title: An RNA-Seq transcriptome and splicing database of neurons, glia, and vascular cells of the cerebral cortex

city: Dallas

first: kenian

last: chen

address: 5323 Harry Hines Boulevard

department: Population and Data Science

institute: UTsouthwestern

summary:
Purpose: To better understand the function of the various cell types of the brain, we prospectively purified neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these 8 cell types by RNA sequencing and used a highly sensitive algorithm to detect alternative splicing events in each gene. Our analysis identified thousands of new cell type enriched genes and splicing isoforms that will provide novel markers for cell identification, new tools for genetic manipulation, and numerous insights into the biology of the brain.
Method Part1:To purify astrocytes, we took advantage of a BAC transgenic mouse line expressing EGFP under the control of regulatory sequences in Aldh1l1-BAC. This line has been previously characterized to have complete astrocyte-specific labeling throughout the brain. Cells from a litter of 8-16 P7 Aldh1l1-EGFP transgenic mice of both genders were pooled together as one biological replicate. The cortices were dissected out and meninges were removed. The tissue was enzymatically dissociated to make a suspension of single cells as described previously. Briefly, the tissue was incubated at 33 °C for 45 minutes in 20 ml of a papain solution containing Earle’s balanced salts (EBSS, Sigma, St. Louis, MO, E7510), D(+)-glucose (22.5mM), NaHCO3 (26mM), DNase (125U/ml, Worthington, Lakewood, NJ, LS002007),  papain (9 U/ml, Worthington, Lakewood, NJ, LS03126), and L-cysteine (1mM, Sigma, St. Louis, MO, C7880). The papain solution was equilibrated with 5% CO2 and 95% O2 gas before and during papain treatment. Following papain treatment, the tissue was washed three times with 4.5ml of inhibitor buffer containing BSA (1.0mg/ml, Sigma, St. Louis, MO, A-8806), and ovomucoid (also known as trypsin inhibitor, 1.0 mg/ml, Roche Diagnostics Corporation, Indianapolis, IN 109878) and then mechanically dissociated by gentle sequential trituration using a 5ml pipette. Dissociated cells were layered on top of 10ml of high concentration inhibitor solution with 5mg/ml BSA and 5mg/ml ovomucoid and centrifuged at 130g for 5 minutes. The cell pellet was then resuspended in 12 ml Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen, Carlsbad, CA 14287) containing 0.02% BSA and 12.5U/ml DNase and filtered through a 20um Nitex mesh (Sefar America Inc., Depew NY, Lab Pak 03-20/14) to remove undissociated cell clumps. This yields a single cell suspension. Cell health is assessed by trypan blue exclusion. Only single cell suspensions with >85% viability were used for purification experiments. 1μg/ml propidium iodide (PI, Sigma, St. Louis, MO, P4864) was added to the single cell solution to label dead cells. Cells were sorted on a BD Aria II cell sorter (BD Bioscience) with a 70μm nozzle. Dead cells and debris were gated first by their low forward light scatter and high side light scatter and secondly by high PI staining. Doublets were removed by high side light scatter. Cell concentration and flow rate were carefully adjusted to maximize purity. Astrocytes were identified based on high EGFP fluorescence. FACS routinely yielded >99% purity based on reanalysis of sorted cells.
Method Part2:To purify neurons, a single cell suspension was prepared as described above and incubated at 34 °C for one hour to allow expression of cell surface protein antigens digested by papain, and then incubated on two sequential panning plates coated with BSL-1 to deplete endothelial cells (10 minutes each), followed by a 30 minute incuation on a plate coated with mouse IgM anti-O4 hybridoma (Bansal et al., 1989. 4ml hybridoma supernatant diluted with 8ml DPBS/0.2% BSA) to deplete OPCs, and then incubated for 20 minutes on a plate coated with rat anti-mouse CD45 (BD Pharmingen 550539, 1.25ug in 12ml of DPBS/0.2% BSA) to deplete microglia and macrophages. Finally cells were added to a plate coated with rat anti-mouse L1CAM (30ug in 12ml of DPBS/0.2% BSA, Millipore, Billerica, MA, MAB5272) to bind neurons. The adherent cells on the L1CAM plate were washed 8 times with 10-20 ml of DPBS to remove all antigen-negative nonadherent cells, and then removed from the plate by treating with trypsin (Sigma, 1,000U/ml, T-4665) in 8ml Ca2+ and Mg2+ free EBSS (Irvine Scientific, Santa Ana, CA, 9208) for 3-10 minutes at 37°C in a 10% CO2 incubator. The trypsin was then neutralized with 20ml of fetal calf serum (FCS) solution containing 30% FCS (Gibco, 10437-028), 35% Dulbecco’s modified eagle medium (DMEM, Invitrogen, 11960-044), and 35% Neurobasal (Gibco, 21103-049). The cells were dislodged by gentle squirting of FCS solution over the plate and harvested by centrifugation at 200g for 10 minutes.
Method Part3:To purify microglia and oligodendrocyte-lineage cells, the mice were first perfused with 10ml PBS to remove macrophage contamination from the brain. A single cell suspension was then prepared as described above and incubated 20 minutes on a plate coated with rat anti-mouse CD45 (BD Pharmingen 550539, 1.25ug in 12ml of DPBS/0.2% BSA) to harvest microglia, and then incubated sequentially on four BSL1 coated plates (8 minutes each) to deplete endothelial cells and remaining microglia. The remaining cells were next incubated for 30 minutes on a rat anti-PDGFRα (10ug in 12ml DPBS/0.2% BSA, Fitzgerald, Acton, MA, 10R-CD140aMS) coated plate to harvest OPCs, and then incubated on an additional PDGFRα plate and mouse A2B5 monoclonal antibody ascites (American Type Culture Collection, Rockville, MD) coated plate for 30 minutes each to deplete remaining OPCs. The cell suspension was next incubated on an anti-MOG hybridoma coated plate for 30 minutes to harvest myelinating oligodendrocytes, followed by an additional anti-MOG hybridoma coated plate for 30 minutes to deplete any remaining myelinating oligodendrocytes. Finally, the cell suspension was incubated on an anti-GalC hybridoma coated plate for 30 minutes to harvest newly formed oligodendrocytes. For purification of RNA, the cells were lysed while still attached to the panning plate with Qiazol reagent (Qiagen 217004), and total RNA was purified as described below.
Mehtod Part4:To purify endothelial cells, we took advantage of Tie2-EGFP transgenic mice available from Jackson labs. These mice express EGFP under the pan-endothelial Tie2 promotor (Daneman et al., 2010; Motoike et al., 2000). Single cell suspension was prepared and FACS was performed as described above.
Method Part5: RNA-Seq was performed on the polyadenylated fraction of RNA isolated from purified cell samples.Two biological replicates were used for each phase.100 ng total RNAs were used for each sequencing library. RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).The samples were then sequenced using the Illumina HiSeq 2000 sequencer.
Method Part6: Read mapping and Transcriptome construction were done by using optimized pipeline which integrate Tophat followed by Cufflinks.
Result:We purified neurons, astrocytes, oligodendrocyte precursor cells (OPCs), newly formed oligodendrocytes (NFOs), myelinating oligodendrocytes (MOs), microglia, endothelial cells and pericytes from mouse cortex and used RNA-Seq to generate a high resolution transcriptome database of > 22,000 genes. We identified thousands of novel cell type-enriched genes that have not been previously identified. These include novel transcription factors, ligands, receptors, enzymes, and signaling molecules. We then used a novel splice mapping algorithm to identify thousands of cell type-specific alternative splicing events.
Conclusion:We generated a transcriptome database for these 8 cell types by RNA sequencing and used a highly sensitive algorithm to detect alternative splicing events in each gene. Our analysis i

overall_design: mRNA profiles of purified cell samples from mice were generated by RNA-sequencing,  in duplicate, using Illumina HiSeq 2000.

first: Ye

last: Zhang

first: Kenian

last: Chen

first: Steven

last: Sloan

first: Anja

last: Scholze

first: Christine

last: Caneda

first: Nadine

last: Ruderisch

first: Shuyun

last: Deng

first: Richard

last: Daneman

first: Ben

last: Barres

first: Jia

last: Wu


title: Gene expression profiling of wild type and CNS2-deficient Treg cells in the resting state

status: Public on Aug 14 2014

city: New York

first: Aaron

last: Arvey

state: NY

country: USA

institute: Memorial Sloan-Kettering Cancer Center

type: Expression profiling by high throughput sequencing

summary: Resting regulatory T cells (CD4+GFP+CD44lowCD62Lhigh) were sorted from the littermates of male Foxp3gfp and Foxp3ΔCNS2-gfp mice. Poly(A)-tailed RNA were amplified and sequenced on an Ion Torrent platform.

overall_design: RNA-seq of WT and CNS2KO regulatory T cells in the resting state

first: Yong

last: Feng

first: Alexander

middle: Y

last: Rudensky


title: Genome wide analysis of gene expression in LPS stimulated splenic B cell.

city: Gifu

first: Takashi

last: MaruYama

address: 1-1 Yanagido

country: Japan

institute: Gifu University

summary: Analysis of class switch recombination, maturation or differenciation of B cells at gene expression levels. The hypothesis tested in the present study was that TLR signaling in B cells plays an pivotal role for class switch, maturation and differenciation.

overall_design: Total RNA obtained from cultured splenic B cells. Gene expression compared between control and cKO B cells.

