protein Transcripts

city: Urbana

first: Lila

middle: O.

last: Vodkin

state: IL

address: 1201 W. Gregory Dr.

department: Crop Sciences

institute: University of Illinois

summary: The plant cell wall performs a number of essential functions including providing shape to many different cell types and serving as a defense against potential pathogens. The net pattern mutation creates breaks in the seed coat of soybean (Glycine max) because of ruptured cell walls. Using RNA-Seq, we examined the seed coat transcriptome from three stages of immature seed development in two pairs of isolines with normal or defective seed coat phenotypes due to the net pattern. The genome-wide comparative study of the transcript profiles of these isolines revealed 364 differentially expressed genes in common between the two varieties that were further divided into different broad functional categories. Genes related to cell wall processes accounted for 19% of the differentially expressed genes in the middle developmental stage of 100-200 mg seed weight. Within this class, the cell wall proline-rich and glycine-rich protein genes were highly differentially expressed in both genetic backgrounds. Other genes that showed significant expression changes in each of the isoline pairs at the 100-200 mg seed weight stage were xylem serine proteinase, fasciclin-related genes, auxin and stress response related genes, TRANSPARENT TESTA 1 (TT1) and other transcription factors. The mutant appears to shift the timing of either the increase or decrease in the levels of some of the transcripts.  The analysis of these data sets reveals the physiological changes that the seed coat undergoes during the formation of the breaks in the cell wall.

overall_design: Examination of soybean isolines in two different genetic background at three different seed weight stages: Seed coats of Clark standard (CS, wild type) & Clark defective (CD, seed coat mutant), Harosoy Standard (HS) & Harosoy defective (HD) at 50-100mg, 100-200mg and 400-500mg.

first: Anupreet

last: Kour

first: Anne

middle: M

last: Boone


title: iCLIP and NGS to map protein-RNA interactions for U1-snRNP proteins in Trypanosoma brucei

city: Giessen

first: Lee-Hsueh

last: Hung

state: Hessen

address: Heinrich-Buff-Ring 58

department: Biochemistry

institute: University of Giessen

summary: Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei.

overall_design: 3 (2) biological replicates of U1C (U1-70K) -specific co-immunoprecipitated RNA after UV-crosslinking

first: A

last: Bindereif

last: Preusser

first: L


title: Splicing of polyA transcripts in Hep3B cells

status: Public on May 15 2014

city: Oakland

first: Elizabeth

last: Theusch

state: CA

address: 5700 Martin Luther King Jr Way

institute: CHORI

summary: We were interested in identifying novel splice junctions in Hep3B cells, especially in genes involved in cholesterol metabolism like HMGCR, so we sequenced polyA-tailed RNA from Hep3B cells.

overall_design: 1 polyA-selected RNA sample was sequenced from Hep3B cells


title: INO80 complex in the core regulatory network governing ESC self-renewal [ChIP-Seq]

summary: Embryonic stem cell (ESC) self-renewal and pluripotency is controlled by the coordinated action of transcription factors and chromatin regulators. Compared to the pluripotency transcription factors, the function of the chromatin regulators, especially the ATP-dependent chromatin remodelers, remains poorly understood in ESCs. Here, we show that INO80, a SWI/SNF family chromatin remodeling complex, is essential for ESC self-renewal, pluripotency, somatic cell reprogramming, and embryonic development. Ino80, the ATPase of the complex, forms an auto-regulatory loop with the ESC master transcription factors Oct4, Nanog, and Sox2. More importantly, it co-occupies the enhancer regions of most key pluripotency genes with the master transcription factors, and positively regulates their expression by maintaining an open chromatin structure. Our data suggests that INO80 is an integral component of the pluripotency transcription network, and plays a critical role in both the maintenance and establishment of pluripotency

overall_design: Identification of Ino80 localization in mouse embryonic stem cells

first: Guang

last: Hu

first: Ying

last: Du


title: An Integrated Approach Reveals Regulatory Controls on Bacterial Translation Elongation

first: Arvind

last: Subramaniam

address: 1100 Fairview Ave N

department: Basic Sciences Division

institute: Fred Hutchinson Cancer Center

summary: Ribosomes elongate at a nonuniform rate during translation. Theoretical models and experiments disagree on the in vivo determinants of elongation rate and the mechanism by which elongation rate affects protein levels. To resolve this conflict, we measured transcriptome-wide ribosome occupancy under multiple conditions and used it to formulate a whole-cell model of translation in E. coli. Our model predicts that elongation rates at most codons during nutrient-rich growth are not limited by the intracellular concentrations of aminoacyl-tRNAs. However, elongation pausing during starvation for single amino acids is highly sensitive to the kinetics of tRNA aminoacylation. We further show that translation abortion upon pausing accounts for the observed ribosome occupancy along mRNAs during starvation. Abortion reduces global protein synthesis, but it enhances the translation of a subset of mRNAs. These results suggest a regulatory role for aminoacylation and abortion during stress, and our study provides an experimentally constrained framework for modeling translation.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: 7 samples for ribosome profiling and 4 samples for total mRNA profiling

first: Brian

last: Zid

first: Erin

middle: K

last: O'Shea


title: Modeling Fanconi Anemia pathogenesis and therapeutics using integration-free patient iPSCs

city: La Jolla

first: Nongluk

last: Plongthongkum

state: California

address: 9500 Gilman Drive, MC0412, PFBH402

department: Bioengineering

institute: University of California, San Diego

summary: To elucidate the transcriptional and epigenetic alterations underlying the neurogenic defects of FA-NSCs, we conducted gene expression microarray analysis and global DNA methylation profiling. The gene expression pattern of gene-corrected NSCs (C-FA-NSCs) resembled that of control-NSCs but clustered distantly from FA-NSCs (Fig. 6F and Table S1). Hierarchical clustering based on DNA methylation levels in the promoter region (+/-1.5kb from TSS) of genes whose expression levels were rescued in C-FA-NSCs, placed C-FA-NSCs closer to control-NSCs and away from FA-NSCs (Fig. 6G), although this pattern was not seen at the whole genome level (Fig. S4C). This suggests that FANCA gene correction leads to specific methylation changes in a subset of promoters.

overall_design: Examination of the methylomes of NSCs derived from Fanconi Anemia iPSCs before and after gene correction by targeted bisulfite sequencing with padlock probes

first: Guang-Hui

last: Liu

first: Keiichiro

last: Suzuki

first: Mo

last: Li

first: Jing

last: Qu

first: Nuria

last: Montserrat

first: Carolina

last: Tarantino

last: Gu

first: Fei

last: Yi

first: Xiuling

last: Xu

first: Weiqi

last: Zhang

first: Sergio

last: Ruiz

first: Kun

first: Shigeo

last: Masuda

first: Emmanuel

last: Nivet

first: Yuji

last: Tsunekawa

first: Rupa

last: Soligalia

first: April

last: Goebl

first: Emi

last: Aizawa

middle: Y

last: Kim

first: Jessica

first: Ilir

last: Dubova

first: Ruotong

last: Ren

first: Chris

last: Benner

first: Antonio

last: del Sol

first: Juan

last: Bueren

last: Trujillo

first: Jordi

last: Surralles

first: Enrico

last: Cappelli

first: Carlo

last: Dufour

first: Concepcion

last: Esteban

last: Belmonte


title: High resolution mapping of modified nucleobases in DNA using excision repair enzymes

city: Aurora

first: Jay

middle: R.

last: Hesselberth

state: CO

department: Biochemistry and Molecular Gentetics

institute: University of Colorado School of Medicine

summary: Data from Excision-seq experiments to map deoxyuridine and pyrimidine dimers in S. cerevisiae and E. coli

sample_organism:
Escherichia coli
Saccharomyces cerevisiae

overall_design: For uracil mapping in pre-digestion Excision-seq, uracil was excised from genomic DNA from dut ung yeast and bacteria yielding double-stranded DNA fragments. Adapters were ligated to these fragments for Illumina library preparation. Using this method, the number of reads at a genomic location corresponds to the quantity of dU at that location. In post-digestion Excision-seq, uracil-containing library fragments are destroyed by UDG treatment, thus coverage is inversely proportional to uracil content. For pyrimidine dimer Excision-seq, yeast were irradiated with high doses of UVC light to generate a large number of DNA modifications.  Using the excision repair enzyme UVDE from S. pombe, we excised pyrimidine dimers, cutting the phosphodieseter bond to release small double stranded fragments. These fragments were repaired with either CPD photolyase or 6-4 photolyase to generate independent libraries for each modification type. Illumina adapters were ligated to these fragments for library preparation. Using this method the number of reads at a genomic location corresponds to the quantity of the 3' pyrimidine of the dimer.


title: HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors [Mnase-seq]

status: Public on May 14 2014

city: Montpellier

first: Philippe

last: Pasero

address: 141 rue de la Cardonille

country: France

institute: Institute of Human Genetics - CNRS

summary: In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3 cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors.

