title: Transcriptome differences in the rumen of beef steers with variation in feed intake and gain

status: Public on Jul 22 2015

city: Clay Center

first: Amanda

last: Lindholm-Perrry

state: NE

address: State Spur 18D

department: NEMRU

institute: USMARC

summary: RNA sequencing (RNA-Seq) was performed on rumen papillae from 16 steers with variation in gain and feed intake.

overall_design: Sixteen rumen papillae samples were sequenced by Cofactor Genomics (St.Louis, MO).

first: Rebecca

last: Kern

last: Lindholm-Perry


title: RNA-seq based transcriptome of Nesterenkonia sp. AN1

city: Pretoria

first: Habibu

last: Aliyu

state: Gauteng

address: Lynnwood

department: Genetics

country: South Africa

institute: Centre for Microbial Ecology and Genomics

type: Expression profiling by high throughput sequencing

summary: The transcript profiles of Nesterenkonia sp. AN1 grown at 5 ºC (Cold) and 21 ºC (Topt) were acccessed to evaluate the cold reposnse of this Antarctic Nesterenkonia strain. The strain was grown in triplicates at the optimum growth temperature of 21 ºC and a test temperature of 5 ºC. Total RNA was extracted from two replicate samples for each treatment condition and the total RNA was enriched for mRNA. RNA-seq was done using Illumina Miseq platform at Inqaba Biotech, South Africa. The reads were mapped against the genome sequence of Nesterenkonia sp. AN1 (obtained from NCBI database) and assesed for differeential gene expression using CLC Genomics Workbench 7.5.

overall_design: Evaluation of RNA-seq data for Nesterenkonia sp. AN1 at 5 ºC (Cold) and 21 ºC (Topt) in two biological replicates

first: Pieter

last: De Maayer

first: Don

last: Cowan


title: Viral nucleases reveal an mRNA degradation-transcription feedback loop in mammalian cells

status: Public on Jul 23 2015

city: Berkeley

first: Britt

last: Glaunsinger

state: California

address: 565 Li Ka Shing Center

department: Plant and Microbiology

country: USA

institute: UC Berkeley

summary: Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, termed SOX, that cleaves the majority of mRNAs within a cell.  Cleaved fragments are subsequently degraded by the cellular mRNA degradation machinery.  Here, we reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering levels of RNA polymerase II (RNAPII) transcription in the nucleus.  Measurements of both RNAPII recruitment to promoters and nascent mRNA synthesis revealed that the majority of affected genes are transcriptionally repressed in SOX-expressing cells. The transcriptional feedback does not occur in response to the initial endonuclease-induced cleavage, but instead to degradation of the cleaved fragments by cellular exonucleases.  In particular, Xrn1 catalytic activity is required for transcriptional repression. Notably, viral mRNA transcription escapes decay-induced repression, and this escape requires Xrn1.  Collectively, these results indicate that mRNA decay rates impact transcription in mammalian cells, and that gamma-herpesviruses have incorporated this feedback mechanism into their own gene expression strategy.

overall_design: NIH 3T3 cells were mock, WT, or ∆HS infected with MHV68 in duplicate and 4sU-labeled RNA isolated. 4sU-labeled RNA was submitted for sequencing and reads aligned to the mouse genome or MHV68 viral genome. Differential cellular gene expression was determined between mock and WT infected, mock and ∆HS infected, as well as differential viral gene expression between WT and ∆HS.

first: Emma

last: Abernathy

first: Ravi

last: Alla


title: Genome-wide epigenetic cross-talk between DNA methylation and H3K27me3 in zebrafish embryos

city: Darlinghurst

first: Ozren

last: Bogdanovic

state: NSW

address: 384 Victoria Street

department: Genomics and Epigenetics

country: Australia

institute: Garvan Institute of Medical Research

type: Genome binding/occupancy profiling by high throughput sequencing

type: Methylation profiling by high throughput sequencing

summary: DNA methylation and histone modifications are epigenetic marks implicated in the complex regulation of vertebrate embryogenesis. The cross-talk between DNA methylation and Polycomb-dependent H3K27me3 histone mark has been reported in a number of organisms and both marks are known to be required for proper developmental progression. Here we provide genome-wide DNA methylation (MethylCap-seq) and H3K27me3 (ChIP-seq) maps for three stages (dome, 24hpf and 48hpf) of zebrafish (Danio rerio) embryogenesis, as well as all analytical and methodological details associated with the generation of this dataset.

overall_design: DNA methylation (MethylCap-seq) and H3K27me3 (ChIP-seq) profling of zebrafish embryogenesis


title: MicroRNA-203 represses selection and expansion of oncogenic HRas transformed tumor initiating cells

city: Aurora

first: Kent

middle: Augustus

last: Riemondy

state: Colorado

department: Biochemistry and Molecular Genetics

institute: University of Colorado School of Medicine

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: Effects on the transcriptome of adult mouse pancreas (principally acinar cells) by the inactivation of the Ptf1a gene in vivo

city: Dallas

first: Galvin

last: Swift

state: TX

address: 6000 Harry Hines Blvd

department: Molecular Biology

institute: U TX Southwestern Medical Center

summary: RNA-seq analysis documented mRNA changes in total pancreatic RNA preparations 14 days after Ptf1a inactivation.

overall_design: pancreas mRNA profiles of Tamoxifen treated adult control mice [Ptf1a(CreER/+)] and Ptf1a conditional knockout mice [Ptf1a(CreER/fl)] were generated by deep sequencing using an Illumina Hiseq 2500.

first: Chinh

last: Hoang

middle: H

first: Ana

last: Azevedo-Pouly

first: Raymond

middle: J

last: MacDonald


title: Comparative transcriptome analysis reveals that ECM-Receptor Interaction contributes to the venous metastases of hepatocellular carcinoma

city: Shanghai

first: Hong

last: Zhang

address: Ming Dao Building  138 Yi Xue Yuan Road

department: Institute for Biomedical Sciences

country: China

institute: Fudan University

summary: Hepatocellular carcinoma (HCC) is the most common kind of liver cancer in the world. Portal vein tumor thrombus (PVTT) is one of the most serious complications of HCC and strongly correlated to a poor prognosis for HCC patients. However, the detailed mechanisms of PVTT development still remains to be explored. In this study, we presented the large scale transcriptome analysis of 11 HCC with PVTT patients by RNA-sequencing. The dysregulated genes between HCC and PVTT suggested that the ECM-Receptor Interaction was correlated to the venous metastases of HCC. Among all the recurrent alternative splicing events, we identified exon 6 skipping of RPS24, which is likely to be a cancer driver. We also identified five common fusion genes between HCC and its corresponding PVTT samples, including ARID1A-GPATCH3, MDM1-NUP107, PTGES3-RARG, PRLR-TERT and C9orf3-TMC1. All of the findings broaden our knowledge in PVTT development and may also contribute to the diagnosis and treatment for HCC patients with PVTT.

first: Junyi

last: Ye

first: Xiaoling

last: Weng

first: Fatao

last: Liu

first: Lin

last: He

first: Daizhan

last: Zhou

first: Yun


title: Transcriptomic differences between day and night in Acropora millepora provide new insights into metabolite exchange and light-enhanced calcification in corals

city: Arcachon

first: Anthony

last: Bertucci

address: Place du Dr Peyneau

country: France

institute: University of Bordeaux

summary: The metabolic bases of the interaction between the coral Acropora millepora and its dinoflagellate symbiont were investigated by comparing gene expression levels under light and dark conditions at the whole transcriptome level. Among the differentially expressed genes identified, a suite of genes involved in cholesterol transport was found to be up-regulated under light conditions, confirming the significance of this compound in the coral symbiosis. Although ion transporters likely to have roles in calcification were not differentially expressed in this study, expression levels of many genes associated with skeletal organic matrix composition and organization were higher in light conditions. This implies that the rate of organic matrix synthesis is one factor limiting calcification at night. Thus, LEC during the day is likely to be a consequence of increases in both matrix synthesis and the supply of precursor molecules as a result of photosynthetic activity.

overall_design: Branch tips from three adult colonies of Acropora millepora were sampled at midday and midnight

first: Sylvain

last: Forêt

first: Eldon

middle: E

last: Ball

first: David

last: Miller


title: Transcriptional activation without chromatin marking in developmentally regulated genes [ChIP-seq]

city: Barcelona

first: Enrique

last: Blanco

address: Dr. Aiguader 88

department: Gene Regulation, Stem Cells and Cancer

country: Spain

institute: Center for Genomic Regulation (CRG)

summary: We have conducted ChIP-Seq of H3K4me1/H3K27ac in order to perform a comparative study between wing imaginal discs and eye imaginal discs from Drosophila melanogaster.

overall_design: Examination of two histone modifications associated with gene regulation in wing/eye imaginal discs cells

first: Sílvia

last: Perez-Lluch

first: Hagen

last: Tilgner

first: Joao

last: Curado

first: Montse

last: Corominas

first: Roderic

last: Guigó


title: Analysis of H3K9ac in fly imaginal discs [ChIP-seq]

summary: We have analyzed ChIP-Seq of H3K9ac and H3K27me3 in wing imaginal discs and eye imaginal discs from Drosophila melanogaster.

overall_design: Examination of two histone modifications in wing/eye imaginal discs cells


title: Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana

city: Lincoln

first: Jean-Jack

middle: M.

last: Riethoven

address: 1901 Vine Street

department: Beadle Center for Biotechnology

institute: University of Nebraska-Lincoln


title: Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana [ChIP-Seq]

summary: Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained largely transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which even under normal conditions exhibited lower H3T3ph levels in pericentromeric regions, and a few transposons and repeat sequences showed modest transcriptional activation. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5’ genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s).

overall_design: Columbia-0 and double mutant at3g03940/at518190 knockdown plants were grown in 12 hr light for 3 weeks in pots with soil covered with miracloth to prevent soil contamination of leaf tissues. Control was kept in normal watered state, for other samples (peg) drought stress was induced by treatment with 30% Polyethylene glycol (PEG 6,000) for 5 hours. Pulldowns on H3, H3K4, and H3T3 were performed on all samples with 3-4 replicates.

