title: Genome-wide measurement of chromatin accessibility by RED-seq

city: Singapore

first: Benson

last: Chen

address: 60 Biopolis Street, Genome, #04-01

department: Genome Institute of Singapore

country: Singapore

institute: A*STAR

summary: Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. DNA accessibility is primarily determined by the presence or absence of nucleosomes. At present, methods for mapping the positions and occupancy of nucleosomes genome-wide (MNase-seq) have uncovered the nucleosomal landscapes of different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays. Here we describe a new genome-wide method, RED-seq (deep sequencing analysis of Restriction Enzyme Digestion), that measures chromatin accessibility in an unbiased way. Using this method, we identified differences in chromatin accessibility between populations of cells, not only in broadly nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the more widespread regions of nucleosomal occupancy. Furthermore, RED-seq identified both large differences in chromatin accessibility in distinct cell lineages and subtle changes observed during differentiation of mouse embryonic stem cells (ESCs). Most notably, using RED-seq, we detected differences in accessibility among sequences occupied by distinct nucleosome variants. Therefore, RED-seq provides unique insights into genome-wide chromatin dynamics.

overall_design:
Examination of chromatin accessibility during ESC differentiation.
We developed a new throughput method, REDseq, to measure chromatin accessibility by restriction enzyme digestion. For proof of concept, we made several REDseq libraries and compared chromatin accessibility in different cell lines. First, we compared chromatin accessibility between chromatin, sample 3 (ESC_chromatin) and naked DNA, sample 4 (ESC_DNA). Next, we examined chromatin accessibility between undifferentiated (sample 1, ESC) and well-differentiated cells (sample 2, iMEF). In order to test the sensitivity of our new method, we tried to detect the change in chromatin accessibility during ESC differentiation. ESCs were differentiated by Oct4 knockdown (sample 6, ESC_Oct4KD) or GFP knockdown as control (sample 5, ESC_GFPKD). The knockdown experiments were performed by esiRNA transfection in sample 5 and sample 6. We used E14, a ESC cell line, in this study and the immortalized MEF were isolated from C57BL/6 mouse strain.

first: Thomas

last: Fazzio

first: Poshen

middle: B


title: Identification of microRNA-like RNAs in the filamentous fungus Trichoderma reesei by Solexa sequencing

status: Public on Dec 31 2014

city: Shenzhen

first: Kang

last: Kang

state: Guangdong

address: Nanhai Road No. 3688

department: College of life science

country: China

institute: Shenzhen University

type: Non-coding RNA profiling by high throughput sequencing

summary: MicroRNAs (miRNAs) are small non-coding RNAs capable of negatively regulating gene expression. Trichoderma reesei is an industrial filamentous fungus that can secrete abundant hydrolases for cellulosic biofuels. Recently, microRNA-like RNAs (milRNAs) were discovered in several filamentous fungi rather than T. reesei. The purpose of this study was to explore the presence of milRNA in T. reesei, to characterize the differential expression of T. reesei milRNA under cellulose induction, and to reveal the target genes of milRNA involved in cellulase production. Two small RNA libraries of cellulose induction (IN) or non-induction (CON) were generated and sequenced using Solexa sequencing technology. A total of 664,463 and 529,545 unique sequences, representing 1,271 and 1,021 unique small RNAs, were obtained from the IN and CON samples, respectively. Thirteen milRNAs were finally identified in T. reesei using the hairpin structure analysis. The milRNAs profiles obtained in deep sequencing were validated by RT-qPCR assay. The miRanda program predicts a number of potential targets for T. reesei milRNAs, including several hydrolases and carbon catabolite repressor Cre1.The presence and differential expression of T. reesei milRNAs, along with their predicted targets indicate that milRNAs might play a regulatory role in cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application.

overall_design: Evaluation of the small RNA expression in Trichoderma reesei under cellulase induction (IN) or non-induction (CON) conditions

first: Jiasheng

last: Zhong

first: Liang

last: Jiang

first: Gang

last: Liu

first: Yuan

last: Gou

first: Qiong

last: Wu

first: Jun

last: Luo

first: Deming


title: Identification of targetted gene loci by MIXL1 in the AML cell line U937

city: Houston

first: Lalitha

last: Nagarajan

state: TX

address: 6767 Bertner

department: Genetics

country: USA

institute: Unviersity of Texas MD Anderson Cancer Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: While the role of the paired-type homeobox transcription factor MIXL1 in early ebryonic development has been previously established, very little is known about the role of MIXL1 in adult hematopoiesis. To determine what role MIXL1 plays in adult hematopoietic cells, we used an established enforced expression line for FLAG-tagged MIXL1 in U937, then isolated targetted regions of the genome by ChIP-Seq.  Through this analysis, we identified new putative direct transcirptional targets for MIXL1 in AML and hematopoiesis.

overall_design: In attempt to identify the downstream role of MIXL1 in adult hematopoietic cells, we used two previously established MIXL1 overexpression lines (1MIXL and 2MIXL) and empty vector control (Control) clone of the AML cell line U937.  90% RPMI/10% FBS for 48 hours at a starting concentration of 1x105/ml before harvestings.  Protein-DNA complexes were isolated from sonicated, crosslinked whole cell lysates using IgG mouse (IgG Control) or Flag antibodies. The purified DNA from these complexes for each sample was  sequenced and analyzed for ChIP-Seq, then experimental samples (Flag-IP 1MIXL and 2MIXL) were normalized to control samples (Flag-IP or IgG-IP Control) to determine which regions of the genome MIXL1 interacted with.

first: Raymond

last: Aaron

first: Lu

last: Yue

first: Nagarajan

last: Lalitha


title: Somatic activity of the piRNA pathway in the mouse

city: Los Angeles

first: Dario

last: Boffelli

state: CA

address: 700 N San Vicente Blvd

department: Pediatrics

institute: Cedars Sinai

summary: The piRNA pathway is necessary for the suppression of retrotransposon activity in germ cells and the maintenance of germ cell populations, but the existence of the pathway in adult somatic cells has not been demonstrated. This study investigates the activity of the piRNA pathway in somatic cells of the adult mouse. We find that the PIWI protein MIWI2 is expressed in a variety of somatic tissues. Immunoprecipitation of MIWI2 from extracts of spleen cells and Friend virus-transformed mouse erythroblasts (MEL cells) identifies abundant piRNAs encoded by multiple intergenic clusters. In comparison to MIWI2-bound testis piRNAs, the somatic piRNAs are slightly smaller, and markedly deficient in repeat-derived sequences, but they have the same preference for a uracil at the 5’ end; “ping-pong” amplification is absent.

overall_design: Determination of MIWI2-bound piRNAs in mouse erythropoietic cells


title: Deep sequencing of small RNA libraries reveals dynamic expression patterns of miRNAs in multiple developmental stages of Bactrocera dorsalis

city: Chongqing

first: Yong

last: Huang

address: Tiansheng Road

institute: Southwest University

summary: Four developmental stage small RNA libraries including Eggs, Larvae, Pupae and Adults were constructed, where a lot of known miRNAs were identified and many novel miRNAs were predicted. Comparison of their expression profiles in the four libraries suggested that Bactrocera dorsalis miRNAs are dynamically regulated throughout the life cycle.Further analysis of the expression and function of these miRNAs could increase our understanding of regulatory networks in the insect and lead to novel approaches to its control.

overall_design: The total RNAs from eggs, larvae, pupae and adults stages of Bactrocera dorsalis were used to construct small RNA libraries for Solexa sequencing.The data from Solexa sequencer (Illumina) was compared to identify small RNAs in different stages of Bactrocera dorsalis.

first: Wei

last: Dou

first: Jinjun

last: Wang


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

status: Public on Dec 30 2014

first: deqiang

last: sun

address: 2121 W Holcombe Blvd

institute: Texas A&M University

summary: Long non-coding RNAs (lncRNAs) have recently emerged as new players in gene expression regulation. Whether and how lncRNAs might control hematopoietic stem cell (HSC) function remains largely unknown. Here, we profiled the transcriptome of purified long-term HSCs by deep RNA-sequencing and identified thousands of un-annotated transcripts of which 323 are predicted to be lncRNAs. Comparison of their expression in differentiated lineages represented by B cells (B220+) and Granulocytes (Gr1+), revealed that 159 are likely to be HSC-specific. Knockdown of two such non-coding genes (LincHSC-1 and LincHSC-2) indicated that they regulate HSC lineage differentiation, possibly via targeting cell cycle regulators and chromatin modification enzymes. Taken together, we comprehensively identify lncRNAs in HSC and show to examples that play important roles in HSC function.

overall_design: (I) Mouse Granulocytes mRNA profile were generated by deep sequencing, in duplicate, using Illumina Hiseq 2000. (II) To identify LncHSC target gene, mouse progenitor Sca-1+ cells were isolated from 5-FU injected mice and transduced with GFP+ retrovitus construct to knockdown lncHSC expression, and after in vitro culture for 2 days, KSL-GFP+ cells were sorted for mRNA profiling. (III) Mouse B-cells mRNA profile were generated by deep sequencing, in duplicate, using Illumina Hiseq 2000. (IV) Mouse HSC H3K4me1 and H3K27ac ChIP-seq were generated using 50,000-100,000 primary HSCs by deep sequencing using Illumina Hiseq 2000.

first: B

last: Rodriguez

first: D

last: Sun

first: W

last: Li

first: M

last: Goodell


title: Anti-ZFX ChIP-seq in a human medulloblastoma cell line

city: New York

first: Colin

last: Palmer

state: New York

address: 701 W. 168th Street

department: Microbiology & Immunology

institute: Columbia University

summary: The Hedgehog (Hh) signaling pathway regulates normal development and cell proliferation, whereas its aberrant activation causes tumor formation. Hh-induced tumors can arise from different tissues and can be indolent or highly aggressive, such as basal cell carcinoma (BCC) of the skin and neural progenitor-derived medulloblastoma (MB), respectively. Little is known about cell-intrinsic factors that control the development of such diverse Hh-dependent tumors. Transcription factor Zfx is required for the self-renewal of several stem cell types, whereas its role in malignant transformation remains controversial. We found that the deletion of Zfx prevented BCC formation and significantly delayed MB development caused by Hh activation in vivo. In contrast, Zfx was dispensable for the development of Hh-independent brain tumor glioblastoma. We used genome-wide expression and chromatin binding analysis in a human MB cell line to identify direct, evolutionarily conserved targets of Zfx. These targets included the Hh signal transducer Smoothened (Smo), suggesting that Zfx may directly control Hh pathway activation in tumors. Two additional targets of Zfx, Dis3L and Ube2j1, were also required for the growth of MB cells in vitro. These results identify a common cell-intrinsic regulator of diverse Hh-induced tumors, and suggest Zfx and Zfx-controlled genes as possible therapeutic targets in these malignancies.