overall_design: MNase-seq analysis of nucleosome position in wt, sir2 and rpd3 cells, aligned against genomic DNA (sacCer3; *sorted_s3.bed) and rDNA sequences (*rdna_nucleosomes.bed)

first: Kazumasa

last: Yoshida

first: Julien

last: Bacal

first: Ismael

last: Padioleau

first: Armelle

last: Lengronne

first: Philipe


title: ChIP-seq mapping of Drosophila TFIIIC (dTFIIIC220; CG7099) and SMC containing complexes in Kc167 cells

city: Palo Alto

first: Kevin

last: Van Bortle

address: 3165 Porter Dr

department: Genetics

institute: Stanford University

summary: We map the genome-wide binding profiles for dTFIIIC220, as well as the Drosophila cohesin and condensin complexes in the late-stage embryonic cell line Kc167

overall_design: Examination of several proteins in the Kc167 cell line

first: K

first: N

last: Takenaka

first: V

last: Corces


title: HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors [ChIP-seq]

overall_design:
BrdU-IP-seq analysis of origin activity in wt, sir2 and rpd3 cells, aligned against genomic DNA (sacCer3) and rDNA sequences
Please note that the wt WCE #1 and #2 samples are whole-cell extracts from wild-type cells arrested in HU that were used to calculate  the log ratio of the BrdU IP #1 and #2 batches, respectively.


title: Expression data from embryonic day 15.5 atrioventricular canal regions were isolated from Scx-/- and Scx+/+ mice.

city: Oxford

first: Damien

last: Barnette

state: Oxfordshire

address: Sherrington Building

country: United Kingdom

institute: University of Oxford

summary:
Purpose: Our lab has previously shown that Scleraxis (Scx) is require for proper valve development in vivo. In order to fully explore gene networks regulated by Scx during the vital stages of valve remodeling , high throughput RNA-squencing was performed.
Results:There were a total of 18,810 genes were detected. A total of 864 genes were differentially expressed Scx null AVC regions: 645 being upregulated and 217 downregulated.

overall_design: In this data set, we include expression data from atrioventricular canal (AVC) regions from Scx null and wild-type littermate controls at embryonic day 15.5.  A total of 6 samples were analyzed; 3 valve regions from E15.5 Scx-/- mice, and 3 from E15.5 Scx+/+ wild-type littermate controls. Differential expression read counts are ranked based on p-value (<0.05).

first: Joy

last: Lincoln

middle: N


title: The ABRF Next-Generation Sequencing Study (ABRF-NGS): Multi-platform and cross-methodological reproducibility of transcriptome profiling by RNA-seq [Ion Torrent Proton]

city: New York

first: Christopher

last: Mason

address: 1305 York Ave

department: Physiology and Biophysics

institute: Weill Cornell Medical College

summary: Next-generation sequencing (NGS) technology applications like RNA-sequencing (RNA-seq) have dramatically expanded the potential for novel genomics discoveries, but the proliferation of various platforms and protocols for RNA-seq has created a need for reference data sets to help gauge the performance characteristics of these disparate methods.  Here we describe the results of the ABRF-NGS Study on RNA-seq, which leverages replicate experiments across multiple sites using two reference RNA standards tested with four protocols (polyA selected, ribo-depleted, size selected, and degraded RNA), and examined across five NGS platforms (Illumina’s HiSeqs, Life Technologies’ Personal Genome Machine and Proton, Roche 454 GS FLX, and Pacific Biosciences RS).  These results show high (R2 >0.9) intra-platform consistency across test sites, high inter-platform concordance (R2 >0.8) for transcriptome profiling, and a large set of novel splice junctions observed across all platforms.  Also, we observe that protocols using ribosomal RNA depletion can both salvage degraded RNA samples and also be readily compared to polyA-enriched fractions.  These data provide a broad foundation for standardization, evaluation and improvement of RNA-seq methods.

overall_design:
Two reference RNA standards tested with four protocols (polyA selected, ribo-depleted, size selected, and degraded RNA), and examined across five NGS platforms (Illumina’s HiSeqs, Life Technologies’ Personal Genome Machine and Proton, Roche 454 GS FLX, and Pacific Biosciences RS).

Please note that the samples were named following the ABRF-Platform-Site-Sample-Replicate# format. For example, ABRF-454-CNL-A-1 means Sample A was run on 454 platform at Cornell and this is the first replicate, and  ABRF-454-CNL-A-2 means the same exact sample was ran with same machine at same location and is 2nd replicate.


title: The ABRF Next-Generation Sequencing Study (ABRF-NGS): Multi-platform and cross-methodological reproducibility of transcriptome profiling by RNA-seq [Illumina HiSeq 2500]


title: Conserved expression of lincRNA during human and macaque prefrontal cortex development and maturation

city: Leipzig

first: Zhisong

last: He

address: Deutscher Platz 6

institute: Max Planck Institute for Evolutionary Anthropology

summary: 78 tissue samples from prefrontal cortex (PFC) in human and macaque

sample_organism:
Macaca mulatta
Homo sapiens

platform_organism:
Macaca mulatta
Homo sapiens

overall_design: The data from human and macaque PFC samples with different ages were used to estimate gene expression changes, including both protein-coding genes and lincRNAs, in PFC along lifespan in the two species.

first: Hindrike

last: Bammann

first: Dingding

last: Han

first: Xi

last: Jiang

first: Gangcai

last: Xie

first: Philipp

last: Khaitovich


title: Global analysis of p53-regulated transcription reveals its direct targets and unexpected regulatory mechanisms (GRO-Seq)

city: Boulder

first: Mary

middle: Ann

last: Allen

state: Co

address: UCB 596  JSCBB

department: BioFrontiers

institute: University of Colorado

summary: GRO-seq of HCT116 cells treated with Nutlin to activate p53

overall_design: HCT116 cells were treated with Nutlin or DMSO for 1 hour

middle: A

first: Robin

middle: D

last: Dowell

first: Joaquin

last: Espinosa


title: Identification of transcriptome SNPs for assessing allele specific gene expression within a super-hybrid rice Xieyou9308

city: hangzhou

first: Rongrong

last: Zhai

address: No. 359, Tiyuchang Rd

country: China

institute: China National Rice Research Institute

summary: We identified 9325 reliable single nucleotide polymorphisms (SNPs) distributed throughout the genome, of which nearly 68% were represented in R9308 versus Xieqingzao B by CT and GA SNPs. This may indicate that DNA methylation, a heritable epigenetic mark, exists in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17%) showed significant allelic biases during at least one stage, suggesting that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed allelic expression biases towards the R9308 allele at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed allele expression biases towards the R9308 allele. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play an important role in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study would provide valuable information for further understanding molecular mechanisms of heterosis.

overall_design: Root mRNA profiles of a super-hybrid rice Xieyou 9308 and its parents at tillering and heading stages were generated by deep sequencing, in duplicate, on Illumina Hiseq 2000 platform.

first: Rong-rong

first: Shi-hua

last: Cheng


title: The ABRF Next-Generation Sequencing Study (ABRF-NGS): Multi-platform and cross-methodological reproducibility of transcriptome profiling by RNA-seq [454 GS FLX Titanium]


title: Global analysis of p53-regulated transcription reveals its direct targets and unexpected regulatory mechanisms


title: Proteomic and Genomic Analysis of transcription factor TFII-I reveals insight into the response to cellular stress

city: Athens

first: Giorgio

middle: L

last: Papadopoulos

state: Attiki

address: 34 Fleming Street

department: Molecular Oncology

country: Greece

institute: BSRC 'Alexander Fleming'

summary: We expressed biotin-tagged transcription factor TFII-I (GTF2I) in K562 cells and subjected crosslinked chromatin to streptavidin mediated pull down. We subjected the isolated DNA to Illumina sequencing.  We identified 18920 TFII-I binding peaks. Nearest gene analysis revealed 5886 genes associated with a TFII-I binding peak in proliferating K562 cells.

overall_design: We generated two K562 single cell clones stably expressing bio-tagged TFII-I, and K562 cells expressing only the BirA protein biotin ligase from E.coli. These three clones were subjected to streptavidin mediated pull down and Illumina sequencing.

first: Jorg

last: Bungert

first: John

last: Strouboulis


title: Specific mitotic chromatin association of the major notch effector RBPJ and its implication for transcriptional memory

city: Philadelphia

first: Kyoung Jae

last: Won

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: From the cell-based investigation, RBPJ is one of the few proteins retained on chromatin during cell division. ChIP-seq experiments were performed to understand the binding pattern of RBPJ between interphase and mitosis and to identify the genes requiring RBPJ binding for the maintenance of transcriptional memory. Our results indicate that ~60% of RBPJ occupancy in interphase is retained on mitotic chromatin, and that accounts for 80% of RBPJ in mitosis. The gene ontology analysis reveals that the genes involved in stem cell maintenance, development and differentiation-related pathways correlated with sites of RBPJ occupancy. GO analysis also suggests that RBPJ plays a role in the metabolism and processing of non-coding RNAs. Motif analysis of RBPJ binding sites reveals that not only RBPJ motif but also CTCF motif are enriched around RBPJ binding sites. From these results, we propose that RBPJ can function as a mitotic bookmark, marking genes for efficient transcriptional activation or repression upon exit from mitosis, and may play a role in higher order chromatin structure by collaborating with CTCF.

overall_design:
To compare the genomic RBPJ localization in mitotic and interphase cells, mouse F9 cells were harvested and labeled as cycling cells (containing 95% interphase and 5% mitosis cells); nocodazole treated F9 cells were harvested and labeled as mitotic cells. Cell samples were proceeded to ChIP-seq experiments, and each of the experiment contains a set of ChIP DNA product: input as the background control and IP as the RBPJ binding product. Background noise was substracted and the obtained signal was used for the comparison of interphase and mitosis by statistical analysis.