first: Zhen

last: Wang

first: Armando

middle: C

last: Mollano

first: Jianping

last: Xu

first: Chi

last: Zhang

middle: M

first: Heriberto

last: Cerutti


title: Small RNAs during E. histolytica stress response

city: Stanford

first: Gretchen

middle: Marie

last: Ehrenkaufer

state: CA

address: Grant S-143, 300 Pasteur Dr.

department: Microbiology and Immunology

institute: Stanford Uninversity

summary: We analyzed ~27nt small RNAs from Entamoeba histolytica trophozoites in basal conditions and after heat shock or oxidative stress

overall_design: E. histolytica trophozoites were treated with 1mM H2O2 for 1hr, or heat shocked at 42°C for 1hr and RNA was isolated and small RNA populations were compared to small RNA populations from untreated trophozoites

first: Hanbang

first: Neil

last: Hall

first: Upi

last: Singh


title: Robust axonal regeneration occurs in the injured CAST/Ei mouse central nervous system

city: Los Angeles

first: Giovanni

last: Coppola

address: 1524 Gonda, 695 Charles Young Drive South

department: Psychiatry and Neurology

institute: UCLA

summary: Axon regeneration in the central nervous system (CNS) requires reactivating injured neurons’ intrinsic growth state and enabling growth in an inhibitory environment. Using an inbred mouse neuronal phenotypic screen, we find that CAST/Ei mouse adult dorsal root ganglion neurons extend axons more on CNS myelin than the other eight strains tested, especially when pre-injured.  Injury-primed CAST/Ei neurons also regenerate markedly in the spinal cord and optic nerve more than those from C57BL/6 mice and show greater spouting following ischemic stroke. Heritability estimates indicate that extended growth in CAST/Ei neurons on myelin is genetically determined, and two whole-genome expression screens yield the Activin transcript Inhba as most correlated with this ability. These screens are presented here.

overall_design: Biological quadruplicate - Mouse tissue - Naïve Dorsal Root Ganglia (DRG) and 5 day post sciatic nerve crush DRG - x9 strains.

first: Michael

last: Costigan


title: Small RNAs during E. invadens stage conversion

summary: We analyzed ~27nt small RNAs from Entamoeba invadens trophozoites, 24h cysts, 72h cysts, and excysting cells (8h)

overall_design: E. invadens trophozoites were induced to encyst by incubation in low glucose media, and parasites harvested at 0, 24 and 72h. A subset of the 72h cysts were induced to excyst, and parasites harvested at 8h. Total RNA was extracted for each sampleand small RNA libraries constructed and sequenced as below


title: Risk Variants for Exfoliation Syndrome Modulate Antisense Promoter Activity Within the LOXL1 Locus

city: Durham

first: Gregory

last: Crawford

state: NC

address: 101 Science Drive

department: Center for Genomic and Computational Biology

institute: Duke University

summary: The goal of this study was to identify functional variants that play a role in exfoliation syndrome (XFS) pathogenesis. We sequenced the entire LOXL1 locus in 50 South African XFS cases and 50 matched controls. The most strongly associated variants in this dataset were found in a ~7kb region spanning the LOXL1 exon 1/intron 1 boundary. DNase hypersensitivity analysis was used to demonstrate that this region, which lies upstream of the LOXL1 antisense RNA (LOXL1-AS1), contains regulatory activity.

overall_design: determining of chromatin structural changes in 3 cell types.

first: Lingyun

last: Song

first: Alexias

last: Safi


title: Opposing Roles for C/EBPα and Notch in Irradiation-induced Hematopoietic Stem Cell Defects

city: Cleveland

first: Jihye

last: Kim

state: Ohio

address: 9500 Euclid Ave.

department: Quantitative Health Sciences

institute: Cleveland Clinic

summary: Ionizing radiation (IR) has long been associated with reduced hematopoietic function and increased malignancies, although the mechanisms behind this relationship remain poorly understood. The carcinogenic effect of IR has been commonly attributed to the direct induction of DNA damage. We demonstrate that IR exposure results in long-term, somatically heritable, cell-intrinsic reductions in HSC self-renewal that is mediated by C/EBPα and reversed by Notch, both of which are associated with human leukemias. Remarkably, restoration of HSC self-renewal prevents selection for C/EBPα loss of function in previously irradiated HSC pools. We propose that environmental insults prompt HSC to initiate a program limiting their self-renewal to prevent damaged HSC from contributing to hematopoiesis. This "programmed mediocrity" is advantageous for the localized insults animals have evolved to deal with, but becomes tumor promoting when the entire HSC compartment is damaged, such as during total body irradiation, by increasing selective pressure for adaptive oncogenic mutations

overall_design: Examination of mRNA levels in in vitro and in vivo Hematopoietic Stem Cell that exposed to IR Ionizing radiation (IR) or control. Each group has three replicates.

first: Courtney

last: Fleenor

first: Andrii

middle: I

last: Rozhok

first: Vadym

last: Zaberezhnyy

first: Divij

last: Mathew

first: Aik-Choon

last: Tan

first: Irwin

middle: D

last: Bernstein

first: James

last: DeGregori


title: The LIN28B/let-7 axis is a novel therapeutic pathway in Multiple Myeloma

status: Public on Jul 21 2015

city: Boston

first: Salomon

last: Manier

state: Massachusetts

address: 77 Ave Louis Pasteur

department: Medical Oncology

institute: DFCI

summary: MYC is a major oncogenic driver of Multiple Myeloma (MM) and yet almost no therapeutic agents exist that target MYC in MM. Here we report that the let-7 biogenesis inhibitor LIN28B correlates with MYC expression in MM and is associated with adverse outcome. We also demonstrate that the LIN28B/let-7 axis modulates the expression of MYC, itself a let-7 target. Further, perturbation of the axis regulates the proliferation of MM cells in vivo in a xenograft tumor model. RNA sequencing and gene set enrichment analyses of CRISPR-engineered cells further suggest that the LIN28/let-7 axis regulates MYC and cell cycle pathways in MM. We provide proof-of-principle for therapeutic regulation of MYC through let-7 with an LNA-GapmeR containing a let-7b mimic in vivo, demonstrating that high levels of let-7 expression repress tumor growth by regulating MYC expression. These findings reveal a novel mechanism of therapeutic targeting of MYC through the LIN28B/let-7 axis in MM that may impact other MYC dependent cancers as well.

overall_design: RNA sequencing of MOLP-8 cells transduced with lentiCRISPRv2 scrambled control or containing a sgRNA against LIN28B. Both control and LIN28B KO cells were sequenced in triplicate.

first: Irene

last: Ghobrial


title: The Gonococcal Transcriptome During Infection of the Lower Genital Tract in Women

first: Ryan

last: McClure

address: 650 Albany St.

institute: Boston University

summary: Samples were collected from infected female patients and RNA seq was used to determine the transcriptome of Neisseria gonorrhoeae both during infection and during growth in chemically defined media (CDM).

overall_design: RNA seq of 4 different infecting strains of N. gonorrhoeae during infection and 3 of these same strains in CDM

first: Caroline

last: Genco


title: Imprinted expression is different in visceral yolk sac and cystic embryoid body endoderm

city: Vienna

first: Quanah

last: Hudson

address: Lazarettgasse 14, AKH BT 25.3

country: Austria

institute: CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences

summary: Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin mono-allelic expression of a small subset of mammalian genes. Imprinted genes have been categorized into two groups: multi lineage (ML) imprinted genes that can show imprinted expression in both embryonic and extra-embryonic lineages, and genes that show extra-embryonic lineage (EXEL) specific imprinted expression restricted to tissues like the placenta and visceral yolk sac (VYS) endoderm. Many genes showing EXEL imprinted expression are silenced by lncRNAs that act over long distances. Thus the analysis of this form of gene silencing is likely to be pivotal for understanding new mechanisms of long range gene silencing by lncRNAs. It has been reported that ES cells differentiated into cystic embryoid bodies (cystic EBs) contain VYS endoderm and here we investigate if cystic EBs could serve as an in vitro model for the analysis of EXEL imprinted expression. Unexpectedly we found that cystic EBs lack EXEL imprinted expression, while retaining normal imprinted expression of ML genes. This shows that cystic EBs do not model VYS imprinted expression and also argues against previous claims that cystic EBs contain VYS endoderm. We further characterized cystic EBs by performing RNA-seq and whole genome bisulphite sequencing. By comparison to various embryonic tissues we found that cystic EBs more resemble embryonic liver, which contains definitive endoderm, than the extra-embryonic endoderm of the VYS.

overall_design: Examination of gene and retrotranssposone expression and DNA methylation in extraembryonic, embryonic tissues and ES cell diferentiated as cystic EBs

first: Tomasz

last: Kulinski


title: p53 in colorectal cancer cells

city: Munich

first: Sabine

last: Huenten

address: Thalkirchner Strasse 36

country: Germany


title: Genome-wide measurement of spatial expression in mutants of Drosophila Melanogaster

first: Peter

middle: Acuña

last: Combs

address: Gilbert Rm 309, 371 Serra Mall

department: Biology

institute: Stanford University

summary: We sequenced mRNA from transverse slices of embryos from a variety of D. melanogaster mutants (bicoid over-expression, bicoid knockdown, hunchback knocdown, and zelda mutant) at the blastoderm stage to determine genome-wide patterns of gene expression.

overall_design: mRNA from transverse sections of single D. melanogaster embryos mutant for patterning TFs was sequenced.

middle: A

first: Michael

middle: B

last: Eisen


title: Genome-wide p53-occupancy in colorectal cancer cells.

summary: In order to comprehensively identify genes directly regulated by p53, a genome-wide chromatin-immunoprecipitation analysis (ChIP) followed by next generation sequencing (ChIP-seq) was performed after activation of a conditional p53 allele in SW480 cells.