overall_design: Analysis of ZFX binding to chromatin and genomic DNA in the human medulloblastoma cell line DAOY

middle: J

first: Boris

middle: V

last: Reizis


title: Expression Analysis of Normal and Neoplastic Mouse Pancreatic Ductal Organoids

city: Cold Spring Harbor

first: David

middle: A.

last: Tuveson

state: NY

address: 1 Bungtown Rd.

department: Cancer Center

institute: Cold Spring Harbor Laboratory

type: Expression profiling by high throughput sequencing

summary: This study used Illumina strand-specific, paired-end RNA-sequencing to examine gene expression differences between normal and neoplastic mouse pancreatic ductal cells grown as three-dimensional, organoid cultures. The study analyzed 19 independently derived organoid lines. These 19 lines included 7 biological replicate "mN," or mouse normal, organoid lines, derived from the pancreatic ductal cells of wild-type C57Bl/6J or C57Bl/6N mice. In addition, the study analyzed 6 biological replicate "mP," or mouse PanIN, organoid lines, derived from PanIN-containing mouse pancreata from the KrasLSL-G12D; Pdx1-Cre mouse model. (The presence of PanIN in the pancreata from which these organoid lines were derived was confirmed by examining histological sections from the same pancreata). The final 6 lines examined in this study were biological replicate "mT," or mouse tumor, organoid lines, derived from mouse pancreata containing pancreatic ductal adenocarcinoma (PDAC) from the KrasLSL-G12D; Trp53LSL-R162H; Pdx1-Cre mouse model. We genes differentially expressed in the PanIN- or tumor-derived mouse organoid lines, relative to the mouse normal organoids, that may reflect expression changes required for pancreatic tumorigenesis.

overall_design: Gene expression comparison of 7 mouse normal-derived, 6 mouse PanIN-derived, and 6 moues tumor-derived pancreatic ductal organoid lines.

middle: A

first: Lindsey

last: Baker


title: shRNA knockdown of YAP1 in HCC364 cells, various drug conditions

city: San Francisco

first: Saurabh

last: Asthana

address: 1450 Third Street, Room HD276

department: Helen Diller Family Comprehensive Cancer Center

institute: UCSF

summary: Through a genetic screen in BRAF mutant tumor cells, we show that the Hippo pathway effector YAP acts as a parallel survival input to promote resistance to RAF-MEK inhibitor therapy. Our data uncover YAP as a novel mechanism of resistance to RAF-MEK targeted therapy. The findings unveil the synthetic lethality of YAP and RAF-MEK co-suppression as a promising strategy to enhance response and patient survival.

overall_design: RNAseq analysis of HCC364 (lung adenocarcinoma) cells in the context of drug treatment with PLX4720 (vemurafenib, a BRAF inhibitor) or Trametinib (a MEK inhibitor) alongside shRNA knockdown of the gene YAP1


title: RNA-Seq Analysis in hES/ iPS cell-derived neuronal samples

first: Liheng

address: 1150 Saint Nicholas Avenue, Room 234

department: Department of Medicine

institute: Columbia University Medical Center

summary: We characterized the gene expression by Hierarchical Clustering and one-matrix clustering in hESC, day 12 progenitors, day 25-day 27, day82 differentiated hypothalamic neurons from hESCs and day 45 neurons derived from iPSCs generated from controls (2 independent) and BBS (Bardet-Biedl Syndrome, 3 independent) subjects.

overall_design: RNA was isolated from cells of 13 samples (1 hESC, triplicate for day 12 progenitors, 1 day 25 neuron sample, duplicate for day 27 neuron samples, 1 day 82 neuron sample, five day 45 neuron samples made from 5 independent iPSC lines ) using RNeasy Micro Kit (QIAGEN). Quality control of the RNA was carried out with the Agilent Bio-analyzer, Qubit 2.0 at the MPSR of Columbia University. 100 ng of RNA with RIN ≥ 9 were used for generating mRNA-focused libraries using TruSeq RNA Sample Preparation Kit v2 and sequencing on an Illumina 2000/2500 V3 Instrument offered by the Columbia Genome Center.

first: Dieter

last: Egli

first: Rudolph

middle: L

last: Leibel


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

summary: Long non-coding RNAs (lncRNAs) have recently emerged as new players in gene expression regulation. Whether and how lncRNAs might control hematopoietic stem cell (HSC) function remains largely unknown. Here, we profiled the transcriptome of purified long-term HSCs by deep RNA-sequencing and identified thousands of un-annotated transcripts of which 323 are predicted to be lncRNAs. Comparison of their expression in differentiated lineages represented by B cells (B220+) and Granulocytes (Gr1+), revealed that 159 are likely to be HSC-specific. Knockdown of two such non-coding genes (LncHSC-2 and LncHSC-1) indicated that they regulate HSC lineage differentiation, possibly via targeting cell cycle regulators and chromatin modification enzymes. Taken together, we comprehensively identify lncRNAs in HSC and show to examples that play important roles in HSC function.

overall_design:
(I)   Mouse Granulocytes mRNA profile were generated by deep sequencing, in duplicate, using Illumina Hiseq 2000
(II)  To identify LncHSC target gene, mouse progenitor Sca-1+ cells were isolated from 5-FU injected mice and transduced with GFP+ retrovitus construct to knockdown lncHSC expression, and after in vitro culture for 2 days, KSL-GFP+ cells were sorted for mRNA profiling.
(III) Mouse B-cells mRNA profile were generated by deep sequencing, in duplicate, using Illumina Hiseq 2000

first: Deqiang

first: Benjamin

first: Margaret


title: Exosomal miR-1290 and miR-375 as prognostic markers in castrate resistant prostate cancer

city: Milwaukee

first: Liang

state: Wisconsin

address: 8701 Watertown Plank Rd.

department: Pathology

institute: Medical College of Wisconsin

summary: Extracellular microRNAs (miRNAs) embedded in circulating exosomes may serves as prognostic biomarkers in cancer. This study was performed to identify and evaluate plasma exosomal miRNAs for prognostication in castration resistant prostate cancer (CRPC). RNA sequencing was performed to identify candidate exosomal miRNAs associated with overall survival in a screening cohort of 23 CRPC patients. Candidate miRNAs were further evaluated for prognosis using qRT-PCR in a follow-up cohort of 100 patients. Cox regression and Kaplan–Meier survival curve analysis were used to evaluate prognostic value of miRNA candidates with and without incorporation of clinical prognostic factors (age, Gleason score and time from androgen deprivation therapy to clinical progression). In the screening cohort, we obtained ~6.80 million mappable RNA reads per patient. Of those with normalized read counts ? 5, 43% were mapped to miRNAs for a total of 375 known and 57 novel miRNAs. Cox regression analysis identified an association of miR-1290, -1246, and -375 with overall survival (FDR<0.1). Of those, higher levels of miR-1290 and -375 were verified to be significantly associated with poor overall survival (p<0.004) in the follow-up cohort. The miR-1290/-375-based prediction model showed better performance with time-dependent area under the curve (AUC) =72% compared to clinical variable-based model with AUC=65%. Plasma exosomal miR-1290 and miR-375 are promising prognostic biomarkers for CRPC patients. Prospective validation is needed for further development of these candidate miRNAs.

first: Xiaoyi

first: Tiezheng

last: Yuan

first: Meihua

last: Liang

first: Meijun

last: Du

first: Shu

last: Xia

first: Rachel

last: Dittmar

first: Dian

first: William

last: See

first: Brian

last: Costello

first: Fernando

last: Quevedo

first: Winston

last: Tan

first: Debashis

last: Nandy

first: Graham

middle: H

last: Bevan

first: Sherri

last: Longenbach

first: Zhifu

first: Yan

last: Lu

first: Tao

first: Stephen

middle: N

last: Thibodeau

first: Lisa

last: Boardman

first: Manish

last: Kohli


title: RNA-Seq of single cells from the Arabidopsis root

city: Rehovot

first: Idan

last: Efroni

address: POB 12

department: Plant Sciences

country: Israel

institute: Hebrew University

summary: The definition of cell identity is a central problem in biology. Single-cell RNA-seq provides a wealth of information regarding the developmental state of individual cells. However, better methods are needed to map the identity of single cells, especially during identity transitions. We have developed a quantitative classification method that is robust to expression noise and can detect primary and chimeric identities from single-cell RNA sequencing profiles. The method uses existing transcriptome repositories of grouped cell-types to define a set of optimal cell-identity markers, which are then used to define a cell identity metric. This metric accurately classified diverse cell identities in Arabidopsis root tips and human glioblastoma cells. We demonstrate the strength of the approach to resolve a dynamic developmental process by analyzing the identity of single cells captured from regenerating Arabidopsis roots following removal of their stem-cell-niche. We discover that, apart from new niche formation at the vicinity of the cut site, cells that are distant from the injury site also undergo a transient, partial collapse of identity during the regeneration and reorganization of the root, demonstrating the usefulness of a quantitative cell identity metric.

overall_design: 4 Technical replicates (pooled-and-split single QC cells), 23 cells from the QC,  3 cells from the Stele, and 4 cells from the stele, 16h following root tip decapitation

first: Pui-Leng

last: Ip

first: Tal

last: Nawy

first: Alison

last: Mello

first: Kenneth

middle: D

last: Birnbaum


title: SMARCAD1 Regulates Nucleosome Positioning and Maintenance of the Naïve Pluripotent State

city: La Jolla

first: Pengfei

last: Yu

address: 9500 Gilman Drive MC 0419

department: Bioengineering

institute: University of California San Diego

summary: Genome-wide maps of nucleosome positioning  in mouse ES cells with control shRNA and on Smarcad1 KD. MNase-seq data for human colo829 cells are also included.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We used the strategies of Mnase-Seq to detect the nucleusome positions features in E14 and Smarcad1-knock down cells. With global comparison, we described the fuction of Smarcad1 in switch pluripotent states.