Please note that processed data (*bed) was generated from *rep1 sample (i.e. no processed-data for rep2 sample).

first: Robert

last: Lake

first: Pei-Fang

last: Tsai

first: Inchan

last: Choi

first: Kyoung-Jae

first: Hua-Ying

last: Fan


title: Gene Expression Changes in the Development of the Soybean Seed-Cotyledon Stage

city: Davis

last: Harada

address: One Shields Avenue

department: Plant Biology

institute: University of California

summary: We investigated soybean seed development because (1) soybean seeds are a major source of food and fuel, (2) soybean seeds have been an excellent system for studying the basic processes controlling seed development for over three decades, and (3) new soybean genomic resources, including the sequence of the soybean genome and the gene expression profiles for all seed compartments, tissues, and cell types, can be used to gain new insights into the regulatory processes required for seed differentiation. We sequenced messenger RNA populations of specific soybean seed compartments, which will provide new insights into gene expression that are important for “making a soybean seed.”

overall_design: Ten compartments of the Cotyledon stage of the soybean seeds were analyzed. Three biological replicates were collected for each compartment.

middle: J

middle: B

last: Goldberg

first: Ryan

last: Kirkbride

first: Julie

last: Pelletier

first: Tina

last: Wang

first: Ssu-Wei

last: Hsu

first: Shu-Heng

last: Chang

first: Russell

middle: W

last: Baden

first: Rie

last: Uzawa

first: Alexander

last: Olson

first: Lourenz

last: Balayan

first: Erik

last: Garber

first: Meryl

last: Hashimoto

first: Uranus

last: Chau


title: 3' RNA-Seq (3'SEQ) analysis gene expression profiling at G1-S transition in human embryonic stem cells (hESCs) and hESC-derived endoderm cells

first: Edward

last: Chuong

address: 596 UCB

institute: University of Colorado Boulder

summary: In this report, we examine transcripts on a genome-wide level between the synchronized cell cycles of hESCs and hESC-derived endoderm cells. We found 10347, 10299 and 10362 genes expressed in the G1, G1/S, and S phase of hESCs, respectively; 10333, 10227 and 10215 genes expressed at the G1, G1/S and S phase of derived endoderm. We compared the transcriptome between these data sets and identified genes with differentiated phase expression between hESCs and hESC-derived endoderm cells.

overall_design: Examination of the transcriptome at the G1-S transition in hESCs compared to hESC-derived endoderm cells.

first: Hui

last: Zhu

last: Baker


title: Identification of Novel Post-Transcriptional Features in Olfactory Receptor Family mRNAs

status: Public on May 13 2014

first: Miles

middle: F

last: Wilkinson

address: 9500 Gilman Dr

institute: The University of California, San Diego

summary: Olfactory receptor (Olfr) genes comprise the largest gene family in mice.  Despite their importance, most Olfr mRNAs are uncharacterized.  Using RNA-seq analysis, we annotated the repertoire of mouse Olfr mRNAs expressed in the olfactory epithelium and found that they have several atypical features suggesting how post-transcriptional regulation impacts their expression.  First, many Olfr mRNAs are intronless and those with introns typically have a single intron at the 5’ end.  Second, Olfr mRNAs, as a group, have dramatically higher average AU content and lower predicted secondary structure than do control mRNAs.  Third, Olfr mRNAs have a higher density of AU-rich elements (AREs) than control mRNAs.  Fourth, Olfr mRNAs have much shorter 3’ UTR regions and fewer predicted binding sites for neurally expressed miRNAs than control mRNAs.  Fifth, Olfr mRNAs have a significantly greater number of upstream open reading frames (uORFs) in their 5’ UTR than control mRNAs.  All of these novel properties correlated with higher Olfr expression.  Finally, we identified striking differences in the mRNAs from the two classes of Olfr genes, a finding consistent with their independent evolutionary origin.  Our study suggests that the Olfr gene family has encountered unusual selective forces that have driven these unique post-transcriptional regulatory features.

overall_design: Examine transcriptome of Olfr genes in mouse olfactory epithelium

first: Eleen

last: Shum


title: CFIm25 links alternative polyadenylation to glioblastoma tumour suppression

city: Portland

first: Zheng

last: Xia

state: Oregon

address: 3181 SW Sam Jackson Park Rd

department: Department of Biomedical Engineering

institute: Oregon Health & Science University

summary:
Purpose: To identify all of the APA targets of CFIm25 on a global scale and develop an algorithm that can idenitify APA events from standard RNA-seq data

Methods: RNA from HeLa cells treated with control siRNA and CFIm25 siRNA were subject to RNA-Seq. Using a custom-designed algorithm to mine RNA-seq data for novel APA events regulated by CFIm25.

Results: We identified over 1,400 genes with shortened 3’UTRs after CFIm25 knockdown.  Importantly, we show that as a consequence of APA, many of these mRNAs have greatly enhanced protein expression due to the loss of destabilizing features within the 3’UTR.

Conclusions: Our study underscored the critical function of the CFIm complex members in governing APA  and establish a previously unknown link between APA and metabolic pathways important for tumor progression.

overall_design: Hela cell line mRNA profiles of control treated and CFIm25 Knockdown were generated by RNA-Seq using Illumina GAIIx.

first: Chioniso

last: Masamha

first: Todd

middle: R

last: Albrecht

first: Wei

last: Li

first: Ann-Bin

last: Shyu

first: Eric

last: Wagner


title: Identification of rapidly-induced genes in the peanut (Arachis hypogaea) response to water deficit and abscisic acid

city: guang zhou

first: ling

last: li

address: South China Normal University, Guangzhou, P.R. China

institute: south china normal universty

summary:
Purpose: to screen the candidate genes involved in the peanut drought stress response, we conducted global transcriptome analysis of peanut plants challenged with water deficit and ABA, using the Illumina HiSeq2000 sequencing platform.
Methods: a sequences library of Yueyou7 were constructed at first. Then the profile of diffentialy expressed genes (DEGs) under three different treatments (control, water deficit without ABA, and water deficit with ABA)  were conducted based on above sequence library. For sequencing library construction, plants were grown under normal conditions, as described previously , Seeds were planted in soil and kep in the greenhouse at temperature of 25-30℃ and water well. Three tissues (leaves, roots, and stems) were collected at three development stages (four-leaf, flowering and podding stages), respectively. Then all of these tissues were mixed to extract the total RNA for sequence library construction.  For DEGs study, two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 µmol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel. After treatments,  Total RNA was extracted from 100 mg of plant material, and RNA integrity was checked by gel electrophoresis. Also RNA quality was checked and RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) and Nanodrop ND-1000 (Thermo Scientific, Waltham, USA).
Results: we generated 4.96×107 raw sequence reads and assembled the high quality reads into 92,390 unique genes. Compared with the control, we found that 621 genes (≥1.5 fold change) responded rapidly to water deficit and 2665 genes (≥1.5 fold change) responded rapidly to ABA. We found 279 genes that overlapped between water deficit and ABA responses, 264 genes that showed the same trend in expression while 15 genes expression that showed opposite trend. Among the identified genes, 257 showed high induction by ABA (>5 fold), and 19 showed high induction by drought (>5 fold). In addition, we identified 100 transcription factor genes among the ABA-inducible genes but only 22 transcription factor genes among the water deficit-inducible genes.
Conclusions: we identified genes differentially expressed in the early response to water deficit or ABA. These genes were annotated with GO functional categories for water deficit (33 categories) or ABA (31 categories). We found that only 19 genes were highly induced by water deficit, but 257 genes were highly induced by ABA. Our previous work has examined many of these genes and our future work will reveal their functions and relationships. These data will facilitate functional genomic studies and have established a biotechnological platform for examination of the early drought- and ABA-responsive transcriptome regulatory network in peanut.

overall_design: Two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 µmol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel.

first: Xiaoyun

first: Jiabao

last: Lu

first: Shuai

last: Liu

first: Xu

first: Yingying

last: Lin

first: Ling


title: Global transcriptomic changes following N-deprivation in Nannochloropsis oceanica IMET1: Illumina sequencing

city: Qingdao

first: Jian

last: Xu

state: Shandong province

address: No.189 Songling Rd, LaoShan District

department: BEGC

institute: QIBEBT-CAS

summary: Limited systems-level understanding of oil synthesis in wild oleaginous algae has hindered the development of microalgal feedstock. Nannochloropsis is a small unicellular microalgae widely distributed in oceans and fresh water. In many large-scale and pilot-scale outdoor cultivation facilities, Nannochloropsis strains have been found to be capable of robust growth when supplied with flue gases, naturally accumulating large quantities of oils in a stationary phase, and exhibiting resistance to environmental contaminants. The rich genomic resources, compact genomes, resistance to foreign DNA invasion, wide ecological adaptation, large collections of natural strains and the demonstrated ability to grow on a large scale suggested Nannochloropsis can serve as research models and platform strains for economical and scalable photosynthetic production of fuels and chemicals. To untangle the intricate genome-wide networks underlying the robust biomass accumulation and oil production in Nannochloropsis, we applied high-throughput mRNA-sequencing and reconstructed the structure and dynamics of the genome-wide functional network underlying robust microalgal triacylglycerol (TAG) production in Nannochloropsis oceanica, by tracking the genome-wide, single-base-resolutiontranscript change for the complete time-courses of nitrogen-depletion-induced TAG synthesis.