overall_design: SW480/pRTR-p53-VSV cells were subjected to ChIP-Seq analysis after 16 hours doxycycline-treatment.

first: H

last: Hermeking

first: S

last: Hünten


title: Genome-wide RNA-expression analysis after p53 activation in colorectal cancer cells.

summary: In order to comprehensively identify RNA-expression changes after p53-activation, total RNA was isolated and subjected to next generation seqencing (RNA-Seq) after activation of a conditional p53 allele in SW480 cells.

overall_design: SW480/pRTR-p53-VSV cells were subjected to RNA-Seq analysis after 48 hours doxycycline-treatment.


title: E. histolytica and E. invadens

first: Gretchen

last: Ehrenkaufer

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

sample_organism:
Entamoeba histolytica
Entamoeba invadens

overall_design: Refer to individual Series


title: A microfluidic device for epigenomic profiling using 100 cells

city: Iowa City

first: Kai

state: IA

address: 3292 CBRB, 285 Newton Road

department: Internal Medicine

institute: The University of Iowa

summary: We developed the microfluidic-oscillatory-washing-based ChIP-Seq (MOWChIP-Seq) protocol. We achieved genome-wide mapping of histone modifications (H3K4me3 and H3K27ac) with as few as 100 cells. Moreover, the automated microfluidic platform dramatically reduced assay time and has a potential for future scale-up.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of 2 different histone modifications (H3K4me3 and H3K27ac) in 2 cell types (GM12878 and mouse Embryonic day 14.5 Fetal Liver Lineage-Sca1+c-Kit+ cells) with chromatin samples of various sizes (10K, 1K, 600, and 100 cells). These samples were aliquoted from a stock generated by sonicating 10K cells. Examination of 2 different histone modifications (H3K4me3 and H3K27ac) in GM12878 cells by directly starting with 100 and 600 sonicated cells (labeled as "direct").

first: Zhenning

last: Cao

first: Changya

last: Chen

first: Bing

last: He

first: Chang

last: Lu


title: Tbx3 controls Dppa3 levels to regulate exit from pluripotency towards mesodermal lineage

first: avinash

last: waghray

state: MA

address: 185 Cambridge Street, CPZN Room 4254A

department: Center for Regenerative Medicine

institute: Massachussets General Hospital

summary: Tbx3, a member of the T-box family, plays important roles in development, stem cells, nuclear reprogramming and cancer. Loss of Tbx3 induces differentiation in mouse embryonic stem cells (mESCs). However, we show that mESCs exist in an alternate stable pluripotent state in the absence of Tbx3. In-depth transcriptome analysis of this mESC state reveals Dppa3 as a direct downstream target of Tbx3. Also Tbx3 facilitates the cell fate transition from pluripotent cells to mesoderm progenitors by directly repressing Wnt pathway members required for differentiation. Wnt signaling regulates differentiation of mESCs into mesoderm progenitors and helps maintain a naïve pluripotent state. We show that Tbx3, a downstream target of Wnt signaling, fine-tunes these divergent roles of Wnt signaling in mESCs. In conclusion, we identify a signaling-TF axis that controls the exit of mESCs from a self-renewing pluripotent state towards mesoderm differentiation.

overall_design:
ChIPSeq and RNASeq (population and single cell) was performed on the indicated cell lines. Replicates are indicated as needed. The mm9 genome assembly was used.
For single cell mRNA-Seq preparation, SSEA1+DAPI- mESCs were sorted and collected.

first: Avinash

last: Waghray

first: Ihor

middle: R

last: Lemischka


title: Chaperoning 5S RNA assembly by structural probing

status: Public on Jul 20 2015

city: PARIS

first: Simon

last: Lebaron

address: 4 avenue de l'observatoire

institute: Université Paris Descartes

summary: In eukaryotes, three of the four ribosomal RNAs (rRNAs), the 5.8S, 18S and 25S/28S rRNAs, are  processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the  5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein  particle (5S RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its  incorporation into pre-ribosomes. In mammals the 5S RNP is also central regulator of the  homeostasis of the tumour suppressor p53. The nucleolar localisation of the 5S RNP and its  assembly into pre-ribosomes is performed by a specialised complex composed of Rpf2 and Rrs1 in  yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterisation  of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA and within pre-60S ribosomes. We  show that the Rpf2-Rrs1 complex contains a specialised 5S RNA E loop binding module, contacts  the Rpl5 protein and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We  propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the  incorporation of the 5S RNP in pre-ribosomes in the initial conformation prior to its rotation to form  the central protuberance found in the mature large ribosomal subunit.

overall_design: Refer to individual Sample (2). Crac results for Rpf2-HTP and control strain BY4741 are provided

first: Juliana

last: Pipoli


title: ChIP-Sequencing of Shox2 and Nkx2-5 on embryonic heart

city: New Orleans

first: Yiping

state: LA

address: 6400 Freret

department: CMB

institute: Tulane University

summary: ChIP-Sequencing on Shox2-HA E12.5 heart

overall_design: ChIP-Sequencing on Shox2-HA E12.5 heart (contain proximal part of pulmonary vein and R/LSVC, part of ventricle was dissected away). For Shox2, anti-HA(Cat.Ab9110, Lot.  GR146572-6).  For Nkx2-5, anti-Nkx2-5 (SantaCruz. Cat. N-19, Lot. SC-8697, mixed  Lot C0113 and Lot 12514) was used. Please see associated manuscript for more detailed information.

first: Wenduo

last: Ye


title: Generation of Cynomolgus Monkey Chimeric Fetuses Using Embryonic Stem cells

city: Kunming

first: Tianqing

last: Li

address: No. Boda Road, Yuhua Area, Chenggong New Town

institute: Primate Translational Medicine Research Center, Kunming University of Science and Technology

summary: Because of their similarity to humans, non-human primates are important models for studying human disease and developing therapeutic strategies.  Establishment of chimeric animals using embryonic stem cells (ESCs) could help with these investigations, but has not so far been achieved. Here, we show that cynomolgus monkey ESCs (cESCs) grown in adjusted culture conditions are able to incorporate into host embryos and develop into chimeras with contribution in all three germ layers and in germ cell progenitors. Under the optimized culture conditions, which are based on an approach developed previously for naive human ESCs, the cESCs displayed altered growth properties, gene expression profiles and self-renewal signaling pathways, suggestive of an altered naive-like cell state. Thus our findings show that it is feasible to generate chimeric monkeys using ESCs and open up new avenues for the use of non-human primate models to study both pluripotency and human disease.

overall_design: compare gene expression profiles between primed monkey embryonic stem cells (ESCs) and dome-shaped monkey ESCs

first: Yongchang

first: Yuyu

last: Niu

first: Yanjiao

first: Zongyong

last: Ai

first: Yu

last: Kang

first: Hong

last: Shi

first: Zheng

last: Xiang

first: Zhaohui

last: Yang

first: Tao

last: Tan

first: Wei

last: Si

first: Xueshan

last: Xia

first: Qi

last: Zhou

first: Weizhi

last: Ji


title: Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

country: Japan

institute: Kyoto University, Graduate school of medicine

summary: Mechanisms underlying human germ cell development are unclear, partly due to difficulties in studying human embryos and lack of suitable experimental systems. Here, we show that human induced pluripotent stem cells (hiPSCs) differentiate into incipient mesoderm-like cells (iMeLCs), which robustly generate human primordial germ cell-like cells (hPGCLCs) that can be purified using the surface markers EpCAM and INTEGRINα6. The transcriptomes of hPGCLCs and primordial germ cells (PGCs) isolated from non-human primates are similar, and although specification of hPGCLCs and mouse PGCs rely on similar signaling pathways, hPGCLC specification transcriptionally activates germline fate without transiently inducing eminent somatic programs. This includes genes important for naive pluripotency and repression of key epigenetic modifiers, concomitant with epigenetic reprogramming. Accordingly, BLIMP1, which represses somatic programs in mice, activates and stabilizes a germline transcriptional circuit and represses a default neuronal differentiation program. Together, these findings provide a foundation for understanding and reconstituting human germ cell development in vitro.

sample_organism:
Macaca fascicularis
Homo sapiens
Mus musculus

overall_design:
RNAseq analysis of human induced pluripotent stem cells (hiPSC), incipient mesoderm-like cells (iMeLC) and primordial germ cell-like cells (BLIMP1-2A-tdTomato (BT)/TFAP2C-2A-ECFP (AG) expressing cells or EpCAM/CD49f (CSM) positive cells)
RNAseq analysis of mouse embryonic stem cells (mESC), epiblast-like cells (EpiLC) and primordial germ cell-like cells (PGCLC; Blimp1-mVenus (BV) or BV/stella-eCFP (SC) positive cells)
Single cell RNAseq analysis of cynomolgus ESCs and gonadal PGCs

first: Kotaro

last: Sasaki

first: Shihori

last: Yokobayashi

first: Tomonori

last: Nakamura

first: Ikuhiro

last: Okamoto

first: Kazuki

last: Kurimoto

first: Hiroshi

last: Ohta

first: Yoshinobu

last: Moritoki

first: Chiduru

last: Iwatani

first: Hideaki

last: Tsuchiya

first: Kiyotoshi

last: Sekiguchi

first: Tetsushi

last: Sakuma

first: Takashi

last: Yamamoto

first: Takahide

last: Mori

first: Knut

last: Woltjen

first: Masato

last: Nakagawa

first: Takuya

first: Kazutoshi

last: Takahashi

first: Shinya

last: Yamanaka

first: Mitinori

last: Saitou

first: Shinichiro


title: RC3H1 posttranscriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-kB pathway [RNA-Seq]

city: Berlin

first: Markus

last: Landthaler

address: Robert-Rössle-Straße 10

department: Berlin Institute for Medical Systems Biology

institute: Max-Delbrück-Center for Molecular Medicine

summary: The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNFalpha mRNA decay via a 3'UTR constitutive decay element (CDE). Here, we applied PAR-CLIP to human RC3H1 to identify about 3800 mRNA targets with more than 16000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage induced mRNAs, indicating a role of this RNA-binding protein in the posttranscriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of NF-kB pathway regulators such as IkBalpha and A20. RC3H1 uses roquin and Zn-finger domains to contact a binding site in the A20 3'UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with IkB kinase and NF-kB activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-kB pathway.