first: Takayuki

last: Isagawa


title: H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

city: Boston

first: Ruslan

last: Sadreyev

state: Massachusetts

address: 185 Cambridge Street

department: Molecular Biology

institute: Massachusetts General Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The bithorax complex (BX-C) in the fruit fly, Drosophila melanogaster, is a cluster of homeotic genes that determines the identities the body segments.  Expression of these genes is governed by cis-regulatory domains, one for each parasegment, which are arranged on the chromosome in the order of the parasegments they affect.  Stable repression of these domains depends on the functions of the Polycomb Group, including its ability to methylate lysine 27 of histone H3.  To learn whether PcG proteins generate parasegment-specific chromatin signatures, we have used transgenes to mark and isolate nuclei from single parasegments.  These nuclei were profiled for histone modifications and chromatin proteins.  The H3K27me3 profiles across the BX-C in successive parasegments show a striking “stairstep” pattern that reveals sharp boundaries of the BX-C regulatory domains.  The borders of H3K27me3 modification domains are sharp, and align precisely with binding sites for the CCCTC-binding protein (CTCF).  H3K27ac is broadly enriched across active domains, in a pattern complementary to K27me3.  These findings provide a molecular definition of the homeotic domains, and implicate precisely localized H3K27 modification as a central determinant of segment identity.

overall_design: Nuclei from single parasegments of the Drosophila embryo were isolated and ChIP-seq performed for H3K27me3, CTCF, H3K27ac, H3K4me3, Ph, Pol2, Pc, and Suz12. For each sample, two biological replicates were performed.

first: Sarah

middle: K

last: Bowman

first: Aimee

middle: M

last: Deaton

first: Heber

last: Domingues

first: Peggy

middle: I

last: Wang

first: Robert

middle: E

last: Kingston

first: Welcome

last: Bender


title: Ecdysone and Mediator trigger a metabolic switch uncoupling cell cycle from cell growth to end proliferation in Drosophila neural stem cells

city: Vienna

first: Thomas

middle: Rainer

last: Burkard

address: Dr. Bohrgasse 7

country: Austria

institute: IMBA

summary: Stem cells are highly abundant and proliferate rapidly during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. Here, we show that changes in energy metabolism induced by the steroid hormone Ecdysone initiate an irreversible cascade of events leading to cell cycle exit in Drosophila neural stem cells. We show that the timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple cell cycle progression from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Neuroblasts isolated from brain tumors fail to undergo this shrinkage process and this may explain why they are immortalized. Our findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation control in stem cells.

overall_design: Comparison of transcriptomes of Drosophila melanogaster central brain NBs from wild-type larval NBs, wild type pupal NBs and med27 RNAi pupal NBs.

first: Catarina

middle: F

last: Homem

first: Victoria

last: Steinmann

middle: R

first: Juergen

middle: A

last: Knoblich


title: Discovery of biomarkers predictive of GSI response in triple negative breast cancer and adenoid cystic carcinoma

city: Charlottesville

first: Chongzhi

last: Zang

state: VA

institute: University of Virginia

summary: Next generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple negative breast cancers (TNBC, 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with pacitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in TNBC patients. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD low tumors without NOTCH1 mutations were resistant.

overall_design: Gene expression profiling for Notch-sensitive cancer cell lines using RNA-seq, each sample with triplicates

first: Brandon

last: Kremer

first: Len

last: Taing

first: X

last: Liu

first: Jon

middle: C

last: Aster

first: Warren

middle: S

last: Pear


title: Analysis of the global retinal transcriptome of rod photoreceptor-specific Dicer1 conditional knockout mice and control littermates

city: Cleveland

first: Xiaodong

last: Bai

address: 10900 Euclid Avenue

department: Center for RNA Molecular Biology

institute: Case Western Reserve University

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors [ChIP-seq]

city: RTP

first: Raja

last: Jothi

state: NC

address: 111 TW Alexander Drive; A314

department: National Institute of Environmental Health Sciences

institute: National Institutes of Health

summary: Cell type-specific master transcription factors (MTFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that all three subunits of the ubiquitous heterotrimeric CCAAT-binding NF-Y complex are required for the maintenance of embryonic stem cell (ESC) identity, and establish NF-Y as a novel component of the core pluripotency network. Genome-wide occupancy and transcriptomic analyses in ESCs and neurons reveal that not only does NF-Y regulate genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with MTFs. Mechanistically, NF-Y's distinctive DNA-binding mode promotes MTF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a novel function for NF-Y in promoting chromatin accessibility, and suggest that other proteins with analogous structural and DNA-binding properties may function in similar ways.

overall_design: Genome-wide mapping of NF-YA, NF-YB, and NF-YC subunits of the NF-Y complex in mouse ESCs, and microarray gene expression profiling of control knockdown (KD), NF-YA KD, NF-YB KD, NF-YC KD, and NF-YA/NF-YB/NF-YC triple KD ESCs.

first: Andrew

middle: J

last: Oldfield

first: Pengyi

last: Yang


title: The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

first: Colin

middle: Dean

last: Malone

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

department: Skirball Institute - Developmental Genetics

institute: NYU Medical Center

summary: The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where in the absence of RnpS1 the fourth intron of piwi is retained. Within this intron the polypyrimidine tract is disrupted by a transposon-adjacent A/T-rich sequence that confers dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing.

overall_design: Small-RNA libraries from two control samples and four knockdowns in germline or somatic tissues of the Drosophila melanogaster ovary.

middle: D

first: Jean-Yves

last: Roignant

first: Claire

last: Mestdagh


title: DNA methylation and gene expression analysis of an Epithelial-to-mesenchymal transition model

city: L'Hospitalet de Llobregat

first: Manel

last: Esteller

state: Barcelona

address: Hospital Duran i Reynals Av. Gran Via s/n km, 2.7

department: PEBC

country: Spain

institute: IDIBELL

type: Methylation profiling by high throughput sequencing


title: Reinstatement of developmental stage-specific GATA4 enhancers controls the gene expression program in heart disease

status: Public on Aug 13 2014

first: Fei

last: Gu

state: MA

address: 320 Longwood Ave.

department: Cardiology

institute: Childrens hospital boston, Harvard Medical School

summary: Through integration of GATA4 genome-wide occupancy and RNA-seq dataset in embryonic conditional knockout heart, we identified a class of the master cardiac regulator GATA4 bound enhancers. With combinatorial assembly of chromatin signatures, we further refined that a unique type of fetal distal GATA4 enhancers enriched for H3K27ac, H3K4me1, but not for H3K4me3 and RNA polymerase II, are reinstated in adult hypertrophic heart.

overall_design: Study Gata4 and histone modifications in E12.5, Adult, Banding and Sham, and to correlate to gene expression and heart function

first: Aibin

last: He

first: Yong

last: Hu

first: Qing

last: Ma

first: Lilian

last: Ye

first: Axel

last: Visel

last: Pennacchio

first: William

middle: T

last: Pu


title: A relative shift in cloacal location repositions external genitalia in amniote evolution

city: Basel

first: Patrick

last: Tschopp

address: Vesalgasse 1

department: Department of Environmental Sciences

country: Switzerland

institute: University of Basel

summary: Comparative RNA-seq profiling of mouse and anole lizard developing limbs and external genitalia, to assess evolutionary and develomental relationships, between the two tissue types based on transcriptomic data

sample_organism:
Mus musculus
Anolis carolinensis

platform_organism:
Mus musculus
Anolis carolinensis

overall_design: RNA-seq profiling of embryonic limb and external genitalia tissue at different stages of development, in mouse and anole lizard, in duplicates, using Illumina HiSeq

first: Clifford

last: Tabin


title: Transcriptome Sequencing (RNA-seq) of Ara-C Resistant Murine AML Cell Lines Identifies Mechanisms of Resistance

city: Minneapolis

first: David

middle: A.

last: Largaespada

state: MN

address: 2231 6th Street SE

department: Dept. of Genetics, Cell Biology and Development, Cancer Center

institute: The University of Minnesota

summary: An RNA-seq study of altered gene expression and mutations in Ara-C resistant acute myeloid leukemia murine cell lines.  The analysis of the RNA-seq data led to the identification of a large deletion within the Dck coding sequence of the B117H cell line, which produced an alternatively processed form of Dck mRNA.  The RNA-seq analysis also identified the presence of an insertion mutation in Dck in the B140H cell line.  The RNA-seq analysis also identified a number of significant expression changes which did not appear in a previous microarray analysis (GSE18322), as well as identified other mutations which may be contributing to Ara-C resistance.

overall_design: Two highly Ara-C resistant cell lines, B117H and B140H were derived from Ara-C sensitive parental cell lines, B117P and B140P.  Variations in gene expression as well identification of acquired mutations between these Ara-C resistant/sensitive sets were studied using various RNA-seq analysis tools.

first: S

last: Rathe

first: D


title: RNAseq analysis of the global retinal transcriptome of rod photoreceptor-specific Dicer1 conditional knockout mice and control littermates

first: Xiaodong

last: Bai

summary: We report RNAseq analysis of the transcriptome of retinas from mature rod-specific Dicer1 cKO mice and control littermates lacking Cre expression in order to better understand changes in gene regulation that could lead to retinal degeneration in cKO mice.

overall_design: Examine retinal transcriptome of 3 biological replicates for each genotype from 4-week-old animals with tissue collected between 8:00 - 10:00AM

first: Thomas

last: Sundermeier

first: Krzysztof

last: Palczewski


title: Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection [IMR90_Input]

status: Public on Aug 15 2014

city: La Jolla

first: Bing

last: Ren

state: CA

address: 9500 Gilman Drive

institute: UCSD

summary: Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF?-, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication.

overall_design: Chromatin IP background Input DNA for ChIP-seq normalization

first: Roberto

last: Ferrari

first: Dawie

last: Gou

first: Gauri

last: Jawdekar

first: Sarah

last: Johnson

first: Miguel

last: Nava

first: Trent

last: Su

first: Ahmed

last: Yousef

first: Nathan

last: Zemke

first: Matteo

last: Pellegrini

first: Siavash

last: Kurdistani

first: Arnold

last: Berk


title: Specificity, propagation and memory of pericentric heterochromatin in mouse fibroblasts

status: Public on Aug 12 2014

city: Heidelberg

first: Jan-Philipp

last: Mallm

address: Im Neuenheimer Feld 267

country: Germany

institute: DKFZ

summary: We assembled a quantitative map for the abundance and interactions of 16 factors related to PCH in living cells and found that stably bound complexes of the histone methyltransferase Suv39h1/2 demarcate the PCH state. From the experimental data we developed a predictive mathematical model that explains how chromatin-bound Suv39h1/2 complexes act as nucleation sites and propagate a spatially confined PCH domain with elevated histone H3 trimethylation levels via chromatin dynamics. Enrichment of HP1, Suv39h1/h2, H3K9me3 and H3K36me3 was assessed by ChIP-seq in NPCs derived from ESCs showing differential occupation at intergenic major satellite repeats and enrichment of heterochromatin factors.