overall_design: Nannochloropsis oceanica IMET1 cells were grown in liquid cultures under continuous light (approximately 50 µmol photons m-2 s-1) at 25℃ and aerated by bubbling with a mixture of 1.5% CO2 in air. Mid-logarithmic phase algal cells were collected and washed three times with axenic seawater. Equal numbers of cells were re-inoculated in nitrogen replete medium (Control condition or C, i.e. N+) and nitrogen deprived medium (N deficiency or N, i.e. N-) with 50µmol m-2 s-1 light intensity, respectively. Cell aliquots were collected for RNA isolation after being transferred to the designated conditions for 3h, 4h, 6h, 12h, 24h and 48h. Three biological replicates of algal cultures were established under each of the above “C” (i.e. N+) and “N” (i.e. N-) conditions, respectively. In total, 36 samples collected at six time points (3h,4h,6h,12h,24h and 48h) were used for mRNA-Seq library preparation and then submitted to Illumina HiSeq 2000 for sequencing.

first: Jing

last: LI

last: XU


title: HDACs act on ribosomal DNA to control the yeast replication program and the competition between origins for limiting initiation factors

first: Philippe

last: Pasero

type: Genome binding/occupancy profiling by genome tiling array

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

sample_organism:
Saccharomyces cerevisiae
Saccharomyces cerevisiae W303

overall_design: Refer to individual Series


title: The ABRF Next-Generation Sequencing Study (ABRF-NGS): Multi-platform and cross-methodological reproducibility of transcriptome profiling by RNA-seq.

first: Christopher

middle: E

last: Mason

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Two reference RNA standards tested with four protocols (polyA selected, ribo-depleted, size selected, and degraded RNA), and examined across five NGS platforms (Illumina’s HiSeqs, Life Technologies’ Personal Genome Machine and Proton, Roche 454 GS FLX, and Pacific Biosciences RS).


title: SRSF10 regulates alternative splicing and is required for adipocyte differentiation.

status: Public on May 12 2014

city: Tianjin

first: Xuexia

last: Zhou

address: 154 Anshan Road

department: Tianjin Neurological Institute

institute: Tianjin Medical University General Hospital

summary: During adipocyte differentiation, significant alternative splicing changes occur in association with the adipogenic process. However, little is known about roles played by splicing factors in this process. We observed that mice deficient for the splicing factor SRSF10 exhibit severely impaired development of subcutaneous white adipose tissue as a result of defects in adipogenic differentiation. To identify splicing events responsible for this, RNA-seq analysis was performed using embryonic fibroblast cells. Several SRSF10-affected splicing events that are implicated in adipogenesis have been identified. Skipping of lipin1 exon 7 is controlled by SRSF10-regulated cis-element located in the constitutive exon 8. The activity of this element depends on the binding of SRSF10 and correlates with the relative abundance of lipin1a mRNA.  A series of experiments demonstrated that SRSF10 controls the production of lipin1a and thus promotes adipocyte differentiation. Indeed, lipin1a expression could rescue SRSF10-mediated adipogenic defects. Taken together, our results identify SRSF10 as an essential regulator for adipocyte differentiation and also provide new insights into splicing control by SRSF10 in lipin1 pre-mRNA splicing.

overall_design: RNA-seq for wide type (WT) and SRSF10-deficient (KO) mouse MEF cells

first: Wenwu

last: Wu

first: Ying

last: Feng


title: Parallel T-cell cloning and deep sequencing of the transcripts of human MAIT cells reveal stable oligoclonal TCRβ repertoire

city: Singapore

first: Michael

last: Poidinger

address: #04-06, 8A Biomedical Grove

department: Bioinformatics

institute: Singapore Immunology Network

summary: Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize conserved bacterial antigens derived from riboflavin precursors, presented by the non-polymorphic MHC class I-like molecule MR1. Here, we show via transcriptomic analysis that human MAIT cells are remarkably oligoclonal in both blood and liver, display high inter-individual homology, and exhibit a restricted length CDR3β domain of the TCRVβ chain. We extend this analysis to a second sub-population of MAIT cells expressing a semi-invariant TCR conserved between individuals.

overall_design: Study of CDR3 regions of TCRalpha and beta sequences

first: Lucia

last: Mori

first: Gennaro

last: De Libero


title: Drosophila PTB regulates dorso-ventral patterning genes in embryos

status: Public on May 10 2014

first: Joseph

last: Heimiller

address: 347 UCB

department: MCDB

institute: Univ of Colorado Boulder

summary: Drosophila PTB (Polypyrimidine Tract-binding protein dmPTB) regulates dorso-ventral patterning genes in embryos

overall_design: Comparison of wild type (yw genotype) and PTB mutant (heph03429) drosophila embryos

first: Vinod

last: Sridharan

first: Jim

last: Huntley

first: Cedric

last: Wesley

first: Ravinder

last: Singh


title: RNA-seq analysis of IL-10 treatment of wildtype and Ptpn1-/- knockout peritoneal macrophages

city: Shenzhen

first: Andrew

middle: P

last: Hutchins

address: 1088 Xueyuan Rd

institute: Southern University of Science and Technology

summary: We report here the deep sequencing of the mRNA from peritoneal exudate cells (macrophages) purified from wildtype or Ptpn1 (PTP1B) knockout mice, either treated or untreated with IL-10. In periotenal macrophages IL-10 activates the transcription factor STAT3 to execute and anti-inflammatory gene expression programme. The tyrosine phosphatase PTPN1 targets STAT3 for dephosphorylation and leads to the deactivation of STAT3. In this study we examined the role of PTP1B in controlling the normal homeostatic level phosphorylation of STAT3 by comparing the IL-10/STAT3-mediated anti-inflammatory gene expression programme. We find that loss of PTP1B leads to an up-regulation of the activity of STAT3, both at the level of phosphorylation and also in enhanced expression of anti-inflammatory gene products.

overall_design: RNA-seq of wildtype and Ptpn1 (PTP1B) knockout mouse peritoneal macrophages, treated or untreated with IL-10

first: Diego

last: Miranda-Saavedra

first: Kelly

last: Pike

first: Michel

last: Tremblay


title: Rapid proliferation and differentiation impairs the development of memory CD8+ T cells in early life

city: Ithaca

last: Grimson

state: NY

address: 445 Biotechnology Building

department: Molecular Biology & Genetics

institute: Cornell University

summary: Neonates often generate incomplete immunity against intracellular pathogens, although the mechanism of this defect is poorly understood.  An important question is whether the impaired development of memory CD8+ T cells in neonates is due to an immature priming environment or lymphocyte-intrinsic defects.  Here we show that neonatal and adult CD8+ T cells adopted different fates when responding to equal amounts of stimulation in the same host.  While adult CD8+ T cells differentiated into a heterogeneous pool of effector and memory cells, neonatal CD8+ T cells preferentially gave rise to short-lived effector cells and exhibited a distinct gene expression profile.  Surprisingly, impaired neonatal memory formation was not due to a lack of responsiveness, but instead because neonatal CD8+ T cells expanded more rapidly than adult cells and quickly became terminally differentiated.  Collectively, these findings demonstrate that neonatal CD8+ T cells exhibit an imbalance in effector and memory CD8+ T cell differentiation, which impairs the formation of memory CD8+ T cells in early life

overall_design: mRNA profiles of effector CD8+ T cells from neonatal and adult mice

first: Erin

last: Wissink


title: The ABRF Next-Generation Sequencing Study (ABRF-NGS): Multi-platform and cross-methodological reproducibility of transcriptome profiling by RNA-seq [Ion Torrent PGM]

first: Christopher

last: Mason

address: 1305 York Ave

institute: Weill Cornell Medical College

summary: Next-generation sequencing (NGS) technology applications like RNA-sequencing (RNA-seq) have dramatically expanded the potential for novel genomics discoveries, but the proliferation of various platforms and protocols for RNA-seq has created a need for reference data sets to help gauge the performance characteristics of these disparate methods.  Here we describe the results of the ABRF-NGS Study on RNA-seq, which leverages replicate experiments across multiple sites using two reference RNA standards tested with four protocols (polyA selected, ribo-depleted, size selected, and degraded RNA), and examined across five NGS platforms (Illumina’s HiSeqs, Life Technologies’ Personal Genome Machine and Proton, Roche 454 GS FLX, and Pacific Biosciences RS).  These results show high (R2 >0.9) intra-platform consistency across test sites, high inter-platform concordance (R2 >0.8) for transcriptome profiling, and a large set of novel splice junctions observed across all platforms.  Also, we observe that protocols using ribosomal RNA depletion can both salvage degraded RNA samples and also be readily compared to polyA-enriched fractions.  These data provide a broad foundation for standardization, evaluation and improvement of RNA-seq methods.

overall_design:
Two reference RNA standards tested with four protocols (polyA selected, ribo-depleted, size selected, and degraded RNA), and examined across five NGS platforms (Illumina’s HiSeqs, Life Technologies’ Personal Genome Machine and Proton, Roche 454 GS FLX, and Pacific Biosciences RS).