overall_design: We measured global mRNA decay rates in mock and RC3H1/RC3H2-depleted HEK293 cells. Transcription was blocked by Actinomycin D zero, one or two hours before harvesting. Total RNA was isolated in two biological replicates and subjected to polyA selection followed by high-throughput sequencing.

first: Murakawa

last: Yasuhiro

first: Landthaler

last: Markus


title: Biomarker discovery: Quantification of microRNAs and other small non-coding RNAs using next generation sequencing

city: Montreal

first: Juan Pablo

last: Lopez

state: Quebec

address: 6875 La Salle Blvd

department: Human Genetics

country: Canada

institute: McGill University

summary:
Background: Small ncRNAs (sncRNAs) offer great hope as biomarkers of disease and response to treatment. This has been highlighted in the context of several medical conditions such as cancer, liver disease, cardiovascular disease, and central nervous system disorders, among many others. Here we assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing. At each stage, we evaluated the pros and cons of different techniques that may be suitable for different experimental designs. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.
Results: Our results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS. In addition, we describe the strengths and limitations of three commercially available library preparation methods: (1) Novex TBE PAGE gel; (2) Pippin Prep automated gel system; and (3) AMPure XP beads. We describe our bioinformatics pipeline, provide recommendations for sequencing coverage, and describe in detail the expression and distribution of all sncRNAs in four human tissues: whole-blood, brain, heart and liver.
Conclusions: Ultimately this study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate and effective high-throughput sequencing quantification method for various study designs, and overall generating valuable information that can contribute to our understanding of small ncRNAs as potential biomarkers and mediators of biological functions and disease.

overall_design: Quantification of microRNAs and other small non-coding RNAs using next generation sequencing for Biomarker discovery

first: Juan

middle: P

first: Carl

last: Ernst


title: Characterization of mouse histone H3 variants

city: Fukuoka

first: Yasuyuki

last: Ohkawa

address: 3-1-1 Maidashi, Higashi-ku, Fukuoka

department: Medical Institute of Bioregulation, Medical Institute of Bioregulation

institute: Kyushu University

summary: The selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled. We screened human and mouse genomes by in silico hybridization using canonical histone sequences, and found 14 uncharacterized H3-related genes in mouse. Most were similar to the H3.3 variant; their 3′ untranslated regions lacked a stem-loop structure but did have a poly- adenylation signal. Transcriptome analysis revealed tissue-specific expression of these variants. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome distributions of most variants were similar to that of H3.3. Over-expression of some variants altered gene expression patterns in differentiated C2C12 cells, suggesting that these new H3 variants have specific and distinct functions.

overall_design: RNA-Seq and  ChIP-Seq  for GFP fused Histone H3 variants of C2C12 cells in growth or differentiated conditions; 3'-seq analysis for C2C12 cells and mouse tissues

first: Kazumitsu

last: Maehara

first: Akihito

last: Harada


title: Genome-wide miRNA-expression analysis after p53 activation in colorectal cancer cells

first: Sabine

last: Huenten

summary: In order to comprehensively identify miRNA-expression changes after p53-activation a miRNA-Seq analysis was conducted after activation of a conditional p53 allele in SW480 cells.

overall_design:
SW480/pRTR-p53-VSV cells were subjected to miRNA-Seq analysis after 16 hours doxycycline-treatment.
Kompetenzzentrum für fluoreszente Bioanalytik, Regensburg, Germany

last: Hünten

first: Florian

last: Erhard

first: Anne

last: Dueck

first: Norbert

last: Eichner

first: Ralf

last: Zimmer

first: Gunter

last: Meister

first: Heiko

last: Hermeking


title: Genome-wide ChIP-seq binding profiles and linked gene expression arrays [ChIP-seq]

city: Cambridge

first: Evangelia

last: Diamanti

address: Wellcome Trust / MRC Building, Hills Road

department: Haematology

country: United Kingdom

institute: University of Cambridge

summary: Pu.1-mutated murine AML cell line X18.1.1 transfected with inducible Pu.1 (PuER) or empty vector (EV) and clonal lines selected. Cells were induced with OHT for 2 hours prior to collection for ChIP-Seq and expression arrays.

overall_design: Examination of transcription factors Pu.1 and CEBPA and histone mark H3K27Ac by ChIP-Seq.

first: Jonathan

last: Sive


title: Analysis Of The TGFb-Induced Program In Primary Airway Epithelial Cells Shows Essential Role Of NF-kB/RelA Signaling Network In Type II Epithelial Mesenchymal Transition

city: Galveston

first: Allan

last: Brasier

address: 301 Univ. Blvd

department: Medicine

institute: University Texas Medical Branch

summary: The airway epithelial cell plays a central role in coordinating pulmonary response to injury and inflammation.  Here, transforming growth factor-b (TGFb) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins.  This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells.  In this study, we applied next generation sequencing (RNA-seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it. Generalized linear modeling was performed on a two-factor RNA-seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates).  Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT.  Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-kB/RelA.  Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-kB/RelA pathway.  This Type II EMT program was compared to Type III EMT in TGFb stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling.  We confirmed experimentally that TGFb-induced the NF-kB/RelA pathway by observing a 2-fold change in NF-kB/RelA nuclear translocation.  A small molecule IKK inhibitor blocked TGFb-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression.  These data indicate that NF-kB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT.  Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

overall_design: A human small airway epithelial cell line was treated with TGF-Beta to induce the epithelial to mesenchymal transition. TGF-Beta treated and untreated cells were further treated with TNF-alpha for 1 and 12 hours. Three replicates for each treatment and untreated controls were performed for a total of 18 samples.

first: Bing

last: Tian

first: Xueling

last: Li

first: Mridul

last: Kalita

first: Steven

middle: G

last: Widen

first: Jun

last: Wang

first: Suresh

last: Bhavnani

first: Bryant

last: Dang

first: Andrez

last: Kudlicki

first: Mala

last: Sinha

first: Fanping

last: Kong

first: Thomas

last: Wood

first: Bruce

last: Luxon


title: Small RNA Solexa sequencing of Chinese sacred lotus (Nelumbo nucifera Gaertn.)

first: Yun

last: Zheng

state: Yunnan

address: 452 Fengyuan Road

institute: Yunnan Agricultural University

summary: The total RNA were extracted from pooled tissues of leaves and flowers from several plants of sacred lotus using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict phased small interfering RNAs from Chinese sacred lotus (Nelumbo nucifera  Gaertn.).

overall_design: Identification of phased small inferfering RNAs in Chinese sacred lotus by analyzing small RNA sequencing profiles of leaves and flowers using Illumina GAII.

first: Ramanjulu

last: Sunkar


title: RNA-seq analysis of contextual fear conditioning

city: Spokane

first: Lucia

last: Peixoto

state: WA

address: PO Box 1495

department: College of Medicine

institute: Washington State University, Spokane

summary: This dataset constitutes the first RNA-seq study of gene expression following contextual fear conditioning in the mouse hippocampus.

overall_design: 15 total samples were analyzed, including animals trained in a contexual conditioning paradigm (FC) and controls. Tissue was collected at 30 minutes (FC),  as well as 30 minutes after testing for retrieval of the memory (RT). Testing was performed at 24 hours after training over a 5-minute interval. Animals that were handled but not trained were dissected at the same time of day to control for variations due to circadian rhythms (CC3). The protocol was repeated over the course of 5 days to obtain 9 animals (2 hippocampi) per group, so that 5 independent FC experiments were represented in each time point and all animals for each group were dissected at the exactly the same time of day.

first: Shane

last: Poplwaski

first: Ted

last: Abel


title: N6-adenosine methylation in miRNAs

city: Essen

first: Ludger

last: Klein-Hitpass

address: Virchowstr. 173

department: Universitaetsklinikum

institute: Institut fuer Zellbiologie

summary: Methylation of N6-adenosine (me6A) has been observed in rRNA, tRNA, snoRNA, lncRNA and mRNA (ref.), but not yet miRNA. Interestingly, many mRNAs contain miRNA-binding sites and me6A in their 3'-UTR, although they do not appear to overlap (Mayer). We have investigated whether adenosine methylation affects miRNA levels and whether miRNAs are methylated. Indeed, we have obtained evidence suggesting that miRNA can be methylated and that the steady state level of certain miRNAs is affected by the level of the 6meA demethylase FTO.

overall_design: RNA-Seq of small RNAs prepared from FTO knockdown cells. RNA-Seq of small RNA fraction first immunoprecipitated by antibody against me6A

first: Tea

last: Berulava

first: Katrin

last: Rademacher

first: Sven

last: Rahmann

first: Bernhard

last: Horsthemke


title: RC3H1 posttranscriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-kB pathway

first: Markus

last: Landthaler


title: Identify differentially expressed genes between two development stages in Xuhuai goat skeletal muscle through RNA-Sequencing

status: Public on Jul 19 2015

city: Xuzhou

first: Yanhong

last: Wang

address: shanghai road number101

institute: Jiangsu Normal University

summary: The goal of this study was to compare the transcriptome profiles (RNA-seq) of fetal and six month old goat in skeletal muscle. Total RNAs from the Chinese Xuhuai goat longissimus thoracis at fetal and six month old stages were used to construct cDNA libraries for Solexa SBS technology sequencing. After remove reads with adapters, reads in which unknown bases were more than 5% and low quality reads, we obtained 27,512,850 clean reads from the fetal caprine (FC) library and 27,582,908 clean reads from the six month old caprine (SMC) library. The total basepairs were 2,476,156,500 for FC and 2,482,461,720 for SMC. The ratio of clean reads mapped to reference genome was 45.43% for FC and 33.73% for SMC. The ratio of reads mapped to reference gene was 47.77% for FC and 34.03% for SMC. To reveal the molecular events during different development stages, genes that differentially expressed between the two libraries were identified. Clean reads that matched reference genes in each library were used to evaluate the expression level of transcripts. In total, 11663 genes were differentially expressed between the two development stages. There were 5134 genes with at least a two-fold difference in expression between FC and SMC libraries (FDR, 0.001), of which 1070 genes were up-regulated, 4064 were down-regulated in the six month old caprine compared to the fetal caprine muscle tissue, and 4911 were expressed in both library. Genes with less than one fold changes between FC and SMC libraries were excluded from further analysis. The results indicated that there were significant difference between the two development phase in gene-expression. In addition, some muscle related genes such as MyoD, Myf5, Myogenin, Mrf4, myosin heavy chain 3 (MYH3), Myoglobin and DLK1 were differentially expressed.