overall_design: ChIP-seq of HP1, Suv39h1, Suv39h2, H3K9me3, H3K36me3 in NPCs

first: J

middle: P

first: C

last: Bauer

first: K

last: Müller-Ott

first: F

last: Erdel

first: A

last: Rademacher

last: Rippe


title: Transcriptomic Analysis of Airway Smooth Muscle by Next-Generation Sequencing (RNA-Seq): Asthma versus Healthy Controls

city: Amsterdam

first: Ching Yong

last: Yick

address: Meibergdreef 9

department: Respiratory Medicine

country: Netherlands

institute: Academic Medical Center

summary:
Rationale: Asthma and atopy shares common features including Th2-inflammation. However, impairment of airway function seems to be absent in atopy. Increased understanding of the complex cellular and molecular pathways defining the similarities and differences between asthma and atopy may be achieved by transcriptomic analysis (RNA-Seq).
Hypothesis and Aims: As the airway smooth muscle (ASM) layer plays an important role in airway function, we hypothesized that the transcriptomic profile of the ASM layer in endobronchial biopsies is different between atopic asthma patients and atopic healthy controls. First, we examined the differences in transcriptomic profiles of the ASM layer in endobronchial biopsies between atopic mild, steroid-free asthma patients, and atopic and non-atopic healthy controls. Second, we investigated the association between the transcriptomic profiles of the ASM layer and airway function.
Methods: This cross-sectional study included 12 steroid-free atopic asthma patients, 6 atopic, and 6 non-atopic healthy controls. RNA of ASM from 4 endobronchial biopsies per subject was isolated and sequenced (GS FLX+, 454/Roche). Ingenuity Pathway Analysis was used to identify gene networks. Comparison of the numbers of reads per gene in asthma and controls was based on the negative binomial distribution. At the current sample size the estimated false discovery rate was approximately 1%.
Results: Yield of isolated RNA was 30-821ng. We identified 174 differentially expressed genes between asthma and atopic controls, 108 between asthma and non-atopic controls, and 135 between atopic and non-atopic controls. A set of 8 genes was identified, which seems to define asthma patients from non-asthmatic controls regardless of atopy. Four of these genes were significantly associated with airway hyperresponsiveness.
Conclusion: A difference in transcriptomic profile of the airway smooth muscle layer in asthma patients compared to atopic and non-atopic healthy controls may lead to a different regulation of inflammatory pathways and of airway smooth muscle function and development resulting in impaired airway function.

overall_design: This cross-sectional transcriptomics study consisted of 2 visits. At visit 1, asthma patients (n=12), and healthy atopic (n=6) and non-atopic (n=6) controls were screened for eligibility to participate according to the in- and exclusion criteria. Spirometry and a methacholine bronchoprovocation test were performed. At visit 2, FEV1 reversibility was measured and 4 endobronchial biopsies per subject were collected during a bronchoscopy. Airway smooth muscle was collected from the biopsies by laser capture microdissection and total RNA isolated. cDNA was prepared using the Ovation RNA-Seq System (NuGEN). RNA-Seq was performed using the GS FLX+ instrument (454/Roche). Sequence reads were mapped against the human genome (hg19; UCSC). Comparison of the numbers of reads per gene between asthma and healthy controls was based on the negative binomial distribution and carried out with the R package DESeq including correction for multiple testing.

first: Ching-Yong


title: Whole genome bisulfite sequencing of MDCK cells, before and after TGFB-induced epithelial-mesenchymal transition

first: Manel

last: Esteller

overall_design: Sequencing of bisulfite converted DNA from MDCK cells untreated, and after a 30 days treatment with TGF beta

first: Javier

last: Carmona


title: Transcriptional response to stress in serum deprived mouse fibroblasts in the presence of MSK1/2 inhibitor.

city: Goettingen

first: Anna

last: Sawicka

address: Am Fassberg 11

department: Dep. of Molecular Biology

institute: Max Planck Institure for Biophysical Chemistry

summary: We have employed gene expression profiling in order to identify targets of transcriptional response to stress in resting mouse Swiss 3T3 fibroblasts, either untreated (control) or treated with anisomycin for 3 or 6 hours to induce the p38/MAP kinase pathway. In order determine transcriptional effects dependent on MSK1/2 kinase activity, H89 inhibitor was used in the study.

overall_design: Serum starved (72 h 0.2% FCS) mouse 3T3 cells were treated with anisomycin (188.5 nM) for 3 h or 6h (in duplicates) either with or without 15-min pre-treatment with MSK1/2 inhibitor H89 (10 uM). Untreated, serum-starved cells were used as a control. RNA was collected and gene expression profiling using strand-specific RNA-seq was performed.

first: Christian

last: Seiser


title: Transcriptomic characterization of Hepatocellular Carcinoma with CTNNB1 mutation

city: Beijing

first: Tao

last: Cai

address: No. 7 Science Park Road, Zhongguancun Life Science Park

country: China

institute: National Institute Of Biological Sciences, Beijing (NIBS)

summary: we report transcritomic characterization of changes in CTNNB1 mutation positive HCCs in comparison to paratumoral tissues

overall_design: 4 pair of HCC and paratumoral liver tissues are sequenced and compared

first: Liang

last: Chen

first: Xiao

last: Ding

first: Huanwei

last: Huang


title: Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs

city: Solna

first: Joakim

last: Lundeberg

address: Tomtebodavägen 23A

department: Gene Technology

country: Sweden

institute: KTH-Science for Life Laboratory

summary: We extracted RNA from whole cells and RNA from the cytoplasm and performed RNA sequening to compare differences in gene expression level and investigate what is the most appropriate estimate of the amount of mRNA present in a given cell population. The study was based on three human cell lines.

overall_design: Analyze of transcriptome in 3 human cell lines (U-2 OS, A-431, U-251MG). Each cell line was prepared with four biological replicates for total RNA and four for cytoplasmic RNA.

first: Beata

middle: W

last: Solnestam

first: Henrik

last: Stranneheim

first: Jimmie

last: Hällman

first: Max

last: Käller

first: Emma

last: Lundberg

first: Pelin

last: Akan


title: The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites

first: Ruslan

last: Sadreyev

summary: Long noncoding RNAs (lncRNAs) are important regulators of chromatin; however, the mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. We used CHART-seq to map the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1, which localize within the nucleus to paraspeckles and nuclear speckles, respectively. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. Protein mass spectrometry analysis of CHART-enriched material (CHART-MS) identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation affects the localization of NEAT1 to active chromatin sites, implying that DNA sequence itself does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process.

overall_design: Paired-end CHART-seq was performed for a single replicate of each capture oligonucleotide in untreated MCF-7 cells to establish binding sites of these RNAs, for a total of 6 samples. To investigate the effects of transcriptional inhibition and E2 stimulation on the localization of these RNAs, we performed paired-end CHART-seq with each capture oligonucleotide for two biological replicates of flavopiridol- and vehicle (DMSO)-treated MCF-7 cells and for two biological replicates of E2- and vehicle (ethanol)-treated MCF-7 cells. To establish the overlap of NEAT1 and MALAT1 binding sites with a known component of paraspeckles (NEAT1-containing subnuclear body), we performed paired-end ChIP-seq for the paraspeckle component PSF in MCF-7 cells, as well as a single-end biological replicate.

first: Jason

last: West

first: Christopher

last: Davis

first: Hongjae

last: Sunwoo

first: Matthew

last: Simon

first: Peggy

last: Wang

first: Michael

middle: Y

last: Tolstorukov

first: Robert

last: Kingston


title: small RNAseq analysis of the global retinal transcriptome of rod photoreceptor-specific Dicer1 conditional knockout mice and control littermates

first: Xiaodong

last: Bai

first: Thomas

last: Sundermeier

first: Krzysztof

last: Palczewski


title: Transcriptional response to stress in serum deprived mouse fibroblasts [RNA-Seq]

first: Anna

last: Sawicka

summary: We have employed gene expression profiling in order to identify targets of transcriptional response to stress in resting mouse Swiss 3T3 fibroblasts, either untreated (control) or treated with anisomycin to induce the p38/MAP kinase pathway.

overall_design: Serum starved (72 h 0.2% FCS) mouse 3T3 cells were treated with anisomycin (188.5 nM) for 1 h (in duplicates). Untreated, serum-starved cells were used as a control. RNA was collected and gene expression profiling using strand-specific RNA-seq was performed.

first: Christian

last: Seiser


title: Stress-induced signaling to the nucleosome in serum deprived mouse fibroblasts.

summary: We have employed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze changes in chromatin architecture as well as the occupancy of two RNA Polymerase II (RNAPII) isoforms, initiation-competent (RNAPIIS5ph) as well as elongation-competent (RNAPIIS2ph) upon stress induction. We used resting mouse Swiss 3T3 fibroblasts, either untreated (control) or treated with anisomycin to induce the p38/MAP kinase pathway.

overall_design: Serum starved (72 h 0.2% FCS) mouse 3T3 cells were treated with anisomycin (188.5 nM) for 1 h (in duplicates). Untreated, serum-starved cells were used as a control. Isolated chromatin was subjected to immunoprecipitation with the following antibodies: a-H3S28ph, a-H3K9ac, a-H3K4me3, a-RNAPIIS5ph and a-RNAPIIS2ph. Resulting DNA was sequenced using Illumina platforms.