Please note that the samples were named following the ABRF-Platform-Site-Sample-Replicate# format. For example, ABRF-454-CNL-A-1 means Sample A was run on 454 platform at Cornell and this is the first replicate, and  ABRF-454-CNL-A-2 means the same exact sample was ran with same machine at same location and is 2nd replicate.


title: Small RNA sequencing of control and Su(var)3-7 mutant Drosophila ovary

city: Geneva

first: Denis

last: Basquin

state: Geneva

address: 30 Quai Ernest-Ansermet

department: Genetics & Evolution

country: Switzerland

institute: University of Geneva

summary: Comparison of small RNA fractions derived from piRNA clusters and transposon sequences in control and Su(var)3-7 null mutant.

overall_design: Small RNA profile of 3-day old control and Su(var)3-7 mutant ovaries were generated by high-throughput sequencing on Illumina HiSeq 2000

first: D

first: M

last: Delattre


title: Increased expression of BubR1 protects against aneuploidy in aging oocytes and extends reproductive lifespan

city: Beijing

first: yanchang

last: wei

address: #1 Beichen West Rd

institute: Institute of Zoology, Chinese Academy of Sciences

summary: Chromosome aneuploidy increases in oocytes with maternal age, and is considered the leading cause for the increased incidence of infertility, miscarriage, and birth defects. Using mRNA-Sequencing of oocytes from 12 month old mouse versus 3 month young mouse, we identified a spindle assembly checkpoint gene, BubR1, whose expression was significantly decreased. We employed a mRNA microinjection based approach to increase BubR1 expression in aging oocytes. We find that increased expression of BubR1 protects against aneuploidy and chromosome misalignment in aging oocytes. After in vitro fertilization, the embryos derived from BubR1 increased expression aging oocytes exhibited chromosome stability as robust as those of the young ones. Furthermore, following embryo transfer, these embryos showed greatly improved developmental competency, with comparable levels of full-term development to those of the young ones. These results indicate that the decline in oocyte quality may be reversible and could lead to treatments that prolong female fertility.

overall_design: Examination of the effect of maternal aging on the mRNA expression in the mature oocytes of the female mice. Naturally ovulated mature oocytes (MII stage) were collected from 6 young (3 month) and 6 aging (12 month) female mice (3 oocytes per mice, 18 oocytes for each group).

first: Yanchang

last: Wei

first: Qing-Yuan

last: Sun


title: Methylation QTLs are associated with coordinated changes in transcription factor binding, histone modifications, and gene expression levels [Bisulfite-Seq]

status: Public on May 09 2014

city: Chicago

first: Nicholas

last: Banovich

address: 920 E. 58th Street, CLSC 317

department: Human Genetics

institute: University of Chicago

summary: DNA methylation is an important epigenetic regulator of gene expression. Recent studies have revealed widespread associations between genetic variation and methylation levels. However, the mechanistic links between genetic variation and methylation remain unclear. To begin addressing this gap, we collected methylation data at ~300,000 loci in lymphoblastoid cell lines (LCLs) from 64 HapMap Yoruba individuals, and genome-wide bisulfite sequence data in ten of these individuals. We identified (at an FDR of 10%) 13,915 *cis* methylation QTLs (meQTLs), i.e., CpG sites in which changes in DNA methylation are associated with genetic variation at proximal loci. We found that meQTLs are frequently associated with changes in methylation at multiple CpGs across regions of up to 3 kb. Interestingly, meQTLs are also frequently associated with variation in other properties of gene regulation, including histone modifications, DNase I accessibility, chromatin accessibility, and expression levels of nearby genes. These observations suggest that genetic variants may lead to coordinated molecular changes in all of these regulatory phenotypes. One plausible driver of coordinated changes in different regulatory mechanisms is variation in transcription factor (TF) binding. Indeed, we found that SNPs that change predicted TF binding affinities are significantly enriched for associations with DNA methylation at nearby CpGs.

overall_design: Whole genomic DNA from 10 Yoruba HapMap individuals and spiked in unmethylated lambda phage DNA was bisuflite converted using the  Invitrogen MethylCode Bisulfite Conversion Kit and sequenced using a Illumina HiSeq 2000

first: Xun

last: Lan

first: Bryce

last: van de Geijn

first: Graham

last: McVicker

first: Jacob

last: Degner

first: John

last: Blischak

first: Jonathan

last: Pritchard

first: Yoav

last: Gilad


title: Widespread contribution of transposable elements to the innovation of gene regulatory networks [mouse ENCODE]

city: SAINT LOUIS

first: Daofeng

last: Li

state: MO

address: 4515 McKinley Avenue

institute: Washington University in St. Louis

summary:
Transposable elements (TE) have been shown to contrain functional transcription factor (TF) binding sites for long, but the extent to which TEs contribute TF binding sites is not well know. Here, we comprehensively mapped binding sites for 26 pairs of orthologous TFs, in two pairs of human and mouse cell lines (i.e., leukemia, and lymphoblast), along with epigenomic profiles representing DNA methylation and six histone modifications. We found that on average, 20% of TF binding sites were embedded in TEs. We further identified 710 TF-TE relationships in which certain TE subfamilies enriched for TF binidng sites. TE-derived TF binding peaks were also strongly associated with decreased DNA methylation and increased enhancer-associated histone marks. Most of the TE-derived TF binding sites were species-specific, but we also identified conserved binding sites. Additionally, 66% of TE-derived TF binding events were cell-type specific, associated with cell-type specific epigenetic landscape.

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:
To evaluate the contribution of transposable elements  (TE) to transcription factor (TF) binding landscapes, we profiled ChIP-seq datasets for 26 TFs in two cell lines in human and mouse, generated by the ENCODE and MouseENCODE consortia. The epigenomic profiles were evaluated from six histone modification in each of the cell lines, also generated by the consortia. We added DNA methylation to the epigenomic profiles, using two complementary techniques, MeDIP-seq and MRE-seq.

The human data related to this study are available through GSE56774:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE56774

first: Vasavi

last: Sundaram

first: Yong

last: Cheng

first: Xiaoyun

last: Xing

first: Michael

last: Snyder

first: Ting

last: Wang


title: Next Generation Sequencing of RNAs immunoprecipitated with Cyt c under hyperosmotic stress

city: Cleveland

first: Maria

last: Hatzoglou

state: OH

address: 10900 Euclid Ave

department: Pharmacology

institute: Case Western Reserve University

summary: For the detection of tiRNAs accumulated in Cyt c-IP vs tiRNAs in total cell lysates during hyperosmotic stress in the presence of angiogenin

overall_design: Total cell lysates from mouse embryonic fibroblasts treated with angiogenin under hyperosmotic stress.  Lysates split into two parts: one part serveed as the total RNA, the other part served as Cyt c-IP fraction.  Small RNAs were enriched from both parts and went through deep sequencing followed by bioinformatic analysis.

first: Mridusmita

last: Saikia

first: Marc

last: Parisien


title: Steady-state hydrogen peroxide induces glycolysis via metabolic reroute in P. aeruginosa and S. aureus

status: Public on May 08 2014

city: Hong Kong

first: Xin

last: Deng

address: To Yuen Building

department: Biomedical Sciences

country: Hong Kong

institute: City University of Hong Kong

summary: GAPDHs from human pathogens S. aureus and P. aeruginosa can be readily inhibited by ROS-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment of H2O2 induces rapid metabolic reroute from glycolysis to pentose phosphate pathway (PPP). We conducted RNA-seq analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which reveals profound transcriptional changes including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces a metabolic reroute from glycolysis to PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Given that H2O2 can be produced constitutively by the host immune response, exposure to the steady-state stress of H2O2 recapitulates more accurately bacterial responses to host immune system in vivo.

sample_organism: Staphylococcus aureus subsp. aureus str. Newman

overall_design: RNA-seq in Pseudomonas aeruginosa and Staphylococus aureus under steady state of H2O2

first: Chuan

last: He


title: Molecular architecture of transcription factor hotspots in early adipogenesis

city: Odense M

first: Susanne

last: Mandrup

address: Campusvej 55

department: Biochemistry and Molecular Biology

country: Denmark

institute: University of Southern Denmark

summary: Here, we have used digital genomic footprinting to precisely define protein localization for several adipogenic transcription factors at a genome-wide level. In combination with ChIP-seq data, these analyses reveal novel molecular insight into the organization of transcription factors at hotpot regions, which provides a new framework for understanding transcription factor cooperativity on chromatin.

overall_design: Digital genomic footprinting and gene expression in 3T3-L1 pre-adipocytes by high throughput sequencing.

first: Rasmus

last: Siersbæk

first: Songjoon

last: Baek

first: Atefeh

last: Rabiee

first: Ronni

last: Nielsen

first: Sofie

last: Traynor

first: Nicholas

last: Clark

first: Albin

last: Sandelin

first: Ole

last: Jensen

first: Myong-Hee

last: Sung

first: Gordon

last: Hager


title: mRNA-seq analysis of superficial zone (SFZ) and deeper layers (deep AC) of articular cartilage of adult rats

city: Bunkyo-ku, Tokyo

first: Yoshifumi

last: Mori

department: Orthopaedic Surgery

country: Japan

institute: Tokyo University

summary: We compared gene expression profiles of SFZ and deep AC of articular cartilage through laser microdissection (LMD) using adhesive tape, linear amplification of mRNA, and mRNA-seq analysis.