first: Shu

last: Xiao

first: Chieh-Chun

last: Chen

first: Bharat

last: Sridhar

first: Darina

last: McDee

first: Yaping

last: Li

first: Dong

first: Laura

last: Sloofman

first: Xiaopeng

last: Zhu

first: Bhanu

middle: P

last: Telugu

first: Fei

last: Wang

first: Dana

last: Levasseur

first: Shaying

last: Zhao

first: Tetsuya

last: Tanaka

first: Sheng

last: Zhong


title: LincRNA-p21 Blocks Somatic Cell Reprogramming by Sustaining H3K9me3 and CpG Methylation at Pluripotent Gene Promoters

status: Public on Dec 29 2014

first: Andrew

last: Hutchins

address: 1088 Xueyuan Rd

department: Bioinformatics

institute: Southern University of Science and Technology

summary: Through expression profiling and functional screening we have identified the large intergenic non-coding RNA p21 (lincRNA-p21) as a barrier for reprogramming. LincRNA-p21 is induced by p53 but contrary to its role in the DNA damage response it does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase Setdb1 and the maintenance DNA methyltransferase Dnmt1 in separate complexes that are facilitated by the RNA binding protein hnRNP-K.

overall_design: RNA-seq expression data during MEF to iPSC reprogramming using three shRNAs, targetting either luciferase (control), lincRNA-p21 and hnRNP-K. Days 6, 9 and 12 of the reprogramming timecourse were analyzed

first: Xichen

last: Bao

first: Miguel

last: Esteban


title: A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development

city: Lausanne

first: Bart

last: Deplancke

address: Station 19

department: Institute of Bioengineering (IBI)

country: Switzerland

institute: EPFL

summary: ChIP-seq to map enrichment of histone modifications and RNA polymerase II DNA occupancy in imaginal wing discs at 120 h of development.

overall_design: The following antibodies (Abcam Inc., Cambridge, USA) were used in this study: ab6002: anti-Histone H3 (tri methyl K27); ab28940: anti-Histone H3 (mono+di+tri methyl K79); ab8895: anti-Histone H3 (mono methyl K4); ab6000: anti-Histone H3 (di+tri methyl K4); ab4729: anti-Histone H3 (acetyl K27) antibody; a mix (1:1) of ab5408 and ab817: anti RNA-polymerase II-CTD.


title: Atriplex lentiformis transcriptome profiling

city: Wuhan

first: Qiang

state: Saskatchewan

address: #1 Shizishan Street

institute: Huazhong Agricultural University

summary: To seek further insight into the factors controlling the shift from a 16:3 to 18:3 lipid profile in A. lentiformis, we conducted RNA sequencing (RNA-seq) analysis to assess gene expression in plants grown at 23 ºC and 43ºC

overall_design: Total RNA for RNA-seq analysis were extracted from A. lentiformis leaf tissues with four biological replicates for each growth condition.

first: Jitao

last: Zou


title: Next generation sequencing reveals a controlled immune response to Zaire Ebola virus challenge in cynomolgus macaques immunized with VSVΔG/ZEBOVgp

city: Seattle

first: Michael

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

summary: RNA deep sequencing analysis was perfromed on PBMCs obtained from cynomolgus macaques (CMs) prior to and after immunization with VSV carrying ZEBOVgp (VSVΔG/ZEBOVgp) and at multiple points after ZEBOV challenge

overall_design: We selected two groups of animals either (3 animals in each group) immunized with VSVDG/ZEBOVgp or VSVΔG/MARVgp. A baseline blood draw was taken at day -28, with post immunization and challenge blood draws at days 0, 4, 7, 14 and 35.  Analysis for this study was done on PBMC isolated from whole blood of each animal.

first: Richard

middle: R

last: Green

first: Fredrik

last: Barrenasa

first: Matthew

last: Thomas

first: G

last: Law

first: Sean

middle: C

last: Proll

first: Flora

last: Engelmann

first: Ilhem

last: Messaoudi

first: Andrea

last: Marzi

first: Heinz

last: Feldmann

middle: G


title: Transcriptome analysis of oxdative-stress induced senescence in human astrocytes

status: Public on Dec 27 2014

city: Philadelphia

first: Sager

middle: Jayesh

last: Gosai

state: Pennsylvania

address: 433 S University Ave.

department: Biology

institute: Universiry of Pennsylvania

summary:
Purpose:  Cellular senescence is a cell stress response resulting in permanent growth arrest and the production of an altered pro-inflammatory secretory profile known as the senescecnce-associated secretory phenotype (SASP). The induction of senescence in astrocytes, a cell type responsible for maintaining homeostasis within the central nervous system (CNS) and responding to CNS insults, has been implicated in neurodegenerative disease. However, little is known about the senescent transcriptome in CNS-derived cell types including astrocytes.
Methods: To better understand senescence-associated gene expression changes in astrocytes, we investigated global changes in the astrocyte transcriptome using RNA-seq following the induction of oxidative stress-induced senescence with hydrogen peroxide.
Results:  During senescence, we find evidence of a loss of brain expressed transcripts involved in diverse CNS processes including neuronal differentiation and development, gliogenesis, axonogenesis, and learning and memory as well as a loss of transcripts involved in MHC class II antigen processing and presentation. In addition, we find evidence for induction of the senescent phenotype including a loss of transcripts involved in cell division and an increase in the mRNA level of inflammatory mediators suggestive of a SASP.
Conclusions: Overall, our findings suggest a loss of differentiated function in senescent astrocytes and a gain in neuroinflammatory function as part of the SASP as a potential mechanisms for dysfunction in the aging brain.

overall_design: Examination of transcriptome changes by RNAseq in pre-senescent and senescent astrocytes using 2 biological replicates per condition

first: Brian

last: Gregory

first: Elizabeth

last: Crowe

first: Claudio

last: Torres


title: Assessment of microRNA differential expression and detection in multiplexed small RNA sequencing data

city: Boston

first: Joshua

middle: David

last: Campbell

state: MA

address: 72 East Concord St.

institute: Boston University

summary: Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome.  The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However multiplexing involves a tradeoff between increased number of sequenced samples and reduced number of reads per sample (i.e. lower coverage). To assess the effect of different depths of coverage due to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing 1, 3, 6, 9, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression greater than 1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the Log2 number of reads aligning to microRNA loci (R=0.96). However, most additional microRNAs detected in samples with greater sequencing coverage were in the range of expression which had lower fold change reproducibility. These findings elucidate the tradeoff between increasing the number of samples in a multiplex with decreasing coverage and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.

overall_design: The small RNA of lung tissue samples was collected in a clinical setting and sequenced by multiplexing 1, 3, 6, 9, or 12 samples per lane using the Illumina HiSeq 2000

first: Lingqi

last: Luo

first: Gang

last: Liu

first: Ji

last: Xiao

first: Joseph

last: Gerrein

first: Brenda

last: Guardela

first: John

last: Tedrow

first: Yuriy

middle: O

last: Aleksyev

first: Ivana

last: Yang

first: Mick

last: Correll

first: Mark

last: Geraci

last: Quackenbush

first: Frank

last: Sciurba

first: David

last: Schwartz

first: Naftali

last: Kaminski

first: Stefano

last: Monti

first: Avrum

last: Spira

first: Jennifer

last: Beane

first: Marc

middle: E

last: Lenburg


title: Transcription factor ZFX

first: Colin

last: Palmer

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

overall_design: Refer to individual Series


title: Coordinate Regulation of Enhancer Transcription at Super-Enhancers

status: Public on Dec 25 2014

first: Christopher

last: Benner

state: California

address: 9500 Gilman Dr. MC 0640

department: Medicine

institute: University of California, San Diego (UCSD)

summary: Using GRO-Seq, we find extensive regulation of enhancer RNAs (eRNA) within super-enhancers in response to lipopolysaccharide treatment in macrophages.  Both activation and repression of gene expression are associated with super-enhancers and eRNA transcription dynamics. Treatment of macrophages with LPS targeted specific super-enhancers by attenuating their eRNA expression, leading to expression of key inflammatory genes.  We propose that super-enhancers function as molecular rheostats integrating the binding profiles of key regulators to produce dynamic profiles of gene expression.

overall_design: Nascent transcriptome (GRO-Seq) analysis over a time course (0, 20, 60, 180 min) of Lipopolisaccharide signaling in mouse bone marrow-derived macrophages.

first: Nasun

last: Hah

first: Ruth

last: Yu

first: Michael

last: Downes

first: Ronald

middle: M

last: Evans


title: Distinct gene expression profiles between overt GIST and miniGIST

city: Stanford

first: Xiangqian

last: Guo

address: 300 Pasteur Dr.

department: Department of Pathology

institute: Stanford University

summary: In contrast to the paucity of clinic GIST (Gastrointestinal stromal tumor; 0.0014 % happening rate), there is a more than twenty thousand times higher rate for miniGISTs, about ~30% of human population could be identified to have miniGISTs. Exploring the molecular differences between miniGISTs and overt GISTs will help us to understand the transformation mechanisms along with GIST progression, but C-kit and PDGFRA mutations analysis failed to identify the differences between these two groups.   Conclusion: miniGISTs have significant distinct gene expression profile with overt GISTs by both unsupervised and supervised analysis. Our finding indicated that c-kit and DOG1 expression are the very early events in the genesis of miniGIST and overt GIST, and miniGIST is a molecular distinct group with overt GIST.

overall_design: Within our study, we collected six miniGISTs with diameter less than 1cm from autopsies and seven overt GISTs from clinic, performed the first system-wide gene expression profiling of miniGISTs and overt GISTs by 3’end RNA Sequencing, and further compared the expression profiles between these two groups.