title: The Hippo tranducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation

city: Torino

first: Raffaele

last: Calogero

state: To

address: Via Nizza 52

department: Molecular Biotechnology Center

country: Italy

institute: University of Torino

summary: The Hippo pathway effector YAP1 controls stem cell fate in epithelial tissues, but its role in stem cells of non-epithelial tissues, such as skeletal muscle, is poorly documented. Here we show that sustained YAP1 activity in mouse activated satellite cells in vivo induces rhabdomyosarcoma (RMS) resembling human embryonal RMS (ERMS) with high penetrance and short latency. The transcriptional program of YAP1 in ERMS drives pro-proliferative pathways whilst decreasing MyoD1 and MEF2 pro-differentiation activity to globally maintain the myoblastic phenotype of ERMS. Normalization of YAP1 expression reduced tumor burden and allowed myogenic differentiation of YAP1-driven and RD ERMS xenografts in situ, thereby identifying YAP1 as a potent RMS-causing oncogene and potential target for differentiation therapy.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: A total of four samples were analyzed. Two ChIP-Seq datasets from RD human cells, containing reads connected to TEAD binding and IgG binding as control/background; two ChIP-Seq datasets from YAP-ERMS mouse cells, containing reads connected to TEAD binding and Input reads as control/background

first: Annie

middle: M

last: Tremblay

first: Edoardo

last: Missiaglia

first: Giorgio

middle: G

last: Galli

first: Simone

last: Hettmer

first: Roby

last: Urcia

first: Matteo

last: Carrara

middle: N

last: Judson

first: Khin

last: Thway

first: Gema

last: Nadal

first: Joanna

middle: L

last: Selfe

first: Graeme

last: Murray

first: Cosimo

last: De Bari

first: Peter

last: Zammit

first: Mauro

last: Delorenzi

first: Amy

last: Wagers

first: Janet

last: Shipley

first: Henning

last: Wackerhage

first: Fernando

last: Camargo


title: Promoter proximal pausing in response to stress in serum deprived mouse fibroblasts.

first: Anna

last: Sawicka

summary: We have employed short-capped RNA sequencing (sc-RNA-seq) in order to identify genes whose expression is regulated by promoter proximal pausing of RNA Polymerase II (RNAPI) in response to stress stimulation. We used serum-deprived mouse Swiss 3T3 fibroblasts, either untreated (control) or treated with anisomycin to induce the p38/MAP kinase pathway.

overall_design: Serum starved (72 h 0.2% FCS) mouse 3T3 cells were treated with anisomycin (188.5 nM) for 1 h (in duplicates). Untreated, serum-starved cells were used as a control. We isolated nuclear RNA, performed size fractionation followed by isolation of short-capped RNAs (scRNA). scRNAs were subsequently converted into DNA library and sequenced.

first: Sawicka

last: Anna

first: Seiser

last: Christian


title: RNAseq of memory cocktail versus IL-2 grown OT-I CD8 T cells

first: Rebecca

last: LaRue

address: 117 Pleasant St SE

department: Department of Lab medicine and Pathology

institute: University of Minnesota

summary: Cytotoxic T cells are typically expanded ex vivo for adoptive immunotherapy by culture with IL-2.  This culture period leads to a differentiated phenotype and acquisition of effector function, as well as a loss of in vivo proliferative capability and anti-tumor efficacy. Here, we report antigen-specific and polyclonal expansion of cytotoxic T cells in a cocktail of cytokines and small molecules that leads to a memory-like phenotype in mouse and human cells even during extended culture, leading to enhanced in vivo expansion and tumor control.

overall_design: OT-I CD8 T cells were cultured for 14 days in either IL-2 or a cocktail of memory inducing small molecules and cytokines (IL-7, IL-21, 2-deoxyglucose and TWS119). Populations were sorted, IL-2 cells were CD44+CD62L-, cocktail cells were sorted into CD44+CD62L-, CD44+CD62L+ and CD44lowCD62L+ populations, and naive OT-I cells were CD44-CD62L+. Total RNA was extracted from each population and prepared for sequencing as three technical replicates.

first: Adam

last: Litterman

first: David

last: Largaespada


title: Combinatorial cis-regulation in Saccharomyces species

status: Public on Aug 11 2014

city: St. Louis

first: Aaron

last: Spivak

state: MO

address: 4444 Forest Park Avenue

institute: Washington University in St. Louis

summary: Transcriptional control of gene expression is a result of complex interactions between the cis-regulatory elements (CRE) at gene promoters.  To understand the regulatory logic of a cell, we need to identify the CRE combinations that regulate gene expression.  We developed a sensitive computational method to identify phylogenetically conserved CRE combinations for any species of interest.  In contrast to previous methods, we do not need to align genomes to identify these combinations.  We applied the method in 7 sensu stricto and sensu lato Saccharomyces species.  80% of the predictions displayed some evidence of combinatorial transcriptional behavior in several existing datasets including 1) ChIP-chip data for co-localization of transcription factors, 2) gene expression data for co-expression of predicted regulatory targets, and 3) gene ontology databases for common pathway membership of predicted regulatory targets.  To establish definitive evidence that these CRE interactions influence TF occupancy, we performed ChIPseq experiments on transcription factors in a wild-type strain and strains in which a predicted cofactor was deleted.  Our experiments showed that TF occupancy at the promoters of the CRE combination target genes depends on the predicted cofactor while occupancy of other promoters is independent of the predicted cofactor.

overall_design: ChIP-seq of 6 myc-tagged Transcription factors in wild-type and predicted co-factor knockout yeast strains in triplicate

first: Gary

last: Stormo


title: Development of an RNAseq-based oligonucleotide microarray to study global gene expression profile in the guinea pig animal model

city: Liverpool

last: Davidsen

state: Merseyside

address: Crown Street

country: United Kingdom

institute: University of Liverpool

summary: In 2008 the GP genome was sequenced to a depth of ~7X full coverage, and last updated in 2010. However, because of the lack of cDNA and protein resources the GP genome is at present poorly annotated. Thus, in order to address this issue we performed an in-depth mRNA sequencing of the lung and skeletal muscles transcriptomes and used this to annotate the available GP genome for transcribed sequences. We then used this information to design and validate a custom Agilent microarray platform (GSE51601).

overall_design: Transcriptome sequencing was performed using Illumina sequencing. Briefly, NCBI and Ensembl transcripts of GP were combined with transcripts constructed from Illumina paired end reads using the TopHat and Cufflinks algorithms [20, 21]. Microarray probe sequences were then chosen based on the combined transcriptome assembly.

first: John

last: Herbert

first: Francesco

last: Falciani


title: The Nuclear Exosome is Active and Important during Budding Yeast Meiosis

status: Public on Aug 09 2014

city: Babraham

first: Jonathan

last: Houseley

state: Cambridgeshire

address: Babraham Institute

department: Epigenetics Programme

institute: Babraham Institute

summary: We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation. Lack of TRAMP activity stabilises ~1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC).

overall_design: One sample each of Cbc2-associated RNA from wild-type and trf4-deleted cells at 6 hours of meiosis

first: Stephen

last: Frenk

last: Oxley


title: Changes in cellular microRNA expression induced by porcine circovirus type 2-encoded proteins.

status: Public on Aug 08 2014

city: Seoul

first: Jae-Sang

last: Hong

state: Seoul

address: building life science and biotech west building L204

country: South Korea

institute: Korea University

summary: We employed deep sequencing technology to uncover cellular miRNAs differentially regulated after expression of each of three PCV2-encoded open reading frames (ORFs) in porcine kidney epithelial PK15 cells.

overall_design: Control PK15 vs. PCV2 ORF1, ORF2, ORF3-expressing PK15.

first: Young

last: Lee

first: Nam-Hoon

last: Kim

first: Jun-Seong


title: Global 3' RACE in yeast and humans

city: Zürich

first: Claus

last: Azzalin

address: Otto-Stern-Weg 3

department: Institute of Biochemistry

institute: ETH Zürich

summary: Mpn1 proteins are evolutionarily conserved exonucleases that modify spliceosomal U6 small nuclear RNAs (snRNAs) post-transcriptionally. Mutations in the human MPN1 gene are associated to the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN). Mpn1 deficiency leads to aberrant U6 3’ end processing and accelerated U6 decay through unknown molecular mechanisms. Here we show that in mpn1Δ fission yeast cells U6 is barely bound by the protective Lsm2-8 complex, undergoes extensive oligoadenylation and is degraded by the nuclear RNA exonuclease Rrp6 independently of the poly(A) polymerase Cid14/Trf4. Mpn1 processes U6 in a spliceosome-dependent manner, as mutant U6 molecules that fail to join the spliceosome are not substrates for Mpn1. Moreover, human U6atac, the U6-like snRNA of the minor spliceosome, is a novel substrate for hMpn1. We unveil mechanistic details of a new U6 degradation pathway and further corroborate the notion that inefficient canonical and minor pre-mRNA splicing promotes PN.

sample_organism:
Schizosaccharomyces pombe
Homo sapiens

platform_organism:
Schizosaccharomyces pombe
Homo sapiens

overall_design: The 3' termini of total RNA containing  2'3' cyclic phosphate from Mpn1-proficient and deficient human and yeast cells was sequenced

first: Vadim

last: Shchepachev

first: Charlotte

last: Soneson


title: CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy [ChIP-seq]

city: Giessen

first: Marek

last: Bartkuhn

state: Hessen

address: Aulweg 132

department: Biomedical Informatics and Systems Medicine

institute: Justus-Liebig-University Giessen

summary: H3-ChIP-seq was performed in order to analyze changes in nucleosomal occupancy after depletion of CTCF/P190 and ISWI from Drosophila S2 cells

overall_design: Histone H3 ChIP-seq from Drosophila S2 cells after CTCF/CP190 or ISWI-specific RNAi treatment

first: Rainer

last: Renkawitz


title: RNA-seq data: Baf60c homozygous deletion vs. WT at two time points in cardiac differentiation

status: Public on Aug 10 2014

city: San Francisco

first: Gladstone

last: Bioinformatics

address: 1650 Owens St

department: Data Science and Biotechnology

institute: Gladstone Institutes

summary: This project compares Baf60c homozygous deletions to WT at two time points during cardiac differentiation. This data is used in two separate projects: first to assess the role of Baf60c in regulating cardiac gene expression, and second to assess the repeatability of results from different RNA-seq analysis tools.