overall_design: Examination of Chinese Xuhuai goat mRNAs by deep sequencing

first: YANHONG

last: WANG

first: HONG

last: CHEN


title: A Non-Canonical Nuclear Activity Triggered by Small RNAs and Argonaute Proteins in Human Cells

city: West Haven

first: Francesc

last: Lopez

state: CT

department: Department of Genetics

institute: Yale University

summary: In this study we report the discovery of an unexpected nuclear activity of small RNAs that target an ~8.6 megabase chromosomal domain located on the telomeric q-arm of chromosome 5 from an unbiased genome-wide RNAi screen. Short-hairpin RNAs that target genes within this large domain induce trans-activation of CREB signaling transcriptional reporters and induce an endogenous gene expression signature that includes CREB-target and neuronal-fate genes. Furthermore, CRISPR-guided deletion of AGO-1 and -2 demonstrate their mutually redundant roles in this mechanism, which we named Chromosomally Induced Trans Activation by RNAi (CITAR).

overall_design: We sequenced the transcriptome of HEK293 cells undergoing Chromosomally-Induced Trans Activation by RNAi (CITAR), as measured by transcriptional activation of a CRE-Luciferase reporter (pGL4.29), by shRNAs targeting Chr5q35 loci (TSPAN17 at Chr5:176,074,388, TMED9 at  Chr5:177,019,213 and N4BP3 at Chr5:177,540,556), as well as two negative control shRNAs to account for infection (non-hairpin) and for engagement of the RNAi pathway (ASNA1 encoded on Chr19).

first: Nicole

last: Darricarrère

first: Taavi

middle: K

last: Neklesa

last: López-Giráldez

first: Glenn

middle: S

last: Cowley

first: David

last: Root

first: Craig

last: Crews


title: Fetal microglial in vitro phenotype depends on prior in vivo inflammation

status: Public on Jul 17 2015

first: Martin

last: Frasch

state: QC

address: 3175, Côte-Sainte-Catherine

department: Dépt. obstétrique-gynécologie

institute: Université de Montréal

summary: We sequenced mRNA from 8 microglia extracted from sheep fetuses exposed to LPS or saline solution, to determine genetic expression of the first sheep model of in utero inflammation and second hit inflammation.

overall_design: Examination of genetic expression in naive and second hit microglia.

first: Mingju

last: Cao

first: Marina

last: Cortes

first: Jean-Christophe

last: Grenier

first: Luis

last: Barreiro


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [shRNA]

city: Boston

first: Shannan

last: Ho Sui

address: 401 Park Drive, Level 3 East

department: Biostatistics

institute: Harvard T.H. Chan School of Public Health

summary: Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies.

overall_design: We infected hiF-T cells in biological replicate with a shRNA library (2,900 shRNA targeting 370 distinct epigenetic regulators). This library is a sub-pool of the human 45K shRNA pool (Luo et al., 2008) distributed by the Broad Institute Genomic Perturbations Platform and the RNAi Consortium (TRC). The plasmid for shRNA expression under the control of the constitutive U6 snRNA promoter was the lentiviral vector pLKO.1. shRNA pool production and infection conditions were performed as previously described (Luo et al., 2008). 24h hours after infection as well as at 15 days of repgrogramming, we extracted the genomic DNA and detrmined the abundance of distinct shRNAs based on their representation in the genomic DNA.

first: Davide

last: Cacchiarelli

first: Cole

last: Trapnell

first: Michael

last: J. Ziller


title: RC3H1 posttranscriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-kB pathway [PARCLIP]

first: Markus

last: Landthaler

overall_design: One PAR-CLIP Sample with human HEK293 cells

first: Yasuhiro

last: Murakawa


title: Discovery of novel and differentially expressed microRNAs between fetal and six month old skeletal muscle in goat

first: Yanhong

last: Wang

summary: MicroRNAs (miRNAs) are small (∼22 nucleotides) noncoding ribonucleic acids (RNAs) that regulate gene expression by binding to their complementary sequences. Recent years, a great deal of miRNAs which highly-enriched in skeletal muscle have been identified, which can influence multiple facets of muscle development and function through their regulation of key genes controlling myogenesis. However, to date no miRNAs have been reported to modulate muscle development in goat. Total RNAs from the xuhuai goats longissimus thoracis at fetal and six month old stages were used to construct small RNA libraries for Solexa SBS technology sequencing. In the small RNA profile, a total of 15,627,457 clean reads were obtained from the fetal goat library and 15,593,721 clean reads from the six month old goat library. There are 471 conserved miRNAs overlapped in both libraries, of which 343 miRNAs were differential expressed. We identified 122 novel miRNAs in the fetal caprine library and 53 novel miRNAs in the six month old-caprine library.

overall_design: Examination of Chinese Xuhuai goat miRNAs by deep sequencing

first: YANHONG

last: WANG

first: HONG

last: CHEN


title: Genome-wide expression profiling reveals dominant roles of the cob in maize ear development and low nitrogen response at the silking stage

city: Shanghai

first: Renyi

last: Liu

state: Shanghai

address: 3888 Chenhua Road

department: Bioinformatics

institute: Shanghai Center for Plant Stress Biology

summary: Coordinated functioning of the cob and florets of the maize ear confers grain yield. However, comprehensive molecular differentiation of the cob and florets and their responses to low nitrogen (LN) stress remain elusive. We compared transcriptional profiles of the cob and peripheral florets of a maize hybrid at the silking stage at two N levels in the field and found 1,864 differentially expressed genes between the cob and florets, with 1314 genes up-regulated in the cob and 550 genes up-regulated in florets. The cob was featured by preferential expression of 161 genes, with striking enrichment of genes that are involved in transport facilitation and energy metabolism, consistent with the physiological role of the cob in C/N storage and transfer during ear development. The florets were characteristic of preferential expression of 108 genes, with enrichment of genes that are involved in the cellular process and lipid biosynthetic process. LN caused differential expression of 588 genes in the cob and only 195 genes in florets, indicating that the cob dominated the response of the ear to LN at the transcriptional level. Differentially expressed genes under LN were involved in C/N metabolism, transcriptional regulation, development, cell rescue and defense, and signal transduction in the cob and/or florets. The expression level of 106 genes was regulated by LN in both tissues, suggesting a common regulatory network in these two functionally distinct tissues under LN. Compared with P or K deficiency, LN specifically caused differential expression of 14 genes, revealing unique biological consequences in the ear caused by N limitation at a critical reproductive stage. Collectively, our studies identified unique transcriptomic signatures of the cob and florets, revealed the dominance of the cob in response to LN at the silking stage, and provided molecular markers for N nutritional diagnosis and N efficient maize breeding.

overall_design: Using Digital Gene Expression technology to compare the genome-wide gene expression profiles in cob and peripheral florets of a maize hybrid at the silking stage at two N levels in the field.Three biological replicates were generated for each sample.

first: X

last: Pan

first: Y

last: Li

first: C

last: Liao

first: R


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [ChIP-Seq]

overall_design: ChIP-Seq of secondary fibroblasts and reprogramming intermendiates and iPSCs for histone marks H3K4me1/2/3, H3K27ac, H3K27me3, H3K36me3 and input control


title: Comparative genome analysis of grapevine trunk pathogens reveals distinctive expansion of gene families associated with cell wall degradation, secondary metabolism, and induction of cell death

status: Public on Jul 18 2015

city: Berkeley

first: Abraham

last: Morales-Cruz

state: California

address: 1 Cyclotron Road

institute: Joint Genome Institute

summary: The integration of RNA-seq with Augustus & Maker predictions did not only increase the number of protein-coding genes, but also improved the predicted gene structures, evidenced by wider alignment coverage (Fig. S3C) and greater percent identity when aligned to the proteomes of other ascomycetes (Fig. S3D).

overall_design: T. minima was grown in-vitro in Potato dextrose agar and minimal media agar with dormant canes of Vitis vinifera L. cv. Cabernet Sauvignon and V. vinifera L. cv. Merlot with three biological replicates for each media.

first: Barbara

last: Blanco-Ulate

first: Katherine

last: Amrine

first: Dario

last: Cantu


title: MYB36 regulates the transition from proliferation to differentiation in the Arabidopsis root

city: Durham

first: Louisa

last: Liberman

address: 130 Science Dr. BOX 90338

institute: Duke

summary: Stem cells are defined by their ability to self-renew and produce daughter cells that proliferate and mature. These maturing cells transition from a proliferative state to a terminal state through the process of differentiation. In the Arabidopsis thaliana root the transcription factors SCARECROW and SHORTROOT regulate specification of the bi-potent stem cell that gives rise to the cortical and endodermal progenitors. Subsequent progenitor proliferation and differentiation generates mature endodermis, marked by the Casparian Strip: a cell wall modification that prevents ion diffusion into and out of the vasculature. We identified a transcription factor, MYB36 that regulates the transition from proliferation to differentiation in the endodermis. We show that SCARECROW directly activates MYB36 expression, which in turn directly regulates essential Casparian Strip formation genes. In addition, MYB36 represses extra divisions within the endodermis. Our results demonstrate that MYB36 is a critical positive regulator of differentiation and negative regulator of cell proliferation.