first: Shirley

middle: X

last: Zhu

first: Sushama

last: Varma

first: Alayne

last: Brunner

first: Jonathan

last: Fletcher

first: Robert

middle: B

last: West

first: Matt

last: van de Rijn


title: Drosophila melanogaster S2 cells nucleosome occupancy data after simulation by heat killed Salmonella typhimurium

status: Public on Dec 24 2014

city: Tallahassee

last: Dennis

state: FL

address: 319 Stadium Dr

department: Biological Science

institute: Florida State Unversity

summary: In eukaryotes, nucleosomes participate in all DNA-templated events by regulating access to the underlying DNA sequence. However, the dynamics of nucleosomes during a genome response has not been well characterized . We stimulated Drosophila S2 cells with heat-killed Gram-negative bacteria Salmonella typhimurium, and mapped genome-wide nucleosome occupancy at high temporal resolution by MNase-seq using Illumina HiSeq 2500. We show widespread nucleosome occupancy change in S2 cells during the immune response, with the biggest nucleosomal loss occurring at 4hr post stimulation.

overall_design: Drsophila S2 cells at 0hr, 30minutes, 1hr and 4hr post heat-killed Salmonella typhimuriumstimulation

first: Yingxue

last: Ren

first: Daniel

last: Vera

first: Kimberly

last: Hughes


title: MicroRNAome comparison between intramuscular and subcutaneous vascular stem cell adipogenesis

city: Guangzhou

first: Yunxue

address: Beisan street

institute: Sun Yat-sen university

summary: We report the application of the Solexa deep sequencing method for high-throughput profiling files of small RNA in primary porcine vascular stem cell adipogenesis.The sequences and relative expression levels of 208 (98.6% in miRbase 16.0) known and 288 potential porcine miRNAs were identified.  50 miRNAs changed in similar pattern between IVSC and SVSC differentiation, such as miR-210, miR-10b and miR-99a.  On the contrast, 8 miRNAs changed reversely between IVSC and SVSC differentiation, 16 changed only in IVSC differentiation while 48 only in SVSC differentiation.  And 34 miRNAs showed fat-depot specific expression pattern (30 in cells of intramuscular origin and 4 in cells of subcutaneous origin).  Unexpectedly but reasonably, these adipogenesis-related miRNAs mainly functioned by targeting similar pathways in adipogenesis, obesity and syndrome.

overall_design: Comparison of miRNAome between adipogenesis of 2 primary celltypes

first: Yaosheng

last: Chen

first: Delin

last: Mo

first: Yue

last: Zhang

first: Yun

first: Peiqing

last: Cong

first: Shuqi

last: Xiao

first: Zuyong

last: He

first: Xiaohong

last: Liu


title: Bmp4-induced differentiation of EpiSCs depends on Wnt signals

city: Rotterdam

first: Dorota

last: Kurek

address: Dr Molewaterplein 50

country: Netherlands

institute: ErasmusMC

summary: We used RNA-Seq to analyse the interactions between Bmp4 and Wnt at a genome-wide level in EpiSCs treated for 48 hrs with Bmp4 and/or Wnt3a in the presence of Activin and bFGF.

overall_design: Control EpiSC were cultured in the presence of IWP2 for 48h. Cells were cultured with BMP4 with or without IWP2; Wnt3a and Wnt3a with BMP4 for 48h.

first: Alex

last: Neagu

first: Melodi

last: Tastemel

first: Nesrin

last: Tüysüz

first: Johannes

last: Lehmann

first: Harmen

middle: J

last: van de Werken

first: Sjaak

last: Philipsen

first: Reinier

last: van der Linden

last: Maas

first: Wilfred

last: van Ijcken

first: Micha

last: Drukker

first: Derk

last: ten Berge


title: Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape

city: Bielefeld

first: Marta

last: Irla

address: Universitätsstraße 25

department: Genetics of Prokaryotes

country: Germany

institute: Bielefeld University

summary:
Background: Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Methylotrophy in this organism depends on the endogenous plasmid pBM19 although all ribulose monophosphate cycle enzymes are also encoded on the chromosome. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising and valuable candidate for future biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5’‑ends.
Results: Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5’ UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. The first step of the analysis was the correction of 14 wrongly annotated translation start sites (TLSs) on the basis of the primary transcriptome data. Further investigation of the identified 5’ UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis regulatory RNA elements. In addition to this, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.
Conclusion: The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

overall_design: Pooled samples from 16 different cultivation conditions are analysed in whole transcriptome and 5'end enriched transcriptome protocols.

first: Irla

last: Marta

first: Neshat

last: Armin


title: Transcriptome analysis of heart regeneration in adult zebrafish

city: Beijing

first: Shouyu

last: Yan

address: 5#, Yiheyuan Road

department: Institution of Molecular Medicine

institute: Peking University

summary: After heart injury, adult zebrafish can perfectly regenerate its heart without any scar tissue left. We performed 100bp*2, paired-end, strand-specific, polyA-positive RNA-seq on hearts from 2 group of adult zebrafish, in which one was sham group and the other 7 days after heart tip amputation (7dpa). We found pathways involving hydrogen peroxidate related functions significantly up-regulated in 7dpa group, indicating its role in heart regeneration.

overall_design: RNA-seq: hearts from sham or 7dpa adult zebrafish

first: Jing-wei

last: Xiong

first: Shou-yu

first: Xiao-hai

last: Zhou


title: Ets homologous factor regulates pathways controlling response to injury in Calu-3 airway epithelial cells [ChIP-seq]

status: Public on Dec 23 2014

city: Chicago

first: Ann

last: Harris

state: IL

address: 2430 N Halsted Street  Box 211

department: Human Molecular Genetics

institute: Lurie Children's Hospital of Chicago Research Center

summary: Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the majority of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative enhancers, marked by specific histone modifications. EHF occupies many genomic sites that are close to genes involved in intercellular and cell-matrix adhesion.

overall_design: Examination of EHF binding plus two histone modifications in Calu-3 cells. There are two replicates of the EHF ChIP-seq plus an input sample (control). There is one replicate each of the two histone modifications, plus a second input sample (control).

first: Sara

last: Fossum

first: Michael

last: Mutolo

first: Rui

last: Yang

first: Hong

last: Dang

first: Wanda

last: O'Neal

last: Knowles

first: Shih

last: Leir


title: Age-dependent changes in the LPS-induced transcriptome of bovine dermal fibroblasts occurs without major changes in the methylome

city: Burlington

first: David

last: Kerr

state: VT

address: 500 Main Street 205 Terrill Hall

department: Animal Science

institute: University of Vermont

type: Methylation profiling by high throughput sequencing


title: MIRA-Seq analysis of bovine dermal fibroblasts from the same individuals at two different ages

summary: We report the results of MIRA-Seq-based high-throughput profiling of the bovine fibroblast methylome from three different individuals at two different ages (5 and 16 months of age) to determine the stability of methylation with age.

overall_design: Examination of methylation in one cell line (fibroblasts) of three individuals at two ages (5 and 16 months).

first: Benjamin

last: Green

first: Stephanie

last: McKay


title: Anatomy and transcript profiling of gynoecium development in female sterile Brassica napus mediated by one alien chromosome

first: wenqin

last: fu

state: Hubei

address: No.1,Shizishan Street

department: College of Plant Science and Technology

summary: The gynoecium is one of the most complex organs of angiosperms specialized for seed production and dispersal, but only several genes important for ovule or embryo sac development were identified by using female sterile mutants. The female sterility in oilseed rape (Brassica napus) was before found to be related with one alien chromosome from another crucifer Orychophragmus violaceus. Herein, the developmental anatomy and comparative transcript profiling (RNA-seq) for the female sterility were performed to reveal the genes and possible metabolic pathways behind the formation of the damaged gynoecium. Using Brassica_ 95k_ unigene as the reference genome, a total of 28,065 and 27,653 unigenes were identified to be transcribed in S1 and H3, respectively, suggesting the  newly initiated transcriptions in S1. Further comparison of the transcript abundance between S1 and H3 revealed that 4540 unigenes showed more than two fold expression difference. Gene ontology and pathway enrichment analysis of the Differentially Expressed Genes (DEGs) revealed that a number of important genes and metabolism pathways were involved in the development of gynoecium, embryo sac, ovule, integuments as well as the interactions between pollen and pistil. DEGs for the ovule development were detected to function in the metabolism pathways regulating brassinosteroid (BR) biosynthesis, adaxial/abaxial axis specification, auxin transport and signaling. A model was proposed to show the possible roles and interactions of these pathways for the sterile gynoecium development. The results provided new information for the molecular mechanisms behind the gynoecium development at early stage in B. napus.

overall_design: Gynoecium samples in 1.5 to 3mm long flower buds were collected from Brassica napus (H3) and its female sterile addition line (S1). Total RNA from the two samples were extracted and sequencing was performed using Illumina HiSeq™ 2000.

first: Wen-qin

last: Fu

first: Zhi-gang

last: Zhao

first: Xian-hong

last: Ge

first: Li

last: Ding

first: Zai-yun

last: Li


title: Transcriptomic response of bovine dermal fibroblasts to lipopolysaccharide

summary: We report the responsiveness of bovine dermal fibroblasts to lipopolyssacharide (LPS) within individuals at different ages. Cultures were collected from three different heifers at the ages of approximately 5 and 16 months. Isolated fibroblasts were then exposed to LPS to determine whether there was a differential response within an animal based upon age.

overall_design: Fibroblasts were isolated from 3 cows at the ages 5 months and 16 months. Isolated fibroblasts were treated with LPS for 0 hrs, 2 hrs, and 8 hrs.