overall_design: There are 12 samples total; 3 replicates for each group, times 2 genetic backgrounds (WT and Baf60c homozygous KO), times two timepoints (cardiomyocyte (D10) and cardiac precusor (D5.3)).

first: Benoit

last: Bruneau

first: Alisha

middle: K

last: Holloway

first: Swetansu

last: Hota


title: The Mpn1 exonuclease defines a novel spliceosomal snRNA decay pathway dependent on Rrp6 and Lsm2-8 complex

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

overall_design: Refer to individual Series


title: A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element

city: Vienna

first: Leonie

last: Ringrose

address: Dr. Bohr Gasse 3

institute: IMBA

summary: Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation, by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both ncRNAs inhibit PRC2 histone methyltransferase activity, but in vivo only the reverse strand binds PRC2. Overexpression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that interactions of RNAs with PRC2 are differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of ncRNAs occurs at several hundred PcG binding sites in fly and vertebrate genomes. This work identifies a novel and potentially widespread class of PRE/TREs that switch function by switching the direction of ncRNA transcription.

overall_design: E(Z) and H3K27me3 profile in Drosophila embryos of two transgenic lines (pKC27.vg.fwd' and pKC27.vg.rev') that were crossed to daGAL4 in order to overexpress the forward or the reverse vg PRE/TRE ncRNA from an ectopic site ('site 1' in this study).

first: Veronika

last: Herzog


title: Threonine-4 of the budding yeast RNAP II CTD couples transcription with Htz1-mediated chromatin remodeling

city: Bridgewater

first: Wencheng

last: Li

state: NJ - New Jersey

institute: PTC Therapeutics

summary: The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of repeated YSPTSPS heptapeptides and connects transcription with cotranscriptional events. Threonine-4 (Thr4) of the CTD repeats has been shown to function in histone mRNA 3'-end processing in chicken cells and in transcriptional elongation in human cells. Here, we demonstrate that, in budding yeast, Thr4, although dispensable for growth in rich media, is essential in phosphate-depleted or galactose-containing media. Thr4 is required to maintain repression of phosphate-regulated (PHO) genes under normal growth conditions and for full induction of PHO5 and the galactose-induced GAL1 and GAL7 genes. We identify genetic links between Thr4 and the histone variant Htz1 and show that Thr4, as well as the Ino80 chromatin remodeler, is required for activation-associated eviction of Htz1 specifically from promoters of the Thr4-dependent genes. Our study uncovers a connection between transcription and chromatin remodeling linked by Thr4 of the CTD.

overall_design: RNA-seq of wild type and T4V mutant of budding yeast RNAP II CTD in duplicates

first: Emanuel

last: Rosoninaa

first: Nathan

last: Yurkoa

first: Mainul

last: Hoque

first: Bin

last: Tian

first: James

last: Manley


title: yeast U6 3' RACE

first: Claus

last: Azzalin

overall_design: the 3' termini of U6 or tagged-U6 species from the indicated mutant cells were compared to wt yeast strain


title: human 3' RACE

overall_design: the 3' termini of human U6, U6atac and vtRNA1-1 transcripts from PN patient derived cells and from PN patient cells, compensated with hMPN1 were sequenced.

first: Vadim

last: Shchepachev


title: Chromatin state dynamics during blood formation (ATAC-seq)

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

summary: We develop a new ChIpseq method (iChIP) to profile chromatin states of low cell number samples. We use IChIP to profile the chromatin dynamics during hematopoiesis across 16 different cell types which include the principal hematopoietic progenitors

overall_design: Examination of 2 different histone modifications in 2 cell types.


title: Chromatin state dynamics during blood formation (ChIP-Seq)


title: RNA-seq of 8hr LPS-stimulated bone marrow-derived macrophages from wild type, Ch25h-deficient, and LXRab knockout mice

first: Jason

last: Cyster

state: California

address: 513 Parnassus Avenue

institute: University of California - San Francisco

summary: 25-hydroxycholesterol has been demonstrated to regulate SREBP processing, yet Ch25h-deficient mice have no cholesterol abnormalities.  Using RNA-seq, we find that LPS-stimulated, Ch25h-deficient BMDMs have dysregulated SREBP target genes, demonstrating that 25-hydroxycholesterol is an induced repressor of SREBP during inflammatory settings.

overall_design: mRNA profiles from day 7 M-CSF bone marrow-derived macrophages (wild type, Ch25h-knockout, LXR-double knockout, were generated by deep sequencing on an Illumina HiSeq 2500.  Sequence reads passing a quality filter were aligned to the mouse genome (mm10) using STAR.

first: Eric

last: Dang

first: Andrea

last: Reboldi


title: Chromatin state dynamics during blood formation (RNA-Seq)

overall_design: 3' RNA-seq for digital gene expression quantitation across multiple cell types.


title: Chromatin state dynamics during blood formation


title: CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy [MNase-seq]

first: Marek

last: Bartkuhn

summary: MNase-seq was performed in order to analyze changes in nucleosomal occupancy after depletion of CTCF/P190 and ISWI from Drosophila S2 cells

overall_design: MNase-seq from Drosophila S2 nuclei after CTCF/CP190 or ISWI-specific RNAi treatment

first: Rainer

last: Renkawitz


title: CTCF/CP190 and ISWI dependent regulation of nucleosome occupancy


title: Genome-Wide Profiling of miRNA-mRNA regulatory network in Skeletal Muscle with Aging [miRNA]

city: Daejon

first: Ji Young

last: Kim

address: Aging Research Center, Korea Reseach Institute of Bioscience

institute: KRIBB

summary: Skeletal muscle degenerates progressively, loses mass (sarcopenia) along in years, and leads to reduced physical ability, often causing secondary diseases such as diabetes and obesity. It is known that regulation of gene expression by microRNAs is a key event in muscle development and disease. To understand genome-wide changes in microRNAs and mRNAs during muscle aging, we sequenced microRNAs as well as mRNAs from mouse gastrocnemius muscles at two different ages (6 versus 24-month-old). Thirty-four microRNAs (15 up-regulated and 19 down-regulated) were differentially expressed with age among which were microRNAs such as miR-206 or -434 which were differentially expressed in aged muscle in previous studies. Interestingly, seven microRNAs in a microRNA cluster at imprinted Dlk1-Dio3 locus on chromosome 12 were coordinately down-regulated. In addition, sixteen novel microRNAs were identified. Integrative analysis of microRNA and mRNA expression revealed that microRNAs contribute to muscle aging possibly through the positive regulation of transcription, metabolic process, and kinase activity. Many of the age-related microRNAs were implicated in human muscular diseases. We suggest that genome-wide microRNA profiling helps to expand our knowledge of microRNA function in the muscle aging process.

overall_design: miRNA profiles of gastrocnemius muscle tissues (n=10) were generated by deep sequencing using Illumina Hiseq-2000

first: Ji

first: Young-Kyu

last: Park

first: Tae-Wook

last: Kan

first: Hee-Jin

first: Kwang-Pyo

last: Lee

first: Seon-Young

first: Ki-Sun

last: Kwon


title: Genome-Wide Profiling of miRNA-mRNA regulatory network in Skeletal Muscle with Aging [mRNA]

overall_design: mRNA profiles of gastrocnemius muscle tissues (n=10) were generated by deep sequencing using Illumina Hiseq-2000


title: Genome-Wide Profiling of miRNA-mRNA regulatory network in Skeletal Muscle with Aging


title: A system for genome-wide histone variant dynamics in ES cells reveals dynamic MacroH2A2 replacement at promoters

city: Hsin-Chu

first: Jui-Hung

last: Hung

address: 75 Bo-Ai Street

country: Taiwan

institute: National Chiao Tung University

summary: Our system recapitulates expected characteristics of the well characterized H3.3 histone variant, and show that we gain additional information by using a kinetic approach. Additionaly, our results on the less-studied MacroH2A2 variant revealed differential dynamic profiles of this “repressive” histone variant. Our results represent a novel approach to histone dynamics in mammalian cells, reveal unanticipated dynamic behavior of the MacroH2A2 variant in pluripotent cells, and provide a resource for future studies of tissuespecific histone dynamics in vivo.

overall_design: We use a pulse-chase strategy for carrying out genome-wide measurements of histone dynamics to several histone variants in murine embryonic stem cells and somatic tissues. Genomic binding profiles of histone dynamics in normal ES cells, along with their differentiated counter parts, in MEFs, determined by ChIP-seq.

first: Oliver

last: Rando

first: Christopher

last: Lengner


title: MicroRNAs are required for the feature maintenance and differentiation of brown adipocytes

status: Public on Aug 07 2014

city: Singapore

first: Lei

last: Sun

address: 8 College Road

country: Singapore

institute: Duke-NUS

summary: Brown adipose tissue is specialized to burn lipids for heat generation as a natural defense against cold and obesity. Previous studies established microRNAs as essential regulators of brown adipocyte differentiation, but it remains unknown whether microRNAs are required for the feature maintenance of mature brown adipocytes. To address this question, we ablated Dgcr8, a key regulator of the microRNA biogenesis pathway, in mature brown as well as white adipocytes. The adipose tissue -specific Dgcr8 knockout mice displayed enlarged but pale interscapular brown fat with decreased expression of genes characteristic of brown fat, and the mice were intolerant to cold exposure. In vitro primary brown adipocyte cultures confirmed that microRNAs are required for marker gene expression in mature brown adipocytes. We also demonstrated that microRNAs are essential for the browning of subcutaneous white adipocyte both in vitro and in vivo. Using this animal model, we performed microRNA expression profiling analysis and identified a set of BAT-specific microRNAs that are up-regulated during brown adipocyte differentiation and enriched in brown fat compared to other organs. We identified miR-182 and miR-203 as new regulators of brown adipocyte development. Taken together, our study demonstrates an essential role of microRNAs in the maintenance as well as the differentiation of brown adipocytes.