overall_design: gene expression profiles of SFZ and deep AC obtained from the proximal tibia of two adult rats

first: Taku

last: Saito


title: 4sUDRB-seq: measuring transcription elongation and initiation genomewide

city: Rehovot

first: Gilad

last: Fuchs

address: Rehovot

country: Israel

institute: Weizmann Institute of Science

summary: A new method to measure elongation and intitiation rates

overall_design: Reversal inhibition of transcription with DRB and tagging newly transcribed RNA with 4-thiouridine (4sU)

first: Yoav

last: Voichek


title: Smoking Dysregulates the Human Airway Basal Cell Transcriptome at COPD-linked Risk Locus 19q13.2

city: New York

first: Yael

last: Strulovici-Barel

address: 1300 York Avenue

department: Department of Genetic Medicine

summary:
Rationale: Genome-wide association studies (GWAS) and candidate gene studies have identified a number of loci linked to susceptibility of chronic obstructive pulmonary disease (COPD), a smoking-related disorder that originates in the airway epithelium.
Objectives: Since airway basal cell (BC) stem/progenitor cells exhibit the earliest abnormalities associated with smoking (hyperplasia, squamous metaplasia), we hypothesized that smoker BC have a dysregulated transcriptome linked, in part, to known GWAS/candidate gene loci.
Methods: Massive parallel RNA sequencing was used to compare the transcriptome of BC purified from the airway epithelium of healthy nonsmokers (n=10) and smokers (n=7). The chromosomal location of the differentially expressed genes was compared to loci identified by GWAS and candidate gene studies to confer risk for COPD.
Measurements and Main Results: Smoker BC have 676 known genes differentially expressed compared to nonsmoker BC, dominated by smoking up-regulation. Strikingly, 166 (25%) of these genes are located on chromosome 19, with 13 localized to 19q13.2 (p<10-4 compared to chance), including TGFB1, LTBP4, EGLN2 and NFKBIB, genes associated with risk for COPD.
Conclusions: These observations provide the first direct link of known genetic risks for smoking-related lung disease with the specific population of lung cells that undergoes the earliest changes associated with smoking.

overall_design: The human airway basal cell transcriptome of 7 smokers versus 10 nonsmokers was compared using massive parallel RNA sequencing (Illumina HiSeq 2000).

first: Dorothy

last: Ryan

first: Thomas

last: Vincent

first: Jacqueline

last: Salit

first: Matthew

middle: S

last: Walters

first: Francisco

last: Agosto-Perez

first: Renat

last: Shaykhiev

first: Neil

last: Hackett

first: Jason

middle: G

last: Mezey

first: Ronald

last: Crystal


title: Gene Expression Changes in the Development of the Soybean Seed-Heart Stage

status: Public on May 07 2014

city: Davis

first: John

last: Harada

address: One Shields Avenue

institute: University of California

summary: We investigated soybean seed development because (1) soybean seeds are a major source of food and fuel, (2) soybean seeds have been an excellent system for studying the basic processes controlling seed development for over three decades, and (3) new soybean genomic resources, including the sequence of the soybean genome and the gene expression profiles for all seed compartments, tissues, and cell types, can be used to gain new insights into the regulatory processes required for seed differentiation. We sequenced messenger RNA populations of specific soybean seed compartments, which will provide new insights into gene expression that are important for “making a soybean seed.”

overall_design: Eight compartments of the heart stage of the soybean seeds were analyzed. Three biological replicates were collected for each compartment.

first: Robert

last: Goldberg

first: Ryan

last: Kirkbride

first: Julie

last: Pelletier

first: Tina

last: Wang

first: Ssu-Wei

last: Hsu

first: Shu-Heng

last: Chang

first: Russell

last: Baden

first: Rie

last: Uzawa

first: Alexander

last: Olson

first: Lourenz

last: Balayan

first: Erik

last: Garber

first: Meryl

last: Hashimoto

first: Uranus

last: Chau


title: Culex pipiens pallens and Cx. p. quinquefasciatus with different resistant level

first: Chunxiao

last: Li

address: 20 Dongdajie, Fengtai District

institute: Beijing Institute of Microbiology and Epidemiology

summary:
Culex pipiens pallens and Cx. p. quinquefasciatus are important vectors of many diseases, such as West Nile fever and lymphatic filariasis. The widespread use of insecticides to control these disease vectors and other insect pests has led to insecticide resistance becoming common in these species. High throughput screening using SSH and specific microarray platforms was thought to have identified some resistance-related genes. However, limitations of these methods meant that only a few hundred of the many thousand genes could be screened. It wasn’t until the sequencing of the Cx. quinquefasciatus genome in 2010 that it became possible to screen all 18.9 thousand genes in the mosquito genome for anti-insecticidal activity. We used high throughout Illumina sequencing to identify hundreds of Cx. p. pallens and Cx. p. quinquefasciatus genes that were differentially expressed in response to pesticide exposure. The identification of these genes is a vital first step for more detailed investigation of the molecular mechanisms involved in insecticide resistance in mosquitoes.
In this study, larvae of Cx. pipiens pallens and Cx. pipiens quinquefasciatus were collected from field and transported to the laboratory and reared to adulthood to get F1 generation. Then, half of the F1 generation was conducted to pesticide bioassay. RNA extraction and Illumina sequencing were undertaken in another half of the F1 generation. Therefore, Samples used in Illumina sequencing did not contact any insecticides.

sample_organism:
Culex quinquefasciatus
Culex pipiens pallens

overall_design: Twelve Cx. pipiens pallens and Cx. pipiens quinquefasciatus lavae were undertaken Illumina RNA sequencing.

first: Tongyan

last: Zhao


title: Widespread contribution of transposable elements to the innovation of gene regulatory networks [human ENCODE]

first: Daofeng

department: Genetics

overall_design:
To evaluate the contribution of transposable elements (TE) to transcription factor (TF) binding landscapes, we profiled ChIP-seq datasets for 26 TFs in two cell lines in human and mouse, generated by the ENCODE and MouseENCODE consortia. The epigenomic profiles were evaluated from six histone modification in each of the cell lines, also generated by the consortia. We added DNA methylation to the epigenomic profiles, using two complementary techniques, MeDIP-seq and MRE-seq.

The mouse data related to this study are available through GSE57230:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57230

first: Vasavi

last: Sundaram

first: Yong

last: Cheng

first: Xiaoyun

last: Xing

first: Michael

last: Snyder

first: Ting


title: Differential requirement for Nfil3 during NK cell development

city: Melbourne

first: Sebastian

last: Carotta

address: 1G Royal Parade

country: Austria

institute: WEHI

summary: Natural killer (NK) cells can be grouped into distinct subsets that are localized to different organs and exhibit different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor nuclear factor interleukin 3 (Nfil3; E4bp4) is essential for bone marrow-derived NK cell development but it is not clear whether Nfil3 is equally important for all NK cell subsets nor how it induces NK lineage commitment. Here we show that Nfil3 is required for the formation of Eomesodermin (Eomes)-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL+ Eomes- NK cells develop independent of Nfil3. Loss of Nfil3 during the development of bone marrow-derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3-/- progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cell by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3.

overall_design: RNA-sequencing of natural killer (NK) cell subsets

first: C

last: Seillet

first: N

last: Huntington

first: P

last: Gangatirkar

first: M

last: Minnich

first: G

middle: T

last: Belz

first: S


title: Dominance hierarchy arising from the evolution of a complex small RNA regulatory network

city: Villeneuve d'Ascq

first: Vincent

last: Castric

address: Lille1 university

institute: CNRS

summary: Self-pollen rejection in the Brassicaceae is determined by the diploid genotype of the pollen-producing plant, and it has long been known that the alleles show a dominance hierarchy. How this hierarchy is controlled and evolves has been a classical puzzle since the pre-molecular days of genetics. Here, we uncover the system of at least 17 small RNA (sRNA) producing loci and their multiple target sites that collectively control the dominance hierarchy among alleles of a self-incompatible Arabidopsis species. Our results demonstrate that natural selection shapes a dynamic repertoire of sRNA/targets interactions by jointly acting on sRNA genes, their processing precision and their target sites. It is remarkable that a single gene can evolve such a complex system of regulation among its own alleles.

sample_organism:
Arabidopsis lyrata
Arabidopsis halleri

platform_organism:
Arabidopsis lyrata
Arabidopsis halleri

overall_design: Sequencing of small RNAs from A. halleri floral buds, with this single exception (Al14, from A. lyrata).

first: Martin

last: Figeac


title: Differential gene expression by suppression of either SOX2 or TP63 in KYSE70 human esophageal squamous carcinoma cell line.

city: Boston

first: Shouyong

last: Peng

state: MA

address: 450 Brookline Avenue

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary: SOX2 is a transcription factor essential for pluripotent stem cells, and development and maintenance of squamous epithelium. We previously reported SOX2 an oncogene subject to highly recurrent genomic amplification in squamous cell carcinomas (SCCs)1.  Here we demonstrate in SCCs that SOX2 interacts with another master squamous transcription factor p63, and through ChIP-seq show that genomic occupancy of SOX2 overlaps with that of p63 at a large number of loci and that they cooperatively regulate gene expression including ETV4, which we find essential for SOX2-amplified SCC cell survival. Furthermore, SOX2 binds to distinct genomic loci in SCCs than in embryonic stem cells and the SOX2-p63 coordinate binding is unique to SCC.  In addition, a subset of SOX2 genomic binding sites in SCC that lack p63 co-occupancy are co-occupied by the AP-1 transcriptional complex.  These demonstrate that SOX2’s actions in SCC differ substantially from its role in pluripotency and identify novel SOX2 interactions that will enable deeper characterization of SOX2’s function in SCC.