title: Genome-wide bisulfite sequencing in zygotes identifies demethylation targets and maps the contribution of TET3 oxidation

status: Public on Dec 22 2014

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

country: United Kingdom

institute: Altos Labs

summary: Fertilization triggers a global erasure of 5-methylcytosine from paternal DNA as part of extensive epigenetic reprogramming during the transition from gametic specialization to totipotency. This active removal has been shown to involve oxidation by TET3, but the targeting of this pathway and the wider context of demethylation remain poorly understood. We optimized a novel technique for whole-genome bisulfite sequencing and applied it to wild-type and TET3-deficient zygotes, using SNPs to access paternal alleles. As the great majority of sperm-contributed methylation lies outside the CpG islands (CGIs) and promoters analysed to date, these genome-wide methylation profiles allow paternal methylation trajectories in the zygote to be comprehensively examined for the first time. This global view revealed that in addition to pervasive loss of methylation from intergenic sequences and most repetitive elements, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation, and at gene bodies is also linked to increased transcriptional noise in the early embryo. Our data maps the primary contribution of oxidative demethylation to a subset of gene bodies and single-copy intergenic sequences, and implicates the action of redundant pathways at many loci. We further uncover a novel function for TET3 in protection from de novo methylation at CGIs and promoters. This work adds new breadth to our understanding of the molecular events involved in reprogramming gametic identity following fertilization.

overall_design: Three independent collections of zygotes were performed for each genotype (control and TET3 deletion), giving a total of 225 control and 237 TET3 deletion zygotes, of which 120 and 129 were derived from 129S2/SvHsd studs for control and TET3 samples, respectively. Zygotes were pooled and whole-genome bisulfite libraries were prepared using a post-bisulfite adaptor tagging strategy optimized from (Miura et al. Nucleic Acids Research 2012, 40:e136)

first: Julian

last: Peat

first: Wendy

last: Dean

first: Stephen

last: Clark

first: Sébastien

middle: A

last: Smallwood

first: Gabriella

last: Ficz

first: Jong

middle: K

last: Kim

first: John

last: Marioni

first: Timothy

last: Hore

first: Wolf

last: Reik


title: Characterizing the profiles of histone markers in mouse adipocytes during insulin resistance

first: Evan

last: Rosen

address: 3 Blackfan Cir

department: Endocrinology

institute: Beth Israel Deconess Medical Center

summary: Insulin resistance is a sine qua non of Type 2 diabetes (T2D) and a frequent complication of multiple clinical conditions, including obesity, aging, and steroid use, among others. How such a panoply of insults can result in a common phenotype is incompletely understood. Furthermore, very little is known about the transcriptional and epigenetic basis of this disorder, despite evidence that such pathways are likely to play a fundamental role. Here, we compare cell autonomous models of insulin resistance induced by the cytokine tumor necrosis factor-a (TNF) or by the steroid dexamethasone (Dex) to construct detailed  epigenomic maps associated with cellular insulin resistance.

overall_design: Murine 3T3-L1 adipocytes were treated separately with dexamethasone (Dex; 20nM) or tumor necrosis factor-alpha. To comprehensively assess epigenomic changes caused by Dex and TNF in a time-dependent manner, we profiled cells at early (2 hours), intermediate (24 hours), and late (6 days) points in the development of insulin resistance.

first: Sona

last: Kang

first: Linus

last: Tsai

first: Yiming

last: Zhou


title: Genome-wide binding site profiling of NdgR in Streptomyces coelicolor

city: Daejeon

first: Byung-kwan

last: Cho

address: 291 Daehak-ro

country: South Korea

institute: korea advanced institute of science and technology

summary: We identified genome-wide binding regions of NdgR in Streptomyces coelicolor using chromatin immunoprecipitation sequencing (ChIP-seq). We constructed 6×myc-tagged NdgR strain using homologous recombination with myc-tagging vector. Analysis of the sequencing data aligned to Streptomyces coelicolor genome database (NC_003888).

overall_design: 6x-myc tagged Streptomyces coelicolor M145 was cultured on solid minimal media supplemented with N-acetylglucosamine and asparagine during 36hrs. Anti-myc antibody (9E10) was used for immunoprecipitation (IP). IP-DNA was sequenced using Illumina Genome Analyzer Ⅱx

first: Byung-Kwan

first: Ji-Nu


title: Ets homologous factor regulates pathways controlling response to injury in Calu-3 airway epithelial cells

first: Ann

last: Harris


title: GIST cell cycle dysregulation is required for progression to high-risk disease but not for resistance to kinase inhibitors

first: Xiangqian

last: Guo

summary: Background: To understand the transcriptional consequences of a TP53 modulation in the GIST cell context, we treated GIST430 with increasing doses of the MDM2-inhibitor nutlin-3 (racemate).

overall_design: In this study, 3’ end RNA Sequencing (3SEQ) was used to expression profile GIST430 cell lines treated with DMSO and Nutlin-3, 2.5µM or 10µM for 18h. Then the gene expression profiles between these concentrations were compared.

first: Sebastian

last: Bauer

first: Michael

last: Heinrich

first: Brian

last: Rubin

first: Christopher

middle: L

last: Corless

first: Thomas

last: Mühlenberg

first: Cesar

last: Serrano

first: Barbara

last: Dewaele

first: Carol

last: Beadling

first: Inga-Marie

last: Schaefer

first: Xiangquian

first: Janice

last: Patteron

first: Adrian

last: Marino-Enriquez

first: Cristina

last: Antonescu

first: Anette

last: Duensing

first: Andrea

last: Wallace

first: Tamas

last: Ordog

first: Jerry

last: Call

first: Matt

last: van de Rijn

first: Maria

last: Debiec-Rychter

first: Jonathan

last: Fletcher


title: MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction [II]

city: Zurich

first: Jochen

last: Imig

address: Vladimir-Prelog-Weg 1-5/10

institute: ETH Zurich

summary: Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs.

overall_design: Two replicates of four cDNA libraries were submitted to deep sequencing: an RNA “Input” sample from RNA-12-transfected cells; a sample of the Ago2-immunopurified RNA from cells treated with transfection reagent (“Mock”); a sample of the Ago2-immunopurified RNA from RNA-12-treatment (“Ago2-IP”); and the sample from miR-CLIP-purified RNA (“miR-CLIP”).

first: Andreas

last: Brunschweiger

first: Anneke

last: Brümmer


title: Regulation of Zn and Fe transporters by the GPC1 gene during wheat monocarpic senescence

city: Harpenden

first: Stephen

last: Pearce

state: Hertfordshire

address: West Common

department: Sustainable Soils and Crops

institute: Rothamsted Research

summary: TaGPC1 and TaGPC2 are NAC-domain transcription factors which accelerate the onset of senescence and facilitate nutrient translocation in wheat. We developed knockout mutants of these genes in tetraploid wheat and used RNA-seq to identify the effect of these mutations on the wheat flag leaf transcriptome during monocarpic senescence. Several transporter-related genes were identified which were upregulated during senescence and differentially expressed between genotypes.

overall_design:
Illumina cDNA libraries were constructed from four biological replicates of three genotypes (WT, gpc-a1 and gpc-a1/gpc-b2) at three timepoints (Heading date, 12 days after anthesis and 22 days after anthesis). Reads were aligned to a collection of assembled genomic contigs from flow-sorted chromosome arms (A and B genomes only) provided by the International Wheat Genome Sequencing Consortium. A custom GTF file was generated to identify 139828 gene loci corresponding to transcribed regions of this reference sequence.
Please note that  the contig names provided by URGI (http://wheat-urgi.versailles.inra.fr/Seq-Repository) were used in the analysis.  Most, but not all, of these loci are present in Ensembl (With a modified name, but the same basic information, chromosome arm and unique contig ID) at ftp://ftp.ensemblgenomes.org/pub/plants/release-22/fasta/triticum_aestivum/dna/
Therefore, the contig IDs from URGI (which are available for all our sequences) were used in the processed data file (i.e. the count table and GFF file) and  an additional file describing the corresponding Ensembl names for these was provided (Additional_file_2.xlsx).

first: Jorge

last: Dubcovsky


title: MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction [III]

overall_design: Two replicates of three cDNA libraries were submitted to deep sequencing: a sample from RNA-7-transfected cells; a sample from pre-miR-106a transfected cells; and a control sample.


title: MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction

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

overall_design: Refer to individual Series


title: Ets homologous factor regulates pathways controlling response to injury in Calu-3 airway epithelial cells [RNA-seq]

first: Ann

last: Harris

summary: Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. RNA-seq after EHF depletion or overexpression showed significant alterations in the expression of genes involved in response to wounding. EHF knockdown also targeted genes in pathways of epithelial development and differentiation and locomotory behavior.

overall_design: mRNA profiles from Calu-3 cells treated with negative control (NC) or EHF siRNA, in quintuplicate. mRNA profiles from 3 pcDNA (empty vector control) clones and 3 pcDNA-EHF overexpression A549 clones, 3-4 replicates each.

first: Sara

last: Fossum

first: Michael

last: Mutolo

first: Rui

last: Yang

first: Hong

last: Dang

first: Wanda

last: O'Neal

last: Knowles

first: Shih

middle: H

last: Leir


title: MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction [I]

overall_design: Two replicates of four cDNA libraries were submitted to deep sequencing: an RNA “Input” sample from  RNA-7-transfected cells; a sample of the Ago2-immunopurified RNA from cells treated with transfection reagent (“Mock”); a sample of the Ago2-immunopurified RNA from RNA-7-treatment (“Ago2-IP”); and the sample from miR-CLIP-purified RNA (“miR-CLIP”).


title: A Sensitive Approach to Map Genome-wide 5-Hydroxymethylcytosine and 5-Formylcytosine at Single-base Resolution

city: Ipswich

first: Shengxi

last: Guan

address: 240 County Road

institute: New England Biolabs

summary: Mapping genome-wide 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) at single-base resolution is important to understand their biological functions. We present a cost-efficient mapping method that combines 5hmC-specific restriction enzyme PvuRts1I with a 5hmC enrichment method. The sensitive method enables detection of low abundant 5hmC sites, providing a more complete 5hmC landscape than available bisulfite-based methods. This method generated the first genome-wide 5fC map at single-base resolution. Parallel analyses revealed that 5hmC and 5fC existed with lower abundance and more dynamically in non-CpG context than in CpG context. In the genic region, distribution of 5hmCpG and 5fCpG differed from 5hmCH and 5fCH (H=A, T, C). 5hmC and 5fC were distributed distinctly at regulatory protein-DNA binding sites, depleted in permissive transcription factor binding sites, and enriched at active and poised enhancers. This sensitive bisulfite-conversion free method can be applied to biological samples with limited starting material or low abundance of cytosine modifications.