overall_design: 12 samples analyzed: 3 biological replicates each from 1) wild type (Col-0) whole root, 2) mutant (myb36-1) whole root, 3) wild type (Col-0) sorted endodermis, 4) mutant (myb36-1) sorted endodermis


title: Minocycline counter regulates the global pro-inflammatory response in microglia and protects from retinal degeneration

city: Cologne

first: Thomas

last: Langmann

address: Kerpener Strasse 62

department: Department of Ophthalmology

institute: University of Cologne

summary: Minocycline is a potent modulator of retinal microglia

overall_design: Global mRNA expression analysis of CD1 mouse retinas in control, light damage and light damage plus minocycline conditions


title: H3K36 methylation promotes longevity by enhancing transcriptional fidelity [Yeast RNA-Seq]

city: Philadelphia

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

summary: Epigenetic mechanisms including histone post-translational modifications control longevity in diverse organisms. Relatedly, loss of proper transcriptional regulation on a global scale is an emerging aspect of shortened lifespan, but the specific mechanisms linking these observations remain to be uncovered. Here, we describe a lifespan screen in S. cerevisiae, designed to identify altered amino acid residues of histones that alter yeast replicative aging. Our results reveal that lack of sustained H3K36 methylation is commensurate with increased cryptic transcription in a set of genes in old cells and shorter lifespan. Deletion of the K36me2/3 demethylase Rph1 increases H3K36me3 within these genes and suppresses cryptic transcript initiation to extend lifespan. We show that this aging phenomenon is conserved, as cryptic transcription also increases in old worms. We propose that epigenetic misregulation in aging cells leads to an increase in transcriptional noise that is detrimental to lifespan, and, importantly, this acceleration in aging can be reversed by restoring transcriptional fidelity.

overall_design: This study examines transcription in yeast aging using a WT or Rph1 mutant background over a sequence of time-dependent FACS sorts of old cells. Cryptic transcripts are detected using a small fragment sequencing approach. The youngest WT yeast (designated S1Y, S2Y) are represented in seven biological replicates and one technical replicate, five small fragment and three others; the oldest WT yeast (designated S4O) are also represented in seven biological replicates and one technical replicate, again composed of five small fragment samples and three others. Intermediate WT yeast aging sorts (S2O and S3O) are each represented by five biological replicates and one technical replicate, three small fragment and three others. For mutant yeast, each stage in the time series is represented by five biological replicates and one technical replicate, three small fragment and three others. Experiment 4 is a technical replicate of experiment 3.

first: Payel

last: Sen

first: Weiwei

last: Dang

first: Greg

first: Junbiao

last: Dai

first: Jean

last: Dorsey

first: Xiaohua

last: Cao

first: Wei

first: Kajia

first: Rocco

last: Perry

first: Jun

middle: Y

last: Lee

first: John

last: Wagner

first: Brian

last: Gregory

last: Wasko

first: Daniel

middle: T

last: Carr

first: Matt

last: Kaeberlein

last: Kennedy

first: Jef

last: Boeke

first: Shelley

middle: L

last: Berger


title: Codon usage influences the local rate of translation elongation to regulate co-translational protein folding

city: Dallas

first: Yunkun

state: TX

address: 5323 Harry Hines Blvd

department: Physiology

institute: University of Texas Southwestern Medical Center

summary: Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy and protein folding based on the assumption that codon usage affects translation dynamics.  The role of codon usage in regulating translation, however, is not clear and has been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the use of preferred codons enhances the rate of translation elongation, whereas non-optimal codons slow translation. In addition, codon usage regulates ribosome traffic on the mRNA. These conclusions were supported by ribosome profiling results in vitro and in vivo with substrate mRNAs manipulated to increase signal over background noise.  We further show that codon usage plays an important role in regulating protein function by affecting co-translational protein folding. Together, these results resolve a long-standing fundamental question and demonstrate the importance of codon usage on protein folding.

overall_design: For sample 1 and 2, ribosome-protected fragments (RPF) and mRNA sequencing from WT Neurospora transcriptome were extracted to examine the residence time of each codon to understand the decoding rate. For sample 3 and 4, RPFs of the fully optimized (OPT) Firefly luciferase as well as the chimera [(2-423)OPT-(424-550)WT] Firefly luciferase, both of which were individually targeted to the his3 locus, were extracted to monitor the codon-regulated translational kinetics in vivo. For sample 5-8, RPFs were extracted from in vitro translation reactions covering the OPT construct and three chimera construts: N-OP, (2-223)OPT-(224-550)WT; M-OP, (2-223)WT-(224-423)OPT-(424-550)WT; C-OP (2-423)WT-(424-550)OPT. The RPF reads were compared to each other to monitor the translational kinetics in vitro.

first: Chien-hung

last: Yu

first: Zhipeng

last: Zhou


title: MicroRNA profiling and discovery by deep sequencing cord blood from Down syndrome fetuses

city: Shenzhen

first: Wuxian

last: Li

state: Guangdong

address: 1017, North road, Dongmen

institute: the Second Clinical Medical College of Jinan University

summary: To investigate the expression characteristic of miRNAs during the development of Down syndrome (DS) fetuses and to identify whether another miRNA gene resides in the Hsa21, we employed high-throughput Solexa sequencing technology to comprehensively characterize the miRNA expression profiles of both DS and normal fetal cord blood mononuclear cells (CBMCs). In total, 200 of 395 identified miRNAs were significantly differentially expressed (fold change > 2.0 and P-value < 0.001) and 2 of 181 candidate novel miRNAs were identified as residing within the "Down syndrome critical region" of human chromosome 21 (chr21q22.2-22.3). Additionally, 7 of 14 Hsa21-derived miRNAs genes were detected that three miRNAs (hsa-miR-802, miR-3648, miR-3687) were up-regulated more than 50% and four miRNAs (hsa-miR-99a, let-7c, miR-125b-2, miR-155) were down-regulated in the DS fetal CBMCs compared with the control. Bioinformatics analyses revealed that abnormally expressed miRNAs were major associated with the regulation of transcription, gene expression, cellular biosynthetic process, macromolecule biosynthetic process and nucleic acid metabolic process. The data obtained in our study provides a considerable insight into understanding the expression characteristic of miRNAs in the DS fetal hemopoietic system and the differentially expressed miRNAs may be involved in the hemopoietic abnormalities and the immune defects of DS fetus and newborns.

overall_design: A total of 6 DS and 6 matched control fetal cord blood samples (18-22 weeks of gestation) were collected. Three DS and 3 control cord blood samples were combined to form pooled DS and control cord blood samples, respectively, for small RNA library construction and Solexa sequencing. The remaining samples were used as the validation set to confirm the miRNA differential expression patterns by qRT-PCR.

first: Yong

last: Xu


title: Evidence from mRNA-Sequencing that Acute Olanzapine Infusion is Initiating a Skeletal Muscle Fiber Type Transition In Rat Gastrocnemius

city: Hershey

first: Yuka

last: Imamura Kawasawa

address: 500 University Dr.

department: College of Medicine, Pharmacology

institute: Penn State University

summary: The purpose of this study was to examined the acute actions of the second generation antipsychotic (SGA), olanzapine, on skeletal muscle (gastrocnemius) of Sprague Dawley Rats.  SGAs cause metabolic side effects including leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. These effects are preceded by glucose intolerance and increased FFA flux and metabolism in peripheral tissues. Skeletal muscle is a likely target of glucose intolerance, therefore understanding how olanzapine affects the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects.  Male Sprague-Dawley rats freely fed on normal chow with comparable body weights (vehicle: 373±9g, olanzapine: 388±11g, p=0.34) were infused with vehicle or olanzapine for 24h using a dosing regimen leading to mild hyperglycemia  (vehicle, 98±2mg/dl; olanzapine 127±4mg/dl, p=0.0023). For the olanzapine group, the venous catheter was attached to a syringe pump (Model NE-300) filled with olanzapine (Dr. Reddy’s Laboratories Ltd, Hyderabad, India) in sterile saline (infusion: 1mg/100g BW loading dose for 0.5h and then 0.04mg/100g/h continuously for 23.5h).  Gastrocnemius was then surgically removed under isoflurane anesthesia (carried with 100% O2), and frozen between two aluminum blocks cooled to the temperature of liquid nitrogen and then stored at -80oC until RNA was isolated.  With anesthesia gas flow continuing, the animals were euthanized by cutting the diaphram and removing the heart.  The mRNA was isolated from from these muscles and used for RNA-Seq followed by alignment of the data with the rat genome assembly 5.0.  To determine significant differences in FPKM values between control and olanzapine groups, the DEGexp function of the DEGseq 1.18.0 R package was used with the Likelihood Ratio Test (LRT) and default parameters.  In the uploaded excel file, P values with  p<0.05 and p<0.001 are shown for each row in different columns indicated by the number 1.  The value 0 indicates the row is not significantly different.

overall_design: Comparison of vehicle (n=3) and olanzapine infused (n=3) rats.

first: Christopher

middle: J

last: Lynch

first: Anna

last: Salzberg

last: Kawasawa


title: Genome-wide bisulfite sequencing reveals enhancer as main target of DNA methyltion in neural conversion [Bisulfite-seq]

first: Gang

last: Liu

address: 320 Yueyang Rd, Shanghai

institute: Shanghai Institutes for Biological Sciences

summary: Genome-wide bisulfite sequencing and corresponding transcriptome are analyzed together to seek for methylation target during neural differentiation

overall_design: H1 human embryonic stem cells are able to differentiated into neural stem cells with induction of compound C in 72h. We extracted genomic DNA from 0, 24, 48h samples during induction and measured their DNA methylation level by BS-seq.

first: Jiaxi

first: Yang

last: Bai

first: Lei


title: RNA-seq profiling of lung adenocarcinoma and paired normal tissue

status: Public on Jul 16 2015

city: Seoul

first: Kyu-Tae

last: Kim

address: Irwon-Ro 81

department: Samsung Genome Institute

country: South Korea

institute: Samsung Medical Center

summary: To analyze transcriptome data for the mouse xenograft cells derived from a human lung adenocarcinoma patient, reference RNA-seq data were generated for the tracking of gene expression changes associated with the xeno-engraftment and culture.