overall_design: TotalRNAs were extracted using a Qiagen kit, and 5 μg of total RNAs for each sample were used to prepare the mRNA- Seq library according to the manufacturer’s instruction (NEB). cDNA libraries were prepared and sequenced by Hi-seq in Whitehead Genome Core. 2 replicates of each treatment were analyzed.

last: SUN

first: Yen

last: Lim


title: High resolution ChIP sequencing reveals novel bindings targets and prognostic role for SOX11 in Mantle cell lymphoma (ChIP-Seq)

city: New York

first: Samir

last: Parekh

address: 1470 Madison Ave, 5th FL, Room 114

department: Hematology and Medical Oncology

institute: Icahn School of Medicine at Mount Sinai

summary: SOX11 (Sex determining region Y-box 11) expression is specific for MCL as compared to other Non-Hodgkin’s lymphomas. However, the function and direct binding targets of SOX11 in MCL are largely unknown. We used high-resolution ChIP-Seq to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11 target genes. qCHIP confirmed that SOX11 directly binds to individual genes in these pathways in both MCL cell lines and patients. Interrogation of an eighty-two patient  gene-expression dataset demonstrated that SOX11 mRNA expression was inversely proportional to Ki-67, a marker of cell proliferation. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT signaling and modulates chemotherapy sensitivity to cytarabine in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolina Institute and British Columbia Cancer Agency (BCCA). Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy incorporating cytarabine. Transcriptional regulation of WNT and other biological pathways affects by SOX11 target genes may help explain the impact of SOX11 expression on patient outcomes.

overall_design: ChIP-seq experiment identifies SOX11 direct  binding targets in Granta-519 MCL cell lines


title: Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco, a key control factor of pro-inflammatory signalling

status: Public on Aug 06 2014

city: Leiden

first: Annemarie

middle: H.

last: Meijer

address: Einsteinweg 55

department: Animal Sciences and Health

institute: Institute of Biology, Leiden University

summary: Scavenger receptors on the cell surface of macrophages play an important role in host defence through their ability to bind microbial ligands and induce phagocytosis. Concurrently, signal transduction pathways are initiated that aid in defence mechanisms against the invading microbe. Here we report on the function of scavenger receptor Marco (macrophage receptor with collagenous structure) during infection of zebrafish embryos with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. Morpholino knockdown demonstrates that Marco is required for the rapid phagocytosis of M. marinum following intravenous infection.  Furthermore, gene expression analysis shows that Marco controls the initial transient pro-inflammatory response to M. marinum and remains a determining factor for the immune response signature at later stages of infection. Increased bacterial burden following marco knockdown indicates that this scavenger receptor is important for control of M. marinum growth, likely due to delayed phagocytosis and reduced pro-inflammatory signalling observed under conditions of Marco deficiency

overall_design: Embryos were injected at the one cell stage with a morpholino targeting marco, or with the standard control morpholino from GeneTools for comparison. Subsequently, at 24 hours post fertilization (hpf) the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein, or mock-injected with PBS/2%PVP. After injections embryos were transferred into fresh egg water containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) to prevent melanization and incubated for 4 days at 28°C. After the incubation period, infected and uninfected morphants, mutants and their controls were imaged and groups of 30 embryos were snap-frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.

first: Erica

last: Benard

first: Stefan

last: Roobol

first: Herman

last: Spaink

middle: H


title: Genome-wide analysis of stress response in mouse fibroblasts

city: Goettingen

first: Anna

last: Sawicka

address: Am Fassberg 11

institute: Max Planck Institure for Biophysical Chemistry


title: High resolution ChIP sequencing reveals novel bindings targets and prognostic role for SOX11 in Mantle cell lymphoma (RNA-Seq)

overall_design: RNA-seq experiments studying SOX11-mediated regulation of gene transcription by examining genes differentially expressed following SOX11 depletion in 3 MCL cell lines, Granta-519, Z138 and JEKO-1


title: Gene expression study of Pestalotiopsis fici on rice medium

city: Beijing

first: wang

middle: xiu

address: NO.1 Beichen West Road, Chaoyang District, Beijing

institute: Institute of Microbiology, Chinese Academy of Sciences

summary: we studied the gene expression conditions while Pestalotiopsis fici . The perpose of this study is to find the expressed secondary metabolism genes when P. fici was fermented.

overall_design: Detection of gene expression of P . Fici on rice as substrate after 20 days when Natural products were reached to the peak.

first: Xiuna

last: Wang

first: Xingzhong

last: Liu


title: Genome-wide mapping of REST-bound sites in human CD4+ T cells

city: Bronx

first: Shira

last: Rockowitz

address: 1300 Morris Park Ave

institute: Albert Einstein College of Medicine

summary: We performed ChIP-seq to identify genome-wide REST (NRSF) binding in the human CD4+ T cells. The data were compared to REST occupancy in additional 15 human cell types analyzed by the ENCODE project (GSE32465) in order to study the dynamics and context-dependent functions of REST-chromatin interaction.

overall_design: Identification of REST binding by ChIP-seq in human CD4+ T cells.

first: Wen-Hui

last: Lien

first: Gang

last: Wei

first: Keji

last: Zhao

first: Elaine

last: Fuchs

first: Deyou

last: Zheng


title: A long noncoding RNA protects the heart from pathological hypertrophy

status: Public on Aug 05 2014

city: Philadelphia

first: Sylvia

last: Nurnberg

state: PA

address: 3400 Civic Center Blvd

department: Medicine

institute: University of Pennsylvania

summary: The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription1, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction2. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA–chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1–Hdac–Parp chromatin repressor complex3 to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy3. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized—but not naked—DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt–Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA–chromatin interaction.

overall_design: Overexpression of murine non-coding RNAs in human cell line, comparison of RNA-sequencing with Ribosome Profiling

first: Pei

last: Han

first: Wei

last: Li

first: Chiou-Hong

last: Lin

first: Jin

last: Yang

first: Ching

last: Shang

first: Kevin

last: Jin

first: Weihong

last: Xu

first: Chieh-Yu

first: Chien-Jung

first: Yiqin

last: Xiong

first: Huan-Chieh

last: Chien

first: Bin

last: Zhou

first: Euan

middle: A

last: Ashley

first: Daniel

last: Bernstein

first: Peng-Sheng

last: Chen

first: Huei-Sheng

middle: V

first: Thomas

last: Quertermous

first: Ching-Pin

last: Chang


title: RNA-sequencing analysis of glucose and acetate regulated transcripts in glioblastoma cells

first: Kathryn

last: Wellen

address: 421 Curie Blvd BRB II/III RM 632

department: Cancer Biology

summary: Our studies indicate that glucose and acetate can regulate histone acetylation by altering the acetyl-CoA concentrations in the cell. The purpose of this study was to to determine whether specific gene sets correlated with acetyl-CoA availability. We conclude that 10% of glucose-regulated genes are acetyl-CoA regulated genes (genes suppressed or induced by low glucose and reversed by acetate). Acetate usually regulated gene expression in the same direction as glucose, suggesting that acetyl-CoA is a key mediator of glucose-dependent gene expression.

overall_design: The experiments were performed in quadruplicates for each condition with a total of 12 samples

middle: E

first: Joyce

last: Lee


title: UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells

city: Copenhagen

first: Kristoffer

last: Vitting-Seerup

address: Ole maaloesvej 5

department: Department of Biology

country: Denmark

institute: University of Copenhangen, Denmark

summary: UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre-mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre-mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.

overall_design: Total RNA was extracted from HeLa cells treated with control (CTRL), UBL5 (#57, #58, or #82), or SART1 siRNAs for 48 h and processed for RNA-Seq analysis

first: Niels

last: Mailand


title: Effect of short term diet restriction on gene expression in the bovine hypothalamus using next generation RNA sequencing technology

city: Dunsany

first: Anthony

middle: Gerard

last: Doran

state: Co Meath

address: Grange

department: Animal and Bioscience Department

country: Ireland

institute: Teagasc

summary: Background: Negative energy balance (NEB) is an imbalance between energy intake and energy requirements for lactation and body maintenance, affecting high-yielding dairy cows after calving and is of considerable economic importance due to its negative impact on fertility and health in dairy herds. The hypothalamus is central to the neural control of homeostasis. It is anticipated that the cow hypothalamus experience extensive biochemical changes during the early post partum period in an effort to re-establish metabolic homeostasis. There is variation in the tolerance to NEB between individual cows. In order to understand the genomic regulation of ovulation in hypothalamic tissue during NEB transcriptional patterns between tolerant and sensitive animals was examined. A short term dietary restriction heifer model was developed which induced abrupt onset of anoestrus in some animals (Restricted Anovulatory; RA) while others maintained oestrous cyclicity (Restricted Ovulatory; RO). In addition a third control group (C) received a higher level of normal feeding. Heifers were slaughtered and hypothalamic tissue was collected after 18 days of differential feeding. mRNAseq was performed to examine differential gene expression profiles in hypothalamic tissue between groups.  Results: A total of 15,295 genes were expressed in hypothalamic tissue. The largest difference between groups was observed in the comparison between RA and RO heifers, with 1094 genes shown to be significantly differentially expressed (SDE). Innatedb pathway analysis showed that these SDE genes were associated with 6 canonical pathways (P < 0.01), of which neuroactive ligand-receptor interaction was the most significant. Within the comparisons the main over-represented pathway functions appear to be immune response including neuroprotection (CXCL10, Q1KLR3, IFIH1, IL1 and IL8; RA v C and RA v RO); energy homeostasis (AgRP and NPY; RA v RO); cell motility (CADH1, DSP and TSP4; RO v C) and prevention of GnRH release (NTSR1 IL1α, IL1β, NPY and PACA; RA v RO).     Conclusion: This information assists in understanding the genomic factors regulating the influence of diet restriction on fertility and may assist in to optimise nutritional and management systems that improve reproductive performance.