overall_design: Sensitive mapping of genome-wide 5-hydroxymethylcytosine and 5-formylcytosine in mouse embryonic stem cell at single-base resolution by combining 5-hydroxymethylcytosine specific restriction enzyme PvuRts1I and 5-hydroxymethylcytosine enrichment method (selective chemical labeling or SEAL)

first: Zhiyi

last: Sun

last: Dai

first: Zhenyu

last: Zhu


title: Genomewide analysis of the human p53 transcriptional network unveils a lncRNA tumor suppressor signature (RNA-seq)

city: Pamplona

first: Maite

last: Huarte

state: Navarra

address: c/ Avd Pio XII, 55

country: Spain

institute: Centro de Investigación Médica Aplicada

summary: We report the application of high-throughput sequencing to performed the p53 regulated trancriptome in HCT116 colon cancer cells treated with the DNA damage 5FU. To study the direct targets of p53 we performed ChIP-seq to deterrmined the p53 biding sites and associated with the expression levels. With this study we identified the new genomic regions regulated by p53 and with special attention in those regions that are non coding and are differentially expressed by the DNA damage drug.

overall_design: Description of the p53 transcriptome in HCT116 colon cancer cell line. The RNA-seq libraries were prepared from purified poly-A+ RNA from untreated and 5-Fluorouracil treated p53 +/+ HCT116 cells for 4 and 12h, including two independent samples for the time 12h. Paired-end and strand specific RNA sequencing libraries were prepared according to Illumina instructions and sequenced on HiSeq 2000 (Ilumina) with sequence length of 150 bp. Raw sequencing data were alignment to the human genome (hg19) using Tophat mapper.

first: Yolanda

last: Sanchez

first: Victor

last: Segura


title: Genomewide analysis of the human p53 transcriptional network unveils a lncRNA tumor suppressor signature (ChIP-seq)

overall_design: Description of the p53 transcriptome in HCT116 colon cancer cell line


title: MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction [IV]

first: Jochen

last: Imig

overall_design: Two replicates of two cDNA libraries were submitted to deep sequencing: a sample from siH19-transfected cells and a control sample.

first: Andreas

last: Brunschweiger

last: Brümmer


title: Ets homologous factor regulates pathways controlling response to injury in Calu-3 airway epithelial cells [DNAse-seq]

last: Harris

summary: Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the majority of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative enhancers, marked by specific histone modifications and increased DNase hypersensitivity.

overall_design: Examination of open chromatin region in Calu-3 cells with 1 replicate

first: Gregory

last: Crawford

first: Lingyun

last: Song

last: Fossum


title: Global transcriptional response of Clostridium difficile carrying the ϕCD38-2 prophage

city: Sherbrooke

first: Louis-Charles

last: Fortier

state: Quebec

address: 3201 Jean Mignault

department: Microbiology and Infectiology

country: Canada

institute: Université de Sherbrooke / FMSS

summary: We used RNA-seq to conduct a genome-wide transcriptomic analysis of the C. difficile strain R20291 carrying the newly introduced ϕCD38-2 prophage. A total of 39 bacterial genes were differentially expressed in the R20291 lysogen, 26 of them being downregulated. Several of the regulated genes encode transcriptional regulators and PTS subunits involved in glucose, fructose and glucitol/sorbitol uptake and metabolism. Also of note, the presence of ϕCD38-2 upregulated the expression of a group of regulatory genes located in phi-027, a resident prophage common to most ribotype 027 isolates. The most differentially expressed gene was the conserved phase-variable cell wall protein CwpV, which was upregulated by about 20-fold in the lysogen. This is the first study describing the global response of C. difficile to the presence of a newly introduced prophage.

overall_design: Two strains (WT and lysogen) were subjected for RNA-seq analysis with two biological replicates each.

first: Ognjen

last: Sekulovic


title: RNA-Seq of 40 single cells from the Arabidopsis root FACS sorted GFP positive cells from WOX5:GFP expressing plants

status: Public on Dec 20 2014

city: Rehovot

first: Idan

last: Efroni

address: POB 12

department: Plant Sciences

institute: Hebrew University

summary: 40 QC single cells multiplexed using the CEL-Seq protocol

overall_design: 40 cells from the QC

first: Pui-Leng

last: Ip

first: Tail

last: Nawy

first: Alison

last: Mello

first: Kenneth

middle: D

last: Birnbaum


title: Transcriptional Reversion of Cardiac Myocyte Fate During Mammalian Cardiac Regeneration.

first: Laurie

last: Boyer

address: 77 Massachusetts Avenue

institute: Massachusetts Institute of Technology

summary: Rationale: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. Objective: The objectives of our study were to determine if myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. Methods and Results: We derived a core transcriptional signature of injury-induced cardiac myocyte regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo cardiac myocyte differentiation, in vitro cardiac myocyte explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of cardiac myocyte differentiation processes including reactivation of latent developmental programs similar to those observed during de-stabilization of a mature cardiac myocyte phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13 (IL13), which induced cardiac myocyte cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of IL13 signaling in cardiac myocytes. These downstream signaling molecules are also modulated in the regenerating mouse heart. Conclusions: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.

overall_design: Comparison of transcriptional programs of primary myocardial tissues sampled from neonatal mice and murine hearts undergoing post-injury regeneration, along with in vitro ESC-differentiated cardiomyocytes

first: Joseph

last: Wamstad

first: Caitlin

last: O'Meara

first: Richard

middle: T

last: Lee


title: Characterization of differentiating adipose cells by high-throughput single-cell RNA-Seq

first: Magali

last: Soumillon

address: Divinity Avenue 7

institute: Harvard University, HSCRB

summary: Directed differentiation of cells in vitro is a powerful approach for dissection of developmental pathways, disease modeling and regenerative medicine, but analysis of such systems are complicated by heterogeneous and asynchronous cellular responses to differentiation-inducing stimuli. To enable deep characterization of heterogeneous cell populations, we developed an efficient digital gene expression profiling protocol that enables surveying of mRNA in thousands of single cells at a time. We then applied this protocol to profile 11,116 cells collected during directed differentiation of human adipose-derived stem/stromal cells. The resulting data reveals the major axes of cell-to-cell variation within and between time points and suggests a link between incomplete adipogenesis in vitro and adipocyte dysfunction in vivo.

overall_design: High-throughput single cell RNA-seq method applied to human adipose tissue-derived stromal/stem cells during differentiation towards an adipogenic fate

first: Davide

last: Cacchiarelli

first: Stefan

last: Semrau

first: Alexander

last: van Oudenaarden

first: Tarjei

middle: S

last: Mikkelsen


title: Expression profiles of IAA biosynthesis deficient seedlings of Arabidopsis thaliana

status: Public on Dec 21 2014

first: Hongwei

last: Guo

address: No.5 Yiheyuan Road

department: College of Life Sciences

summary: To gain more information about the Kyn inhibition of TAA1/TARs, we conducted a RNA-seq analysis to compare the genome-wide gene expression profiles of WT seedlings grown on MS medium or MS plus Kyn, and wei8-/- tar2+/- progeny grown on MS. We report that Kyn treatment largely mimics the loss of TAA1/TAR2 functions.

overall_design: RNA seq of 3 samples: Col-0 on MS medium, Col-0 on MS+Kyn medium, wei8 tar2(+/-) on MS medium.

first: Wenrong

last: He

first: Yusi

last: Ji

first: Yun

last: Zhang

first: Mingyu

last: Yang


title: Parkinson's disease genes VPS35 and EIF4G1 interact genetically and converge on alpha-synuclein

status: Public on Dec 19 2014

first: Justin

middle: William

last: Chartron

address: Clark Center E200, 318 Campus Drive

institute: Stanford University

summary: Parkinson's disease (PD) is a common human neurodegenerative movement disorder. Studies of the genetic forms of PD have helped to reveal disease mechanisms. Functional interactions between some Parkinson's disease (PD) genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35 and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1 and alpha-synuclein, a protein with a key role in the pathogenesis of both sporadic and familial PD. We extend our findings from yeast to an animal model and show these interactions are conserved in neurons. We also connect VPS35 impairments to neurodegeneration in alpha-synuclein transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to alpha-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PD genes for future genetic and functional interrogation.

overall_design: Ribosome profiling (RiboSeq) of wild type and VPS35 deletion yeast strains, with or without overexpression of the TIF4631 initiation factor

first: Nripesh

last: Dhungel

first: Simona

last: Eleuteri

first: Ling-bo

last: Li

first: Nicholas

last: Kramer

middle: W

first: Brian

last: Spencer

first: Kori

last: Kosberg

first: Jerel

last: Fields

first: Klodjan

last: Stafa

first: Anthony

last: Adame

first: Hilal

last: Lashuel

first: Judith

last: Frydman

first: Kang

last: Shen

first: Eliezer

last: Masliah

first: Aaron

last: Gitler


title: The RNA editing enzyme ADAR1 is a key regulatory of innate immune responses to RNA

city: Edinburgh

first: Liam

last: Keegan

address: Crewe Road South

department: MRC Human Genetics Unit, MRC IGMM at the University of Edinburgh

institute: University of Edinburgh

summary: The ADAR RNA editing enzymes deaminate adenosine bases to inosines in cellular RNAs, recoding open reading frames. Human ADAR1 mutations cause Aicardi-Goutieres Syndrome (AGS) and Adar1 mutant mice showing an aberrant interferon response and death by embryonic day E12.5 model the human disease. Searches have not identified key ADAR1 RNA editing sites recoding immune/haematopoietic proteins but editing is widespread in Alu sequences. We show that Adar1 embryonic lethality is rescued in Adar1; Mavs double mutant mice in which general antiviral responses to cytoplasmic dsRNA are prevented. We propose that inosine bases are epigenetic marks identifying cellular RNA as innate immune ÒselfÓ. Consistent with this idea we show that an editing-active cytoplasmic ADAR is required to prevent aberrant immune responses in Adar1 mutant mouse embryo fibroblasts. No dramatic increase in repetitive transcripts is observed. AGS mutations in ADAR1 affect editing by the interferon-inducible cytoplasmic ADAR1 isoform.