overall_design: KYSE70 cells with stable expression of either pLKO-Tet-Op-shSOX2 or pLKO-Tet-Op-shTp63 were treated with 50ng/ml of doxycyline for 4 days. Total RNA was extracted, polyA+ selected, reverse transcribed, library constructed and sequencing was performed with Illumina HiSeq 2000. Differencial gene expression between the stable cell lines with Dox-induced and non-Dox treated was analyzed to determine the effects by suppression of either SOX2 or TP63 in KYSE70 cells.

first: Hideo

last: Watanabe

first: Qiuping

last: Ma


title: Nucleosome repositioning links DNA (de)methylation and differential CTCF binding during stem cell development

city: Colchester

first: Vladimir

last: Teif

address: Wivenhoe Park

department: School of Life Sciences

institute: University of Essex

summary: During differentiation of embryonic stem cells, chromatin reorganizes to establish cell type specific expression programs. Here, we have dissected the linkages between DNA methylation (5mC), hydroxymethylation (5hmC), nucleosome repositioning and binding of the transcription factor CTCF during this process. By integrating MNase-seq and ChIP-seq experiments in mouse embryonic stem cells (ESC) and their differentiated counterparts with biophysical modeling, we find that the interplay between these factors depends on their large-scale genomic context. The mostly unmethylated CpG islands have a reduced nucleosome occupancy and are enriched in cell type-independent binding sites for CTCF. The few remaining methylated CpG dinucleotides are preferentially associated with nucleosomes. In contrast, outside of CpG islands most CpGs are methylated and their average density oscillates so that it is highest in the linker region between nucleosomes. Outside CpG islands binding of TET1, an enzyme that converts 5mC to 5hmC, is associated with labile, MNase-sensitive nucleosomes. Such nucleosomes are poised for eviction in ESCs and become stably bound in differentiated cells where the level of TET1 and 5hmCs goes down. In particular, this has a dramatic effect at variable CTCF sites enriched outside CpG islands, that are occupied by CTCF in ESCs but loose CTCF during differentiation. To rationalize this cell type dependent targeting of CTCF binding in competition with the histone octamer, we have developed a quantitative biophysical model, which allowed predicting differences in CTCF binding due to TET1/5hmC/5mC-dependent nucleosome repositioning.

overall_design: MNase-seq experiments for analysis of mononucleosomes were conducted as described previously (Teif et al. Nature Struct. Mol. Biol., 2012). In addition, a dinucleosome fraction also was gel purified and sequenced. Briefly, embryonic stem cells from 129P2/Ola mice were cultured in ESGRO complete medium (Millipore), harvested and resuspended in low salt buffer (10 mM Hepes, pH 8, 10 mM KCl, 0.5 mM DTT) at 4 °C. After disruption of the cells with a douncer the nuclei were collected by centrifugation and washed once with the MNase Buffer (10 mM Tris–HCl, pH 7.5, 10 mM CaCl2), resuspended in the MNase Buffer and digested with 0.5 units MNase per microliter (Fermentas) and incubation for 6–11 minutes at 37 °C. The MNase digestion was stopped by putting the samples on ice and adding EDTA to a concentration of 10 mM. After digestion with 0.1 µg µl–1 RNase A (Fermentas) and removal of protein by phenol and chloroform extraction, the DNA was ethanol precipitated and the resulting DNA pellet was dissolved in H2O. DNA fragments corresponding to mononucleosomes or dinucleosomes were separated on a 2 % agarose gel using an E-Gel electrophoresis system (Life Technologies). The libraries for sequencing were prepared according to the standard Illumina protocol. High-throughput paired-end sequencing of at least 50 bp read length was performed on the Illumina HiSeq 2000 platform at the DKFZ sequencing core facility in Heidelberg, Germany. We obtained about 150 million nucleosome positions per sequencing reaction. ChIP-seq experiments were conducted as described previously (Teif et al. 2012). For each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes, pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 µg of either H3K9me3 (Abcam ab8898) or H3K4me3 (Abcam ab8580) antibody over night. After washes with sonication (10 mM Tris–HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na–deoxycholate), high-salt-buffer (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na–deoxycholate, 0.1% SDS), lithium buffer (20 mM Tris–HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na–deoxycholate) and 10 mM Tris–HCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, DNA was purified and cloned in a barcoded sequencing library for the Illumina HiSeq 2000 sequencing platform (single reads of 50 bp length).

first: Karsten

last: Rippe


title: Methylation QTLs are associated with coordinated changes in transcription factor binding, histone modifications, and gene expression levels

first: Nicholas

last: Banovich

type: Methylation profiling by genome tiling array

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

DNA methylation is an important epigenetic regulator of gene expression. Recent studies have revealed widespread associations between genetic variation and methylation levels. However, the mechanistic links between genetic variation and methylation remain unclear. To begin addressing this gap, we collected methylation data at ~300,000 loci in lymphoblastoid cell lines (LCLs) from 64 HapMap Yoruba individuals, and genome-wide bisulfite sequence data in ten of these individuals. We identified (at an FDR of 10%) 13,915 *cis* methylation QTLs (meQTLs), i.e., CpG sites in which changes in DNA methylation are associated with genetic variation at proximal loci. We found that meQTLs are frequently associated with changes in methylation at multiple CpGs across regions of up to 3 kb. Interestingly, meQTLs are also frequently associated with variation in other properties of gene regulation, including histone modifications, DNase I accessibility, chromatin accessibility, and expression levels of nearby genes. These observations suggest that genetic variants may lead to coordinated molecular changes in all of these regulatory phenotypes. One plausible driver of coordinated changes in different regulatory mechanisms is variation in transcription factor (TF) binding. Indeed, we found that SNPs that change predicted TF binding affinities are significantly enriched for associations with DNA methylation at nearby CpGs.

overall_design: Refer to individual Series


title: Changing the transcriptome by altering ZmPIN1a expression

status: Public on May 06 2014

city: jinan

first: juren

last: zhang

address: shanda nanlv 27

institute: shandong university

summary: A genome-wide transcriptome analysis showed that altering ZmPIN1a expression led to wide-ranging gene expression changes. When comparing overexpression lines A17 with the WT, 2975 genes were differentially expressed with 793 up-regulated and 2182 down-regulated in the leaves, and 2129 genes were differentially expressed with 938 up-regulated and 1191 down-regulated in the roots. GO analysis indicated that these differentially expressed genes participate in multiple biological processes from hormone signaling to metabolism. The local biosynthesis, PAT and signaling of auxin were altered; some was directly employed in plant developments such as AUX/IAA, ARFs and genes related to PAT. The genes involved in ethylene, GA, BR CK and ABA metabolism and signaling were altered as well. These phytohormones were also confirmed as key regulators in plant development and abiotic stress responses. Some of the differentially expressed genes between A17 and WT were identified as Arabidopsis root morphology and development mutant genes. These mutants were abnormal in their plant hormone synthesis and signaling, calcium-mediated signaling, MAP kinase signaling, transcription factors, membrane transporters etc. This finding suggested that ZmPIN1a overexpression led to a change of the auxin signaling transduction and even the metabolism of auxin and the metabolism and signaling of other hormones. These events led to changes in the expression of numerous genes and resulted in the modification of plant morphology, especially the root architecture.

overall_design: Maize (Zea mays L.) inbred line DH4866, its ZmPIN1a sense transgenic lines A17 and antisense transgenic lines a55 were used in this study. The transcriptome by altering ZmPIN1a expression was done by comparing the transcriptome of WT and transgenic lines both in root and leaf of V3 stage plants.

first: Zhaoxia

last: Li

first: Juren

last: Zhang


title: Arabidopsis phytochrome A directly targets numerous promoters for individualized modulation of genes in wide range of pathways [ChIP-seq]

city: Shenzhen

first: Bosheng

state: Guangdong

address: SUStech Huiyuan #1 406

institute: SUSTC (current, may different with previous institution )

summary: The far-red light (FR) photoreceptor phytochrome A (phyA) contains no DNA binding domain but associates with CHS promoter through its chaperon FHY1 and transcription factors to regulate transcription. Here, we performed a genome-wide identification of phyA targets using a combination of phyA ChIP- and RNA-sequencing methods. Our results indicate that the phyA signaling widely impact gene promoters involved in multiple FR-modulated aspects of plant growth. Furthermore, there was an enrichment of hormones- and stresses-responsive elements in the phyA direct target promoters, indicating that a much broader than expected range of transcription factors are involved in the phyA signaling. To verify our hypothesis that phyA regulates genes other than light-responsive ones through interaction with corresponding transcription factors, we examined the phyA action on one of its direct target gene NAC019, encoding an ABAdependent transcription factor. The phyA signaling cascade, consisting of FHY1 and HY5, not only targets two G-boxes on NAC019 promoter for subsequent transcriptional regulation, but also positively coordinates with ABA response for the root elongation inhibition under FR. Our study provides new insights into how plants rapidly fine-tune their growth strategy upon dynamic light environment by escorting photoreceptors to the promoters of hormones- or stresses-responsive genes for individualized modulation.