overall_design: RNA-seq data were generated as a supplementary to GSE69405. The data include RNA-seq of patient normal tissue (matched normal, mN) and of mouse xenograft tissue (xenoT) of the lung adenocarcinoma.

first: Hae-Ock

last: Lee

first: Kyeung

last: Joo

first: Woong-Yang

last: Park


title: H3K36 methylation promotes longevity by enhancing transcriptional fidelity [ChIP-Seq]

first: Gregory

last: Donahue

overall_design: This study examines H3K36me3 in yeast aging using a WT or Rph1 mutant background over a sequence of time-dependent FACS sorts of old cells. There are three sorts: S2Y (young yeast), S2O or S3O (both old yeast). The study has two replicates, F1 and F2. F2 is the combination of two flowcells of sequenced tags. For each sample, we sequenced H3K36me3, H3, and input, and we use H3 as a sonication efficiency normalization in the associated study.

first: Payel

last: Sen

first: Weiwei

last: Dang

first: Greg

first: Junbiao

last: Dai

first: Jean

last: Dorsey

first: Xiaohua

last: Cao

first: Wei

first: Kajia

first: Rocco

last: Perry

first: Jun

first: John

last: Wagner

first: Brian

last: Gregory

last: Wasko

first: Daniel

last: Carr

first: Matt

last: Kaeberlein

last: Kennedy

first: Jef

first: Shelley


title: H3K36 methylation promotes longevity by enhancing transcriptional fidelity [Worm RNA-Seq]

first: Gregory

overall_design: This study examines transcription in worm aging using FUDR treatment. The samples represent a time-series, with one control group without FUDR, and then three treated with FUDR at days 1, 8, and 12. Days 8 and 12 represent old worms. There are no replicates.

last: Liu

last: Boeke

last: Berger


title: H3.3 deposition at endogenous retroviruses and ERV reactivation in Daxx knock-out embryonic stem cells

city: Bethesda

first: Gernot

last: Wolf

state: Maryland

address: 6 Center Drive

department: NICHD

institute: NIH

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

overall_design: Refer to individual Series


title: Brain gene expression associated with facial recognition in Polistes fuscatus paper wasps

city: Ames

first: Ali

last: Berens

address: 251 Bessey Hall

department: Ecology, Evolution, and Organismal Biology

institute: Iowa State University

summary: The goal of this study was to analyze genome-wide brain gene expression during facial and pattern learning for P. fuscatus, the only insect known to possess individual facial recognition. Furthermore, we investigated whether social environment affected a worker's facial learning ability and brain gene expression.

overall_design: PRJNA287145; We sequenced total RNA from 17 individual Polistes fuscatus adult brains from 4 groups: foundresses trained to faces (n=5), foundresses trained to patterns (n=4), workers reared on non-natal conspecific nest and trained to faces (n=4), workers reared on non-natal allospecific nest and trained to faces (n=4).

first: Elizabeth

last: Tibbetts

first: Amy

last: Toth


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [smartseq2]

first: Shannan

last: Ho Sui

overall_design: single cell RNA-seq profiles from 52 unfractionated hiF-T cells after 10 days of reprogramming

first: Davide

last: Cacchiarelli

first: Cole

last: Trapnell

first: Magali

last: Soumillon

first: Michael

last: Ziller


title: H3.3 deposition at endogenous retroviruses in embryonic stem cells

summary: We report DAXX-dependent deposition of histone H3.3 at at endogenous retroviruses in embryonic stem cells.

overall_design: ChIP-seq in conditional Daxx KO ESCs carrying a retrovirally delivered YFP-tagged H3.3 transgene.

first: Warren

last: Wu

first: Brenda

first: Todd

last: Macfarlan


title: H3K36 methylation promotes longevity by enhancing transcriptional fidelity

last: Donahue

sample_organism:
Saccharomyces cerevisiae
Caenorhabditis elegans


title: Sequential DNA methylation and gene expression during human embryonic stem cell inducing into neural progenitor cell

first: Gang


title: ERV reactivation in Daxx knock-out embryonic stem cells

summary: We analyzed expression leveles of transposable elements in conditional Daxx knock-out embryonic stem cells

overall_design: RNA-seq analysis

first: Mohammad

last: Karimi


title: Cdx ParaHox genes acquired distinct developmental roles after gene duplication in vertebrate evolution

city: London

first: Ferdinand

last: Marlétaz

address: Gower Street

department: Genetics, Evolution & Environment

institute: University College London

summary: We examined the degree of regulatory and functional overlap between the three vertebrate Cdx genes using single and triple morpholino knock-down in Xenopus tropicalis followed by RNA-seq.

overall_design: Stage 14 (early neurula) embryos derived from eggs injected with morpholinos against Cdx1, Cdx2, Cdx4 and a mixture of all three plus corresponding uninjected embryos. All in triplicates.

first: Harv

last: Isaacs

first: Peter

last: Holland


title: Brain gene expression associated with visual pattern learning in Polistes metricus paper wasps

summary: The goal of this study was to analyze genome-wide brain gene expression during facial and pattern training for P. metricus. Furthermore, we investigated whether social environment affected a worker's facial learning ability and brain gene expression.

overall_design: PRJNA287152; We sequenced total RNA from 16 individual Polistes metricus adult brains from 4 groups: foundresses trained to faces (n=4), foundresses trained to patterns (n=4), workers reared on non-natal conspecific nest and trained to faces (n=4), workers reared on non-natal allospecific (P. fuscatus) nest and trained to faces (n=4).


title: Latency expression of the Epstein-Barr virus-encoded MHC class I TAP inhibitor, BNLF2a in EBV-positive gastric carcinomas

city: New Orleans

first: Erik

last: Flemington

state: Louisiana

address: 1430 Tulane Avenue, SL-79

department: Tulane Cancer Center/Pathology and Laboratory Medicine

institute: Tulane University

summary: RNA Sequencing performed on EBV positive gastric cancer biopsies and cells lines to study expression of EBV specific genes.

overall_design: Examination of two EBV postitive gastric carcinoma biopsies and two EBV positive gastric cancer cell lines, NCC24 and YCCEL1 by RNA-Seq.

last: Strong

first: Thomas

last: Laskow

first: Hani

last: Nakhoul

first: Eugene

last: Blanchard

first: Yaozhong

last: Liu

first: Xia

last: Wang

first: Melody

last: Baddoo

first: Zhen

last: Lin

first: Qinyan

last: Yin


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [SCRB-Seq]

first: Shannan

last: Ho Sui

state: Massachusetts

summary: Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies.

sample_organism:
Homo sapiens
Mus musculus
synthetic construct

overall_design:
hiF-T cells are secondary reprogrammable cells derived from hiF cells  obtained thorough the directed differentiation of a dox-inducible OKMS  hIPSC line obtained from reprogramming BJ fibroblasts. After directed  differentiation, hiF cells were infected with a lentivirus expressing  the human telomerase and after clonal expansion individual hiF-T  clones were obtained.

The multiplexed data is available at:
https://refinery.stemcellcommons.org/data_sets/GSE69351_DGE/

first: Davide

last: Cacchiarelli

first: Magali

last: Soumillon

first: Michael

last: Ziller


title: Effect of TET1 overexpression on hydroxymethylcytosine levels of HEK293 cells

city: Essen

first: Bernhard

last: Horsthemke

address: Hufelandstrasse 55

department: Universitaetsklinikum Essen

institute: Institut fuer Humangenetik

summary: We compared TET1 overexpressing cells to uninduced cells with endogenous TET1 transcript levels to determine changes of hydroxymethylcytosine levels using RRHP.

overall_design: TET1 overexpression

first: C

last: Grosser

first: B


title: Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome

city: Davis

first: Yoong Wearn

last: Lim

state: CA

address: One Shields Avenue

department: Molecular and Cellular Biology

institute: University of California, Davis

summary: Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4) and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.

overall_design: DNA-RNA immunoprecipitation (DRIP-seq) was performed on control 1, control 3, AGS1 P1, AGS1 P2, AGS2 P1, AGS2 P2, AGS4 P1, AGS4 P2, AGS5 P1 and AGS5 P2. MethylC-seq was performed on control 1, control 2, AGS1 P1, AGS2 P1, AGS4 P1 and AGS5 P1. Reduced representation bisulfite sequencing (RRBS) was performed on GM12697 and AGS2 LCL cell lines. RNA-seq was performed on 2 biological replicates each of control 1, AGS1 P1, AGS2 P1, AGS2 P2, AGS4 P1, AGS4 P2 and AGS5 P1.

first: Yoongwearn

first: Lionel

last: Sanz

first: Xiaoqin

last: Xu

first: Stella

last: Hartono

first: Frederic

last: Chedin


title: Endocardial Brg1 disruption illustrates the EMT origins of semilunar valve disease

first: Brynn

middle: Nicole-Yoshiko

last: Akerberg

state: MA

address: 300 Longwood Ave

department: Cardiology

institute: Boston Children's Hospital

summary: Discreet defects during prenatal semilunar valve (SLV) development frequently progress to pathological states later in life and often require valve replacement surgery. As such, it is challenging to distinguish between the disrupted developmental processes, and their genetic and environmental influences, that trigger valve defects from mechanisms that drive the progression of an anatomically abnormal valve into a disease state. This distinction, which is essential to inform the rationale design of diagnostics and therapeutics, requires carefully characterizing when and where an implicated gene or pathway functions during valve development and/or homeostasis. Disrupted growth, differentiation, and patterning events that trigger SLV disease are coordinated by gene expression changes in endocardial, myocardial, and cushion mesenchymal cells. We explored the roles of chromatin regulation in valve gene regulatory networks via conditional inactivation of the mouse Brg1 associated factor (BAF) chromatin-remodeling complex in the endocardial lineage. Endocardial Brg1-deficient embryos develop thickened and mal-patterned SLV cusps that frequently become bicuspid and myxomatous, including in surviving adults. These SLV disease-like phenotypes originate from deficient endocardial-mesenchymal transformation (EMT) in the proximal outflow tract (pOFT) cushions. Mesenchymal cells of neural crest or other cardiac origins subsequently replace the missing EMT-derived cells but are incompetent to pattern the valve interstitium into regions with distinct extracellular matrix composition. Transcriptomics reveal genes that may promote growth and patterning of SLVs and/or serve as biomarkers of their diseased state. Mechanistic studies of SLV disease genes will distinguish between disease origins and progression; the latter may largely reflect secondary responses to a disrupted developmental system.