overall_design: RNA-seq expression profiling in Adar1 and Adar1/Mavs knockout mice embryos.

first: Robert

last: Young


title: Transcriptome profiling of brown adipose tissue during cold exposure

city: Odense

first: Qin

last: Hao

address: kanslergade 1

country: Denmark

institute: Copenhagen University

summary: We applied a deep-sequencing based method – digital gene expression profiling (DGEP), to investigate gene expression in interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) in acute cold exposure

overall_design: Examination of gene expression level in 3 different adipose tissues in 3 time points, day0, day2 and day4 in cold exposure.

first: Jacob

last: Hansen


title: Impact of regulatory variation from RNA to protein

first: Sidney

last: Wang

address: 920 E. 58th Street, CLSC 317

department: Human Genetics

institute: University of Chicago

summary: Genetic variants that impact gene regulation are important contributors to human phenotypic variation. For this reason, considerable efforts have been made to identify genetic associations with differences in mRNA levels of nearby genes, namely, cis expression quantitative trait loci (eQTLs). The phenotypic consequences of eQTLs are presumably due, in most cases, to their ultimate effects on protein expression levels. Yet, only few studies have quantified the impact of genetic variation on proteins levels directly. It remains unclear how faithfully eQTLs are reflected at the protein level, and whether there is a significant layer of cis regulatory variation acting primarily on translation or steady state protein levels. To address these questions, we measured ribosome occupancy by high-throughput sequencing, and relative protein levels by high-resolution quantitative mass spectrometry, in a panel of lymphoblastoid cell lines (LCLs) in which we had previously measured transcript expression using RNA sequencing. We then mapped genetic variants that are associated with changes in transcript expression (eQTLs), ribosome occupancy (rQTLs), or protein abundance (pQTLs). Most of the QTLs we detected are associated with transcript expression levels, with consequent effects on ribosome and protein levels. However, we found that eQTLs tend to have significantly reduced effect sizes on protein levels, suggesting that their potential impact on downstream phenotypes is often attenuated or buffered. Additionally, we confirmed the presence of a class of cis QTLs that specifically affect protein abundance with little or no effect on mRNA levels; most of these QTLs have little effect on ribosome occupancy, and hence may arise from differences in post-translational regulation.

overall_design: We measured level of translation transcriptome-wide in lymphoblastoid cell lines derived from 72 HapMap Yoruba individuals using ribosome profiling assay, for which we have transcript level, protein level (62 out of 72) and genotype information collected.

first: Yoav

last: Gilad

first: Jonathan

last: Pritchard

first: Alexis

last: Battle

first: Zia

last: Khan

first: Amy

last: Mitrano

first: Michael

last: Ford


title: Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation, and platelet formation and function

status: Public on Dec 18 2014

first: Harmen

middle: Jan George

last: van de Werken

address: Wytemaweg 80

department: Urology

institute: Erasmus MC

summary:
Sp1 and Sp3 belong to the Specificity proteins (Sp)/Krüppel-like transcription factor
family. They are closely related, ubiquitously expressed and recognize G-rich DNA
motifs. They are thought to regulate generic processes such as cell cycle and growth
control, metabolic pathways and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and
combined haploinsufficiency results in hematopoietic defects during the fetal stages.
Here, we show that in adult mice conditional ablation of either Sp1 or Sp3 has minimal
impact on hematopoiesis, while the simultaneous loss of Sp1 and Sp3 results in severe
macrothrombocytopenia and platelet dysfunction. We employed flow cytometry, cell
culture and electron microscopy and show that although megakaryocyte numbers are
normal in bone marrow and spleen, they display a less compact demarcation membrane
system and a striking inability to form proplatelets. Through megakaryocyte
transcriptomics and platelet proteomics we identified several cytoskeleton-related
proteins and downstream effector kinases, including Mylk, that were downregulated
upon Sp1/Sp3 depletion, providing an explanation for the observed defects in
megakaryopoiesis. We show that Mylk is required for proplatelet formation and
stabilization and for ITAM-receptor mediated platelet aggregation. Our data highlights
the specific vs generic role of these ubiquitous transcription factors in the highly
specialized megakaryocytic lineage.

overall_design: Megakaryocyte mRNA profiles of Sp1fl/fl::Sp3fl/fl (WTlox) and Pf4-Cre::Sp1fl/fl::Sp3fl/fl (dKO) mice were generated by deep sequencing, in triplicate.

first: D

middle: I

last: Kulu

first: W

middle: F

last: van IJcken

first: H

first: S

last: Philipsen


title: De novo assembly of the desert tree Haloxylon ammodendron (C. A. Mey.) based on RNA-Seq data provides insight into drought response, gene discovery and marker identification

first: Yan

last: Long

state: China

address: Zhongguancun South street No.12

institute: Chinese Academy of Agricultural Sciences

summary:
Purpose: The goals of this study are to compare the gene expression profiling for drought treated and control plants by using  NGS.
Methods: The four RNA samples were pooled to one, using equivalent quantities of each sample for transctiptome sequencing. Meanwhile, the four RNA samples were used to construct the library for DGE sequencing.
Results: Using Illumina sequencing technology, we generated over two billion bases of high-quality sequence data on H. ammodendron and conducted de novo assembly and annotation of genes without prior genome information. These reads were assembled into 79,918 unigenes (mean length=728 bp).In addition, DGE reads were mapped to the assembled transcriptome for gene expression analysis under drought stress. In total, 1,060 differentially expressed genes were identified.

overall_design: H. ammodendron seedlings grew for one month, and then one set of seedlings were treated with a one-week (7d) stress, and the second set of seedlings was used as a control and received no treatment. Each treatment was with two replicates.

first: Xinjie

last: Tian


title: AUTS2 confers transcriptional activation to PRC1 in the CNS (RNA-Seq)

city: New york

first: Zhonghua

last: Gao

address: 522 1st Avenue, SML 203

department: Biochemistry and Molecular Pharmacology

institute: New York University School of Medicine

summary: Naturally occurring variations of Polycomb Repressive Complex 1 (PRC1) comprise a core assembly of Polycomb group proteins and additional factors that include, surprisingly, Autism Susceptibility Candidate 2 (AUTS2). While AUTS2 is often disrupted in patients with neuronal disorders, the underlying mechanism is unclear. We investigated the role of AUTS2 as part of a previously identified PRC1 complex (PRC1-AUTS2), and in the context of neurodevelopment. In contrast to the canonical role of PRC1 in gene repression, PRC1-AUTS2 activates transcription. Biochemical studies demonstrate that the CK2 component of PRC1-AUTS2 thwarts PRC1 repressive activity and AUTS2-mediated recruitment of P300 leads to gene activation. ChIP-seq of AUTS2 shows that it regulates neuronal gene expression through promoter association. Conditional CNS targeting of Auts2 in a mouse model leads to various developmental defects. These findings reveal a natural means of subverting PRC1 activity, linking key epigenetic modulators with neuronal functions and diseases.

overall_design: mRNA profiles of P1 brain from wild type mice were generated by deep sequencing

first: Danny

last: Reinberg

first: Pedro

last: Lee

first: James

last: Stafford

first: Melanie

last: von Schimmelmann

first: Anne

last: Schaefer


title: Pseudomonas fluorescens transcriptome in flowback water

city: Pittsburgh

first: Kyle

last: Bibby

state: PA

address: 3700 O'Hara Street

department: Civil and Environmental Engineering

institute: University of Pittsburgh

summary: The goal of the study was to examine the influence of flowback water exposure on bacterial survival and biocide tolerance. The transcriptome of P. fluorescens was analyzed using RNA-seq to determine the gene rxpression profile changes occuring upon flowback water exposure. The results indicate that that P. fluorescens induces a well-coordinated genetic response that aids in its survival in flowback water as well as imparts enhanced tolerance against typically used slow acting biocides such as gluteraldehyde but increased susceptibity towards sodium hypochlorite. RNA-seq data demonstrated significant induction of genes involved in osmotic stress, energy production and conversion, membrane integrity, protein transport among others.

overall_design: Examination of P. fluorescens transcriptome exposed to flowback water for one hour and compare with PBS exposed cells.

first: Amit

last: Vikram

first: Daniel

middle: C

last: Lipus


title: Transformation of the intestinal epithelium by the MSI2 RNA binding protein

city: Los Angeles

first: Fan

last: Li

state: CA

address: 675 Charles E Young Drive South

department: Pediatrics

institute: University of California Los Angeles

summary: The MSI2 RNA binding protein has recently emerged as a potent oncogene playing key roles in hematopoietic stem cell homeostasis and malignant hematopoiesis. Here we demonstrate that MSI2 is expressed in the intestinal stem cell compartment, that its expression is elevated in colorectal adenocarcinomas, and that MSI2 loss of function abrogates colorectal cancer cell growth. We thus examined the oncogenic consequences of MSI2 gain of function in the intestinal epithelium with a drug inducible mouse model. Strikingly, MSI2 induction alone was sufficient to phenocopy the majority of morphological and molecular consequences of acute loss of the APC tumor suppressor in the intestinal epithelium. We demonstrate that this phenotype is independent of both the activation of the other oncogenic Musashi family member, Msi1, and of canonical Wnt pathway activation. Transcriptome-wide RNA-binding analysis indicates that MSI2 acts as a pleiotropic inhibitor of known intestinal tumor suppressors including Lrig1, Bmpr1a, Cdkn1a, and Pten. Finally, we demonstrate that inhibition of the PDK-AKT-mTORC1 axis downstream of Pten rescues oncogenic consequences of MSI2 induction. Taken together, our findings identify MSI2 as a central component in an unappreciated oncogenic pathway promoting intestinal transformation.