first: Darragh

last: Matthews

first: Michael

last: Diskin

first: Dermot

last: Morris

first: David

last: Kenny

first: Christopher

middle: J

last: Creevey

first: Sinead

last: Waters


title: Evolution of microRNA transcriptome at the onset of Drosophila metamorphosis

city: Irvine

first: Shu-Dan

last: Yeh

address: 321 Steinhaus Hall

department: Ecology and Evolutionary Biology

country: USA

institute: University of California, Irvine

summary: MicroRNAs (miRNAs) are endogenous RNA molecules that contribute to the regulation of gene expression post-transcriptionally.  Recently, patterns of sequence evolution and evolutionary emergence of miRNAs have been analyzed in detail.  However, the extent to which miRNA expression levels vary within and between species, the role of stabilizing selection versus other evolutionary mechanisms in shaping the temporal dynamics of these changes, and how this variation might unveil the interplay between miRNAs and mRNAs remain poorly understood.  This is especially true during key developmental transitions involving miRNA regulation.  Combining deep-sequencing with multi-species microarrays, we assayed miRNA expression levels immediately before (≥18BPF) and after (PF) the increase in steroid hormone ecdysone that triggers metamorphosis.  We did so both at the intra- and interspecific levels in the Drosophila melanogaster species group.  In contrast to their sequence conservation, ~25% of the analyzed miRNAs differed significantly in expression levels at ≥18BPF and/or PF across D. melanogaster strains.  Additionally, ~33% showed modifications in their pattern of expression bias between developmental timepoints.  Relative to the patterns of evolution, changes in miRNA abundance at PF accumulate linearly over evolutionary time while similar changes at ≥18BPF do not.  Importantly, expression levels of ≥18BPF-enriched miRNAs are the most variable within and between species and therefore the least likely to evolve under stabilizing selection, especially as compared to non-developmentally enriched miRNAs.  Overall, functional attributes such as expression ubiquity are more tightly associated with lower levels of miRNA expression polymorphism at PF than at ≥18BPF.  Furthermore, ≥18BPF- and PF-enriched miRNAs showed opposite patterns of covariation in expression with mRNAs, which was found to denote the type of regulatory relationship between miRNA and mRNA.  By focusing on the miRNA expression levels at the onset of metamorphosis, our results have unveiled contrasting functional patterns of evolutionary divergence during ontogeny.

sample_organism:
Drosophila melanogaster
Drosophila simulans
Drosophila yakuba

overall_design: Total 12 samples are included. Please note that the 'description' field in each sample record contains more details for the biological and technical replicate pairs.

first: Jose

last: Ranz


title: Structural Variant of a Tandem Repeat-containing Silencer Locus Reveals Distinct Pol II and Pol IV-dependent Pathways of siRNA Biogenesis

city: St. Louis

first: Blake

middle: C.

last: Meyers

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

summary: We used a two-component transgene system to study the RNA-dependent DNA methylation (RdDM) and transcriptional gene silencing (TGS) in Arabidopsis. By profiling the small RNA population in  mutants defected in RdDM or RNA polymerase II-transcribed trigger for generating silencing siRNA, we investigated how repetitive loci such as tandem repeats were regulated transcriptionally through the action of RNA polymerase IV.

overall_design: Genome-wide small RNA profiling was done by Illumina TruSeq sample preparation followed by high-throughput sequencing with the Illumina HiSeq 2000 platform. The six samples represent mutants and their corresponding control lines.

middle: C

first: Tzuu-fen


title: A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequence Quality Control consortium

city: Shanghai

first: Leming

last: Shi

address: 2005 Songhu Road

department: School of Life Sciences

institute: Fudan University

summary: We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for sequence discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcriptlevel profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design:
The well-characterized reference RNA samples A (pooled cell lines) and B (human brain) from the MAQC consortium, adding spike-ins of synthetic RNA from the External RNA Control Consortium (ERCC).  Samples C and D were then constructed by combining A and B in known mixing ratios, 3:1 and 1:3, respectively.  All samples were distributed to several independent sites for RNA-Seq library construction and profiling by Illumina HiSeq 2000 and LifeTech SOLiD 5500 platforms.  Also, vendors created their own cDNA libraries that were then distributed to each test site, in order to examine the degree of a ?site effect? that was independent of the library preparation process.  To support an assessment of gene models, samples A and B were also sequenced at independent sites by the Roche 454 platform, providing longer reads.  For comparison to other technologies, these data were also compared to the MAQC-I Affymetrix U133 Plus2 microarray, several current microarray platforms, and also assessed by 20,801 PrimePCR reactions.

Sample A: Universal Human Reference RNA (UHRR) from Stratagene and ERCC Spike-In controls
Sample B: Human Brain Reference RNA (HBRR) from Ambion and ERCC Spike-In controls
Sample C: Mix of A and B (3:1)
Sample D: Mix of A and B (1:3)
Sample E: Ambion ERCC Spike-In Control Mix 1
Sample F: Ambion ERCC Spike-In Control Mix 2

first: Zhenqiang

last: Su

first: Pawel

middle: P

last: Labaj

first: Sheng

last: Li

first: Jean

last: Thierry-Mieg

first: Danielle

first: Wei

first: Charles

last: Wang

first: Gary

last: Schroth

first: Wendell

middle: D

last: Jones

first: Wenzhong

last: Xiao

first: Weihong

last: Xu

first: Roderick

last: Jensen

first: Reagan

last: Kelly

first: Joshua

first: Ana

last: Conesa

first: Hanlin

last: Gao

first: Nadereh

last: Jafari

first: Fei

last: Lu

first: Edward

last: Oakeley

first: Craig

last: Praul

first: Javier

last: Santoyo-Lopez

first: Xin-Xing

last: Tan

first: E

last: Thompson

first: Jo

last: Vandesompele

first: Zhiyu

last: Peng

first: Andreas

last: Scherer

first: Jiri

last: Zavadil

first: Huixiao

last: Hong

first: Yang

last: Liao

first: Robert

last: Setterquist

first: Shashi

last: Amur

first: Scott

middle: S

last: Auerbach

first: Desmond

middle: I

last: Bannon

first: Wenjun

last: Bao

first: Hans

last: Binder

first: Thomas

last: Blomquist

first: Cecilie

last: Boysen

first: Kelli

last: Bramlett

first: Murray

last: Brilliant

first: Pierre


title: Dissecting the dynamic changes of 5-hydroxymethylcytosine in T cell development and differentiation [MeDIP-Seq]

status: Public on Aug 04 2014

first: Tarmo

last: Äijö

address: 162 5th Avenue

department: Center for Computational Biology

institute: Flatiron Institute

summary: The discovery of TET proteins, enzymes that oxidize 5-methylcytosine (5mC) in DNA, has revealed novel mechanisms for the regulation of DNA methylation. We have mapped 5-hydroxymethylcytosine (5hmC) at different stages of T cell development in the thymus and T cell differentiation in the periphery.  We show that 5hmC is enriched in the gene body of highly expressed genes at all developmental stages, and that its presence correlates positively with gene expression.  Further emphasizing the connection with gene expression, we find that 5hmC is enriched in active thymus-specific enhancers, and that genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. Our data constitute a valuable resource that will facilitate detailed analysis of the role of 5hmC in T cell development and differentiation.

overall_design: Map 5hmC in DP T cells and ES cells

first: Ageliki

last: Tsagaratou

first: Anjana

last: Rao


title: Dissecting the dynamic changes of 5-hydroxymethylcytosine in T cell development and differentiation [RNA-Seq]

overall_design: Transcriptome profiling of naïve CD4 and CD8 cells

first: Chan-Wang

last: Lio


title: Proliferation conditions promote intrinsic changes in NK Cells for an IL-10 response

city: Bethesda

first: Yuka

last: Kanno

state: MD

address: 10 Center Drive, Rm13C120

department: NIAMS

institute: NIH

summary: Constitutively found at high frequencies, the role for NK cell proliferation remains unclear. Here, a shift in NK cell function from predominantly producing interferon-γ (IFN-γ), a cytokine with proinflammatory and antimicrobial functions, to producing the immunoregulatory cytokine IL-10, is defined during extended murine cytomegalovirus infection. The NK cells driven to express IL-10 in vivo acquired responsiveness to IL-12 and IL-21 for IL-10 production, but neither cytokine was required for the endogenous response. In vitro studies with IL-2 to support proliferation and in vivo adoptive transfers into murine cytomegalovirus-infected mice demonstrated that NK cell proliferation and further division enhanced the change. During infection, the responding NK cells acquired histone modifications indicative of an open IL-10 gene, and an IL-10 response was proliferation dependent ex vivo if the NK cells had not yet expanded in vivo but independent if they had. Thus, a novel role for proliferation in supporting changing innate cell function is discovered.

overall_design: The chromatin status of ex vivo isolated NK cells during MCMV infection was investigated by ChIP-seq on histone epigenetic marks (H3K4m3, H3K27m3, H3K36m3) on day 0, day 1.5 and day 3.5 post infection.

first: Margarite

last: Tarrio

first: Seung-Hwan

last: Lee

first: Maria

middle: F

last: Fragoso

first: Hong-Wei

last: Sun

first: John

last: O'Shea

first: Christine

last: Biron


title: Dissecting the dynamic changes of 5-hydroxymethylcytosine in T cell development and differentiation [ChIP-Seq]

overall_design: Examine the distribution of the  H3K27Ac in DP T cells. The presence of H3K27Ac in enhancers enable us to distinguish poised(H3Kme1 enriched, but devoid of H3K27Ac)  versus active enhancers (enriched for H3K4me1 and H3K27Ac).


title: The photoreceptor chaperone FHY1 has an independent role in gene modulation and plant development under far-red light

city: Shenzhen

first: Bosheng

last: Li

state: Guangdong

address: SUStech Huiyuan #1 406

department: MCDB

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

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

overall_design: Refer to individual Series


title: HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer.