overall_design: Examination of transcriptional changes (RNAseq) in E14.5 dissected cardiac cushions (two paired samples) of Nfatc1Cre;Brg1F/F mutants and wildtype littermates

middle: N

first: Kryn

last: Stankunas


title: Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements

first: Gregory

last: Crawford

state: NC

address: 101 Science Drive

department: Center for Genomic and Computational Biology

institute: Duke University

summary: Epigenome editing with the CRISPR/Cas9 platform is a promising technology to modulate gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms that direct gene regulation.  Fusions of the nuclease-inactive dCas9 to the KRAB repressor domain (dCas9-KRAB) can effectively silence target gene expression.  We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes. Genome-wide analyses demonstrated that localization of dCas9-KRAB to HS2 specifically induced H3K9 tri-methylation (H3K9me3) at that enhancer and reduced the chromatin accessibility of both the enhancer and its promoter targets.  Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome. This approach enables precise modulation of epigenetic function without modifying the underlying genome sequence.

overall_design:
K562 cells were transduced with in triplicate lentivirus encoding dCas9-KRAB with gRNA targeted to the HS2 globin enhancer.  Cells transduced with dCas9-KRAB without gRNA or dCas9 with gRNA targeted to the HS2 globin enhancer were included as controls.  RNA-seq was used to identify differential expression at on-target and off-target sites.
--------------------------
This dataset is the DNA-Seq component only

first: Pratiksha

last: Thakore

first: Anthony

middle: M

last: D'Ippolito

first: Lingyun

last: Song

first: Alexias

last: Safi

first: Nishkala

last: Shivakumar

first: Ami

last: Kabadi

middle: E

first: Timothy

last: Reddy

first: Charles

last: Gersbach


title: Identification of RNAs bound to MS2 aptamer using MS2-affinity purification coupled with RNA sequencing (MAPS)

city: SHERBROOKE

first: Eric

last: Massé

state: Québec

address: 3201, Jean Mignault

department: Biochemistry

institute: University of Sherbrooke

summary: DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli

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

overall_design: Identification of RNAs co-purified with MS2 aptamer in a rne131 strain. pNM12 empty vector was used as control

first: David

last: Lalaouna


title: Genome-wide reprogramming of transcript architecture by temperature specifies the developmental states of the human pathogen Histoplasma [ribosome profiling]

city: San Francisco

first: Mark

last: Voorhies

address: 513 Parnassus, S472

department: Microbiology and Immunology

institute: University of California

summary: Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel post-transcriptional mechanism of gene regulation in Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements uncovered a class of differential leader transcripts that exhibit tight transcriptional and translational regulation. Further examination of these dually regulated genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature.  This series contains ribosomal footprinting data for Hc G217B.

overall_design: mRNA libraries were prepared with or without ribosome footprinting for two biological replicates each from cells grown in the yeast or hyphal phase (8 samples total).

first: Sarah

last: Gilmore

first: Anita

last: Sil


title: DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli

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


title: Genome-wide reprogramming of transcript architecture by temperature specifies the developmental states of the human pathogen Histoplasma [transcriptional profiling]

summary: Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel post-transcriptional mechanism of gene regulation in Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements uncovered a class of differential leader transcripts that exhibit tight transcriptional and translational regulation. Further examination of these dually regulated genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature.  This series contains expression profiling data for four strains of Hc.

overall_design: Expression profiling by paired-end RNA sequencing was performed for two biological replicates for each of four strains of Histoplasma capsulatum (G217B, G186AR, H88, and H143) from cells grown in the yeast or hyphal phase (16 samples total).


title: MS2-affinity purification coupled with RNA sequencing (MAPS) reveals DsrA sRNA targetome

overall_design: Identification of RNAs co-purified with MS2-DsrA in a rne131 ΔdsrA strain. DsrA (without MS2) was used as control


title: DNA double-strand breaks induced by AID in activated splenic B cells

city: Worcester

first: Richard

last: Baker

address: 55 Lake Avenue North

department: Microbiology & Physiological Systems

institute: University of Massachusetts Medical School

summary: Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.

overall_design: In two separate experiments, sites of Nbs1 binding were identified genomewide and compared in wild-type and AID-deficient splenic B cells undergoing class switch recombination.

first: Lyne

last: Khair

first: Erin

last: Linehan

first: Carol

last: Schrader

first: Janet

last: Stavnezer


title: Proteomics Analysis Reveals Differentially Activated Pathways that Operate in Peanut Gynophores at Different Developmental Stages

status: Public on Jul 15 2015

city: Ji'nan

first: Xingjun

last: Wang

state: Shandong

address: 202#, Gongye Bei Road

department: Biotechnology Research Center

institute: Shandong Academy of Agricultural Sciences

summary: To gain insight into the mechanism of geocarpy, we extracted proteins from aerial gynophores, subterranean unswollen gynophores, and gynophores that had just started to swell into pods. An integrated analysis of proteome and transcriptome data revealed specifically or differentially expressed genes in the different developmental stages at both the mRNA and protein levels.

overall_design: Peanut gynophores mRNA profiles of stage 1 (S1)  (i.e., aerial gynophores that are green or purple and 3-5 cm long), stage 2 (S2)  (i.e., gynophores that have been buried in the soil for about 3 days; the gynophores are white and ovary enlargement is not apparent), and stage 3 (S3) (dark-grown gynophores with 2-3 mm long pods)  were generated by deep sequencing, using Illumina Hiseq 2000.


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [RRBS]

city: Boston

first: Shannan

last: Ho Sui

address: 401 Park Drive, Level 3 East

department: Biostatistics

institute: Harvard T.H. Chan School of Public Health

overall_design: Reduced representation bisulfite sequencing (RRBS) of primary and secondary fibroblasts with reprogramming intermendiates from hiF-T reprogramming

first: Davide

last: Cacchiarelli

first: Cole

last: Trapnell

first: Michael

last: Ziller

first: Rahul

last: Karnik


title: Genetic Correction and Metabolic Rescue of Pluripotent Cells from Patients with mtDNA

city: La Jolla

first: Robert

last: Morey

address: 2880 Torrey Pines Scenic Dr

department: Pathology

institute: UCSD

summary: Mitochondria are vital due to their principal role in energy production via oxidative phosphorylation (OXPHOS)1. Mitochondria carry their own genome (mtDNA) encoding  critical genes involved in OXPHOS, therefore, mtDNA mutations cause fatal or severely debilitating disorders with limited treatment options. 2. Clinical manifestations of mtDNA disease vary based on mutation type and heteroplasmy levels i.e. presence of mutant and normal mtDNA within each cell. 3,4. We evaluated therapeutic concepts of generating genetically corrected pluripotent stem cells for patients with mtDNA mutations. We initially generated multiple iPS cell lines from a patient with mitochondrial encephalomyopathy and stroke-like episodes (MELAS) caused by a heteroplasmic 3243A>G mutation and a patient with Leigh disease carrying a homoplasmic 8993T>G mutation (Leigh-iPS). Due to spontaneous mtDNA segregation in proliferating fibroblasts, isogenic MELAS iPS cell lines were recovered containing exclusively wild type (wt) mtDNA with normal metabolic function. As expected, all iPS cells from the patient with Leigh disease were affected. Using somatic cell nuclear transfer (SCNT; Leigh-NT1), we then simultaneously replaced mutated mtDNA and generated pluripotent stem cells from the Leigh patient fibroblasts. In addition to reversing to a normal 8993G>T, oocyte derived donor mtDNA (human haplotype D4a) in Leigh-NT1 differed from the original haplotype (F1a) at a additional 47 nucleotide sites. Leigh-NT1 cells displayed normal metabolic function compared to impaired oxygen consumption and ATP production in Leigh-iPS cells or parental fibroblasts (Leigh-fib). We conclude that natural segregation of heteroplasmic mtDNA allows the generation of iPS cells with exclusively wild type mtDNA. Moreover, SCNT offers mitochondrial gene replacement strategy for patients with homoplasmic mtDNA disease.

overall_design: Duplicate cDNA libraries of fibroblasts from a Leigh patient and a MELAS patient, two sendai produced iPSC lines from the Leigh patient and three sendai produced iPSC lines from the MELAS patient, three fibroblasts lines produced by differentiating three iPS Leigh patient iPSC lines to fibroblasts, two somatic cell nuclear transfer produced NT-ESC lines from the Leigh patient, two fibroblast lines produced by differentiating two Leigh patient NT-ESC lines, four fibroblasts lines produced by differentiating four MELAS patient iPSC lines with the mutation to fibroblasts, four fibroblast lines produced by differentiating two IVF-ESC lines without mutated mtDNA genomes, four fibroblast lines produced by differentiating two somatic cell nuclear transfer NT-ESC lines without mutated mtDNA genomes, and four fibroblasts lines produced by differentiating two MELAS patient iPSC lines without the mutation to fibroblasts. The sequence reads were mapped to hg19 reference genome and hits that passed quality filters were analyzed for differential expression.

first: Hong

last: Ma

first: Louise

middle: C

last: Laurent

first: Shoukhrat

last: Mitalipov


title: Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency [small RNA-Seq]

overall_design: small RNA sequencing of reprogramming intermendiates from hiF-T reprogramming and reference BJ fibroblasts and HUES64 hESC. RNA samples used for smallRNA sequencing are the same used for mRNA sequencing.