overall_design: 2 wild-type samples, 2 TRE-Msi2 samples

first: Christopher

last: Lengner

first: Brian

last: Gregory


title: Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C

city: Chapel Hill

first: Sara

middle: Rebecca

last: Selitsky

state: NC

address: 120 Mason Farm Rd., 5100H Genetic Medicine Building

department: Genetics

institute: University of North Carolina-Chapel Hill

summary: Small, non-coding RNAs control gene expression post-transcriptionally and play important roles in virus-host interactions. Within the liver, the microRNA (miRNA) miR-122 is essential for replication of hepatitis C virus (HCV), while repression of miR-148a by hepatitis B virus (HBV) may enhance tumorigenesis. Despite their importance to the outcome of these infections, few previous studies have described unbiased profiling of small RNAs in the liver during chronic viral hepatitis. Here, we sequenced small (14-40 nts) RNAs in liver from subjects with chronic hepatitis B and C. We found that small RNAs derived from tRNAs, specifically 5’ tRNA-halves (“5’ tRHs”, ~31-34 nts), are abundant in liver and significantly increased during chronic viral infection in humans and also chimpanzees. In most infected livers, 5’ tRH abundance exceeded that of miRNAs. In contrast, in hepatocellular carcinoma (HCC) tissue from these subjects, tRH abundance was reduced concomitant with decreased expression of the tRNA-cleaving ribonuclease, angiogenin. Although tRHs have been identified in mice, our results show they are abundantly expressed in human tissue, increased in chronic viral infection, and decreased in liver cancer. Our findings highlight the potential biological and clinical relevance of these small non-coding RNAs.

overall_design: Small RNA-seq of liver samples from control subjects (n=4), subjects with chronic hepatitis B (n=4) and hepatitis B associated hepatocellular carcinoma (n=4, 3 out of 4 matched with non-tumor tissue) and subjects with chronic hepatitis C (n=4) and tissue from hepatocellular carcinoma of the same patients. Also, small RNA-seq of AGO2 and IgG pulldown in FT3-7 cells. Sequenced AGO2 pulldown (n=3), IgG pulldown (n=2) and total small RNA from FT3-7 cells (n=3).

middle: R

first: Praveen

last: Sethupathy


title: X Chromosome Reactivation Dynamics Reveal Stages of Reprogramming to Pluripotency

first: Rahul

last: Karnik

address: 419 Broadway

summary: Reprogramming to iPSCs resets the epigenome of somatic cells including the reversal of X chromosome-inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome-reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X-inactivation, while others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.

overall_design: RRBS profiles were generated from female and male ES cells, female iPS cells, SSEA1+ and SSEA1- reprogramming intermediates, and male and female MEFs, for a total of 18 samples.

first: Alex

last: Meissner

first: Kathrin

last: Plath


title: Abundance of isoforms of adenovirus 5-encoded miRNAs.

city: Vienna

first: Maximilian

last: Kauer

address: Zimmermannplatz 10

department: Molecular Biology

country: Austria

institute: CCRI, St.Anna Children Cancer Research Institute

summary: We identified the abundance of isoforms of miRNAs encoded by human adenovirus 5 in total cytoplasmic RNA fractions of infected cells as well as in fractions containing the purified RNA-induced silencing complex (RISC).

overall_design: Human A549 cells were infected with human adenovirus 5 at an MOI of 30 TCID50/cell or were mock-infected. Total RNA was isolated at 30h post-infection. In addition, RNA incorporated in RISC which was isolated by immunoprecipitation using an antibody recognizing all human Argonaute proteins was conducted as well. Purified RNAs were used to generate sRNA libraries with were subjected to RNA sequencing.

first: Florian

last: Bellutti

first: Reinhard

last: Klein


title: RNF17 referees ping-pong in mouse testes [RIP-seq]

status: Public on Dec 17 2014

city: Cold Spring Harbor

first: Vasily

last: Vagin

state: NY

address: 1 Bungtown Rd

institute: Cold Spring Harbor

summary: In mammals, piRNA populations are dynamic throughout male germ cell development. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, however, the piRNA population is transposon-poor and restricted to primary piRNAs derived from pachytene piRNA clusters1-6. The mechanism controlling which piRNAs are present at each developmental stage is poorly understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong (secondary amplification) occurs inappropriately in meiotic cells – aberrantly targeting protein-coding genes and lncRNAs. Our data indicate that RNF17 comprises one component of a “refereeing” mechanism that prevents deleterious activity of the meiotic piRNA pathway by ensuring the selective loading of PIWI proteins with products of meiotic piRNA clusters.

overall_design: Examination of small RNAs isolated from MIWI and MILI IPs of heterozygous and homozygous RNF17 adult testes

first: Kaja

last: Wasik

first: Oliver

last: Tam

first: Yuan

last: Hao

first: Ilaria

last: Falciatori

first: Molly

last: Hammell

middle: V

first: Gregory

last: Hannon


title: RNF17 blocks promiscuous activity of PIWI proteins in mouse testes

summary: PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction.

overall_design: Refer to individual Series


title: β3 agonist administration before reperfusion reduces infarct size  and improves long term cardiac function in small and large animal models of myocardial infarction

city: Madrid

first: David

last: Sanz-Rosa

address: 3, Melchor Fernandez Almagro

department: DEAIC

institute: CNIC

summary:
RNA sequencing and subsequent bioinformatics analyses were performed at early reoxygenation stages in HL-1 cardiomyocytes treated or not with BRL37344
Methods: HL-1 cardiomyocytes were subjected to Hypoxia/Reoxygenation (6h/1h) with/without a β3AR agonist (BRL37344 5µmol/L). mRNA profiles were generated by deep sequencing,  in triplicate, using Illumina GAIIx.The sequence reads that passed quality filters were quantified using BWA aligned reads using RSEM. qRT–PCR validation was performed using SYBR Green assay.
Results: After 6h of hypoxia followed by 1h reoxygenation, 866 genes were differentially expressed upon β3AR stimulation by BRL37344. Among these, 177 were at least 2-fold up or downregulated.
Conclusions: Our results show that Hsp70 plays a key role in the cardioprotection afforded by β3AR agonism in cardiomyocytes during the early window of H/R.

overall_design: mRNA profiles from cardiomyocyte subjected to H/R (6h/1h)  with/without a β3AR agonist  were generated by deep sequencing, in triplicate, using Illumina GAIIx.

first: Jaime

last: Garcia-Prieto

first: Borja

last: Ibanez


title: SEEDSTICK Is a Master Regulator of Development and Metabolism in Arabidopsis Seed Coat

city: Milano

first: IGNACIO

last: EZQUER

state: Italia

address: Via celoria 26

country: Italy

institute: MILANO UNIVERSITY

summary:
Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from the SEEDSTICK mutant in Arabidopsis with a wild type,  to unveil the role of this transcription factor in seed development and decipher the impact of this factor in PAs metabolism
Methods: Total RNA was extracted from two biological replicates from both wild-type and stk mutant inflorescences and siliques until 5 DAP with the Qiagen Kit according to the manufacturer's instructions. DNA contaminations were removed using the PROMEGA RQ1 RNase-Free DNase according to the manufacturer's instructions. RNA quality integrity was analyzed by electrophoresis gel and was validated on a Bioanalyzer 2100 (Aligent, Santa Clara, CA); RNA Integrity Number (RIN) values were greater than 7 for all the samples. In order to confirm that in stk mutant samples STK was not expressed, STK expression was checked by real time PCR with primer RT 780 (5’-TGCGATGCAGAAGTTGCGCTC-3’) and RT 781 (5’-AGTACGCGGCATTGATTTCTTG-3’). Sequencing libraries were prepared according to the manufacturer’s instructions by TruSeq RNA Sample Prep kit (Illumina Inc.) and sequenced on Illumina HiSeq2000 in one lane single-read 50bp. The processing of fluorescent images into sequences, base-calling and quality value calculations were performed using the Illumina data processing pipeline (version 1.8). Raw reads were filtered to obtain high-quality reads by removing low-quality reads containing more than 30% bases with Q < 20. Finally, a quality control of the raw sequence data was performed using FastQC [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/].
Results: A total of 102,278,242 reads passed a quality filter and 85% were mapped back to the Arabidopsis TAIR10 genome. Approximately 90% mapped uniquely to only one location and could be assigned to a single annotated TAIR10 gene. Normalization of expression values was performed using RPKM values. All other parameters were kept at default levels. The CLC Genomic Workbench was also further used to determine all differentially expressed transcripts found in each cDNA library. Baggerley's test and a FDR correction were used for statistical analysis of samples. Our analysis revealed that 156 genes were upregulated, whereas for 90 genes a reduction in their mRNA level was observed in the stk mutant when compared to wild-type . Data analysis revealed a significant enrichment for terms related to the phenylpropanoid metabolic process, flavonoid biosynthetic process as well as cellular amino acid derivative metabolic process.
Conclusion: Our genome-wide transcriptomic analysis suggests that the ovule identity factor STK is involved in the regulation of several metabolic processes providing a strong connection between cell fate determination, development and metabolism. In particular we characterize, through phenotypic, genetic, biochemical and transcriptomic approaches, the role of STK in PAs biosynthesis. Our results indicate that STK exerts this role through the direct regulation of the gene encoding for BANYULS/ANTHOCYANIDIN REDUCTASE (BAN/ANR), which converts anthocyanidins into their corresponding 2,3-cis-flavan-3-ols. Our study also demonstrates that the levels of H3K9ac chromatin modification directly correlate with the active state of BAN in an STK-dependent way. This supports the theory that MADS-domain proteins control the expression of their target genes through the modification of the chromatin states. STK might recruit or negatively regulate histone modifying factors to control their activity. Moreover, we show that STK controls through a complex regulatory network not only directly BAN but also other regulators of this key gene in tannin production

overall_design: mRNA profiles  from both Arabidopsis wild-type and stk mutant inflorescences and siliques until 5 DAP were generated by deep sequencing, in duplicate according to the manufacturer’s instructions by TruSeq RNA Sample Prep kit (Illumina Inc.) and sequenced on Illumina HiSeq2000 in one lane single-read 50bp. In order to confirm that in stk mutant samples STK was not expressed, STK expression was checked by real time PCR with primer RT 780 (5’-TGCGATGCAGAAGTTGCGCTC-3’) and RT 781 (5’-AGTACGCGGCATTGATTTCTTG-3’).

first: Ignacio

last: Ezquer

first: Chiara

last: Mizzotti