city: Aurora

first: Peter

last: Kabos

state: CO

address: 12801 E. 17th Avenue

department: Division of Medical Oncology

institute: University of Colorado School of Medicine

summary: We used a genome-wide approach (High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, or HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. Finally, we show that miRNA targets and networks defined by our analysis are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.

overall_design: Argonaute HITS-CLIP on three representative breast cancer cell lines (each in triplicate).

first: Manoj

middle: M

last: Pillai

first: Austin

last: Gillen

first: Tomomi

last: Yamamoto

first: Enos

last: Kline

first: Joesph

last: Brown

first: Kale

last: Flory

first: Jay

middle: R

last: Hesselberth


title: Modulation of the TNF-induced macrophage response by synovial fibroblasts

first: Laura

last: Donlin

state: NY

department: Arthritis and Tissue Degeneration Program

institute: Hospital for Special Surgery

summary: Here we explored how the human macrophage response to tumor necrosis factor (TNF) is regulated by human synovial fibroblasts, the representative stromal cell type in the synovial lining of joints that become activated during inflammatory arthritis. Genome-wide transcriptome analysis (RNAseq) showed that co-cultured synovial fibroblasts modulate the expression of approximately one third of TNF-inducible genes in macrophages, including expression of target genes in pathways important for macrophage survival and polarization towards an alternatively activated phenotype. This work furthers our understanding of the interplay between innate immune and stromal cells during an inflammatory response, one that is particularly relevant to inflammatory arthritis. Our findings also identify modulation of macrophage phenotype as a new function for synovial fibroblasts that may prove to be a contributing factor in arthritis pathogenesis.

overall_design: Human CD14+ MCSF-differentiated macrophages were cultured with or without synovial fibroblasts in transwell chambers.  TNF was added at Day 0, macrophages were harvested at Day 2. Total of 4 samples: (1) macrophages alone (2) macrophages with fibroblasts (3) macrophages with TNF (4) macrophages with fibroblasts and TNF. Macrophage RNA was purified using RNeasy mini kit (Qiagen).  Tru-seq sample preparation kits (Illumina) were used to purify poly-A transcripts and generate libraries with multiplexed barcode adaptors.  All samples passed quality control on a Bioanalyzer 2100 (Agilent).  Paired-end reads (50 x 2 cycles, ~75x106 reads per sample) were obtained on an Illumina HiSeq 2500.  The TopHat program was used to align the reads to the UCSC Hg19 human reference genome, while the Cufflinks program allowed for measurements of transcript abundance (represented by Fragments Per Kilobase of exon model per Million mapped reads (FPKM)).

middle: T

first: Lionel

middle: B

last: Ivashkiv


title: Dissecting the dynamic changes of 5-hydroxymethylcytosine in T cell development and differentiation [CMS-Seq]

first: Tarmo

last: Äijö

overall_design: Study the distribution of 5hmC in 7 T cell subsets at sequential steps of development, differentiation and specification

first: Ageliki

last: Tsangaratou

first: Anjana

last: Rao


title: Molecular Mechanisms of Master Regulator VqsM mediating Quorum-sensing and Antibiotic Resistance in Pseudomonas aeruginosa

city: Hong Kong

first: Xin

last: Deng

address: To Yuen Building

department: Biomedical Sciences

country: Hong Kong

institute: City University of Hong Kong

summary: The Pseudomonas aeruginosa quorum-sensing (QS) systems contribute to bacterial homeostasis and pathogenicity. Although the AraC family transcription factor VqsM has been characterized to control the production of virulence factors and QS signaling molecules, its detailed regulatory mechanisms still remain elusive. Here, we report that VqsM directly binds to the lasI promoter region, and thus regulates its expression. To identify additional targets of VqsM in P. aeruginosa PAO1, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) which detected 48 enriched loci harboring VqsM-binding peaks in P. aeruginosa genome. The direct regulation of these genes by VqsM has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). A VqsM-binding motif is found by using MEME suite and verified by footprint assays in vitro. In addition, VqsM directly binds to the promoter regions of antibiotic resistance regulator NfxB and the master type III system regulator ExsA. Notably, the vqsM mutant displayed more resistance to two types of antibiotics and promoted bacterial survival in a mouse model, compared to the wild type PAO1 strain. Collectively, this work provides new cues to better understand the detailed regulatory networks of QS systems, T3SS, and antibiotic resistance.

overall_design: Pseudomonas aeruginosa  MAPO1 containing empty pAK1900 or pAK1900-VqsM-VSV

first: Haihua

last: Liang


title: Dissecting the dynamic changes of 5-hydroxymethylcytosine in T cell development and differentiation


title: Next Generation Sequencing Facilitates Quantitative Analysis of Normal Human Kidney Transcriptomes

first: Yi-An

last: Ko

state: Pennsylvania

address: 415 curie blvd

department: Genetics & Gene Regulation

institute: UPeenn

summary: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived normal human kidney transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis

overall_design: The kidney tissue was immediately placed and stored in RNAlater® (Ambion), according to the manufacturer’s instruction. The tissue was manually microdissected under microscope in RNAlater® pool for glomerular and tubular compartment. Dissected tissue was homogenized and RNA was prepared using RNAeasy mini columns (Qiagen, Valencia, CA, US), according to the manufacturer’s instructions. RNA quality and quantity were determined using the Laboratory-on-Chip Total RNA PicoKit Agilent BioAnalyzer. Only samples without evidence of degradation were further used (RNA Integrity Number >6).

first: Nora

last: Ledo

first: Katalin

last: Susztak


title: Association of genes regulated by Ezh2 and trimethylation of histone 3 lysine 27

city: Parkville

first: Jovana

last: Maksimovic

state: Victoria

address: Royal Children's Hospital, Flemington Road

country: Australia

institute: Murdoch Childrens Research Institute

summary: Differentiation of naïve CD4+ T cells into effector (Th1, Th2 and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). EZH2 over-expression and activating mutations are implicated in tumorigenesis and correlate with poor prognosis in several tumor types 35. This spurred the development of EZH2 inhibitors which, by inducing tumor cell growth arrest and cell death, show therapeutic promise in cancer. A role for Ezh2 in suppressing Th1 and Th2 cytokine production and survival has recently been reported.  It is not entirely clear whether Ezh2-PRC2 plays a role in H3K27me3 in cytokine loci in naïve CD4+ T cells and whether H3K27me3 has a non-redundant role in T helper cell lineage differentiation and survival. Here, we investigate the effects of T cell-specific Ezh2 deletion to determine the role that Ezh2-PRC2 plays in regulating the fate of differentiating naïve CD4+ T cells. Loss of Ezh2 altered the expression of 1328 genes in Th0 and 1979 genes in iTreg cells. Gene expression changes were positively correlated in both cell types, indicating that Ezh2 targets similar genes in these cells. As expected, Ifng was one of the genes most increased in expression by following loss of Ezh2. In addition, expression of Tbx21 homolog Eomes, a transcription factor that regulates IFNG production, was also significantly increased. We then performed H3K27me3 ChIP-seq on Ezh2fl/fl and Ezh2fl/fl.CD4Cre Th0 cells. Consistent with cellular phenotype and RNA-seq data, we observed a loss of the H3K27me3 at Eomes, Il4 and Il10 loci . Very low levels of H3K27me3 marks were present at Ifng and Tbx21 loci in differentiated Ezh2fl/fl Th0 cells, suggesting that upon differentiation, upregulation or activation of transcription factors accounts for IFNG overproduction. A significant loss of H3K27me3 was observed >2kb upstream of Gata3 locus , however this did not result in increased transcription . Of the 22381 genes tested for changes in H3K27me3, 1360 showed a statistically significant decrease in Ezh2fl/fl.CD4Cre Th0 cells, compared to wildtype. Furthermore, 404 of these genes also showed a concomitant gain in expression in Ezh2fl/fl.CD4Cre Th0 cells, suggesting that these loci are likely direct Ezh2-PRC2 targets.

overall_design: There are 3 biological replicates each of Ezh2fl/fl.CD4Cre and Ezh2fl/fl in both Th0 and iTreg cells for the RNA-seq experiment.  There are 2 biological replicates each of Ezh2fl/fl.CD4Cre and Ezh2fl/fl in Th0 cells for the ChIP-seq experiment.

first: Yuxia

last: Zhang

first: Sarah

last: Kinkel

first: Esther

last: Bandala-Sanchez

first: Maria

last: Tanzer

first: Gaetano

last: Naselli

first: Jian-Guo

first: Yifan

last: Zhan

first: Andrew

last: Lew

first: John

last: Silke

first: Alicia

last: Oshlack

first: Marnie

last: Blewitt

first: Leonard

last: Harrison


title: Targeted H3R26 deimination specifically facilitates ER binding by modifying nucleosome structure

city: Charlottesville

first: Michael

middle: Joseph

last: Guertin

state: VA

address: 1340 Jefferson Park Ave

department: Biochemistry and Molecular Genetics

institute: University of Virginia

summary: The precise mechanisms by which hormone receptors bind to DNA in the context of chromatin remain unclear. Here we report that the genomic distributions of estrogen receptor (ER) and H3R26 deimination (H3R26Cit) in breast cancer cells are strikingly coincident, linearly correlated, and observed as early as 2 minutes following estradiol treatment. Paired-end MNase ChIP-seq indicates that the charge-neutral H3R26Cit modification facilitates ER binding to DNA by altering the fine structure of the nucleosome. Clinically, we find that PAD2 and H3R26Cit levels correlate with ER expression in breast tumors and that high PAD2 expression is associated with increased survival in ER+ breast cancer patients. Our study suggests that PAD2-mediated histone deimination is fundamental to ER signaling in breast cancer.

overall_design: We performed replicate H3R26Cit ChIP-seq experiments prior to estrogen treatment and 2min, 5min, 10min, 40min, and 160min after E2 treatment. Additionally, we performed H3R26Cit and H3K27ac ChIP on MNase digested chromatin.