overall_design: Four day-old PphyA: phyA-GFP phyA201 transgenic seedlings were irradiated with 3h FR for ChIP with an anti-GFP antibody, which was previously used to detect phyA-GFP upon FR irradiation . Three biologically distinct phyA associated-DNA samples were subjected to library construction and high-throughput Solexa (Illumina) sequencing. An input DNA sample (genomic DNA before antibody immunoprecipitation in ChIP) was sequenced in parallel to exclude false positive signals caused by preferential PCR on certain genomic regions in the process of library construction .

first: Fang

last: Chen

first: Gang

first: Jean-Benoit

last: Charron

first: Mingqiu

last: Daie

first: Xiarong

last: Shi

first: Xing

last: Deng


title: Comparative analysis of TAL1 binding in TSA-treated versus non-treated Endothelial Colony Forming Cells (ECFCs)

city: Ottawa

first: Marjorie

last: Brand

state: Ontario

address: 501 Smyth Road

country: Canada

institute: Ottawa Hospital Research Institute

summary: Endothelial progenitors represent one of the most promising cell-based strategies for vascular repair of ischemic tissue damage, including limb ischemia, myocardial infarction and stroke. We have shown that the transcription factor TAL1 regulates a transcription program that drives the migration and adhesion of ECFCs. Furthermore,  treatment of ECFCs with the HDAC inhibitor TSA increases the expression of TAL1-dependent genes and promotes the migration, chemotaxis and adhesion of ECFCs. Finally, ex vivo treatment with TSA also improves the vascular repair properties of ECFCs in vivo when these cells are transplanted in a mouse model of hindlimb ischemia. The goal of this experiment was to test whether TSA treatment of ECFCs affect TAL1 genomic binding.

overall_design: TAL1 ChIP-sequencing was performed from ECFCs that have been treated or not TSA. As negative controls, we performed Mock-ChIP-seq from the same samples using normal IgG instead of the TAL1 antibody. Overall, we find that there is no change in TAL1 genomic binding in ECFCs upon TSA treatment.

first: Carmen

last: Palii

first: Alexander

last: Griffith

first: Alphonse

last: Chu


title: Genomic binding profile of TAL1 in human Endothelial Colony-Forming Cells

summary: Endothelial colony-forming cells (ECFCs) have been reported as promising cells for regenerative medicine thanks to their angiorepair properties. Transcription factors are primary determinants of the functional capacity of the cells and TAL1 has been shown as a critical regulator of endothelial lineage in both development and adult life. However, only few (three) TAL1 targets have been identified so far in mouse and human endothelial cells. This ChIP-seq experiment was designed to identify genome binding/occupancy of TAL1 by ChIP and high throughput sequencing in primary human endothelial stem/progenitor cells.

overall_design: TAL1 ChIP and IgG ChIP (negative control) were performed in crosslinked ECFCs derived from human umbilical cord blood.

first: Erinija

last: Pranckeviciene

first: Carolina

last: Perez-Iratxeta


title: Arabidopsis phytochrome A directly targets numerous promoters for individualized modulation of genes in wide range of pathways [RNA-seq]

overall_design: The wild-type seedlings and the phyA-1 mutant (both of the Landsberger ecta [Ler] ecotype) were grown in the same conditions used for the phyA ChIP-seq analysis (D4d+FR3h) prior to RNA isolation. Three independent biological replicates were subjected to RNA-seq analysis.


title: Small RNA deep sequencing of cellular subcompartments

city: Baltimore

first: Marikki

last: Laiho

state: MD

address: 1550 Orleans Street, CRB II, Room 444

department: Department of Radiation Oncology and Molecular Radiation Sciences

institute: The Johns Hopkins University School of Medicine

summary: RNA deep sequencing efforts have revealed abundant expression of small RNAs derived from small nucleolar (sno) RNAs. We employ here spatial RNA deep sequencing to assess the expression of 10-40 nt small RNAs in subcellular compartments of HeLa cells. Total cellular, cytoplasmic, nuclear and nucleolar fractions were isolated, RNA was purified and size-fractionated in a two-step process to yield 10-40 nt RNA fractions. cDNA libraries were constructed and sequenced on a Ion Torrent platform. Vast majority of cellular, cytoplasmic and nuclear small RNA reads were identified as miRNAs. We found the expression of eleven ten miRNAs in the nucleolar preparations using a cut-off rate of 10 reads. Several miRNAs had a greater relative abundance in the nucleolus compared to the other compartments. The nucleolar small RNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped both 5’ and 3’ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads from SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as 20 and 25 nt RNAs. This study reveals a rich representation of cell-compartment specific expression of small RNAs and the distinctive unique composition of the nucleolar small RNAs.

overall_design: Examination of small RNAs in cellular subcompartments

first: B

last: Bai

first: M

first: S

last: Wheelan

last: Yegnasubramanian


title: Developmental transcriptome analysis of human erythropoiesis

status: Public on May 05 2014

city: Ann Arbor

first: Lihong

address: 3608 BSRB, 109 Zina Pitcher Place

institute: University of Michigan

summary: We profiled the dynamic, comprehensive transcriptome during human erythroid differentiation in vitro. The erythroid cells at day 4, 8, 11 and 14 differentiation stages were harvested and sequenced by Illumia 72 bp paired-end sequencing format, respectively.

overall_design: Expression profiling of erythroid cells on differentiation days 4, 8, 11 and 14 and performed mRNA-seq on two biological replicates at each stage.

first: Yu-Hsuan

last: Lin

middle: C

last: Sierant

first: Kim-Chew

last: Lim

first: Shuaiying

last: Cui

first: Fan

last: Zhu

first: Yuanfang

last: Guan

first: Maureen

last: Sartor

first: Osamu

last: Tanabe

first: James

last: Engel


title: Nascent elongating transcript sequencing (NET-seq) for Escherichia coli and Bacillus subtilis reveals a consensus pause sequence enriched at translation start sites.

city: San Francisco

first: Matthew

middle: H

last: Larson

address: 499 Illinois St

institute: Grail, Inc.

summary: Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing we identify a 16 nt consensus pause sequence in E. coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP/nucleic-acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and B. subtilis. Our results thus implicate a conserved mechanism unifying known and newly identified pause events.

sample_organism:
Escherichia coli
Bacillus subtilis

platform_organism:
Escherichia coli
Bacillus subtilis

overall_design: Examination of nascent transcripts in E. coli and B. subtilis. 6 samples of E. coli NET-seq, 1 sample of E. coli mRNA-seq, and 1 sample of B. subtilis NET-seq.

first: R

last: Mooney

first: J

middle: M

last: Peters

first: T

last: Windgassen

first: D

last: Nayak

first: C

last: Gross

last: Block

first: W

last: Greenleaf

last: Landick

last: Weissman


title: Arabidopsis phytochrome A directly targets numerous promoters for individualized modulation of genes in wide range of pathways

first: Bosheng

last: Li

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

first: Fang

last: Chen

first: Gang

first: Jean-Benoit

last: Charron

first: Mingqiu

last: Daie

first: Xiarong

last: Shi

first: Xing

last: Deng


title: RNA-Seq based characterization of long non-coding RNA involved in respiratory viruses pathogenesis

city: Seattle

first: Michael

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

summary: We aimed at systematically inferring the regulatory functions of host lncRNAs in response to influenza A virus (IAV) and severe acute respiratory syndrome coronavirus (SARS-CoV) in the mouse model, using a ‘guilt-by-association’ approach which relies on finding which lncRNAs have similar expression profiles to protein-coding genes of known function. To build a large panel of diverse host responses to viral infection, we took advantage of the genetic diversity present in the 8 founder strains of the Collaborative Cross (CC) mouse resource. Extensive pulmonary host-response profiling was performed on mock and viral-infected lungs at 2 and 4 days post-infection using total RNA-Seq. Overall lncRNAs accounted for about 40% of total genes differentially expressed upon infections (5,329 DE lncRNAs). To predict the functions of these lncRNAs, we constructed a co-expression network using the weighted correlation network analysis (WGCNA) and identified modules of co-expressed genes. Several lncRNAs were identified as belonging to gene modules associated with viral replication or weight loss, and enriched in various infection-related biological processes such as immune response. In addition, each lncRNA was individually annotated using ranked list of DE genes based on signed correlation with each LncRNA. We predicted that a few lncRNAs may be implicated in more than 50 biological processes or pathways. Finally, we identified the lncRNAs that were positively or negatively correlated with all their neighbors and may have cis enhancer or inhibitor effects. We validated our prediction by examining additional RNASeq dataset of mice treated with IFN-alpha. Altogether, these results provide a broad categorization of lncRNA functions and identify subsets of lncRNAs with potential key roles for respiratory virus pathogenesis. These data are fully accessible through The MOuse NOn-Code Lung database (MONOCLdb) http://monocldb.viromics.washington.edu/

overall_design: At 2 or 4 days post infection, Collaborative Cross founders mice (n=2-3 for infected conditions, n=2 for mocks) were euthanized and lungs were used for total RNA-Seq.

first: Laurence

last: Josset

first: Nicolas

last: Tchitchek

first: Lisa

last: Gralinski

first: Martin

last: Ferris

first: Richard

last: Green

last: Thomas

first: Fernando

last: Pardo-Manuel de Villena

first: Ralph

last: Baric

first: Mark

last: Heise

first: Xinxia

last: Peng