title: Genome-wide analysis of chromatin and transcription in the ciliates Oxytricha trifallax and Tetrahymena thermophila

city: New York

first: Leslie

last: Beh

state: NY

department: Biochemistry & Molecular Biophysics

country: USA

institute: Columbia University

summary: DNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Through biochemical fractionation of nuclear extracts, we identify a putative DNA 6mA methyltransferase in ciliates, termed MTA1. It contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1 leads to the genome-wide loss of 6mA in vegetative cells and abolishment of the consensus ApT dimethylated motif in vivo. Genes exhibit subtle changes in chromatin organization or RNA expression upon 6mA loss, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We then determine the impact of 6mA on chromatin organization in vitro by reconstructing full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomes in a local, quantitative manner in vitro, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined chromosomes.

sample_organism:
Tetrahymena thermophila
Oxytricha trifallax

overall_design: Datasets: 1) MNase-seq in vivo on chromatin from vegetative Oxytricha cells; 2) MNase-seq in vitro on chromatin reconstituted from Oxytricha histones and native Oxytricha genomic DNA; 3) MNase-seq in vitro on chromatin reconstituted from Xenopus histones and native Oxytricha genomic DNA; 4) MNase-seq in vitro on chromatin reconstituted from Oxytricha histones and PCR-amplified Oxytricha DNA lacking methylation; 5) MNase-seq in vitro on chromatin reconstituted from Xenopus histones and PCR-amplified Oxytricha DNA lacking methylation; 6) Oxytricha DNA N6-methyladenine (6mA) IP-seq using an anti-6mA antibody; 7) Input DNA from Oxytricha 6mA IP-seq experiment; 8) polyA+ RNAseq from vegetative Oxytricha cells; 9) TSS-seq (5'-capped RNAseq) from vegetative Oxytricha cells; 10)  SMRT-seq to identify 6mA modifications in wild type Tetrahymena thermophila native genomic DNA; 11) MNase-seq in vivo on chromatin from MTA1 mutant and matched wild type Oxytricha cells; 12) polyA+ RNAseq from MTA1 mutant and matched wild type Oxytricha cells; 13) SMRT-seq to identify 6mA modifications in MTA1 mutant and matched wild type native genomic DNA; 14) Sequencing of naked MNase-digested genomic DNA

first: Galia

last: Debelouchina

first: Derek

last: Clay

first: Robert

middle: E

last: Thompson

first: Kelsi

last: Lindblad

first: Elizabeth

middle: R

last: Hutton

first: John

last: Bracht

last: Sebra

first: Tom

middle: W

last: Muir

first: Laura

last: Landweber


title: Mapping of microhomologies at CRISPR/Cas9 deletions

status: Public on May 13 2019

city: Oxford

first: Dominic

last: Owens

address: John Radcliffe Hospital

department: MRC WIMM

country: United Kingdom

institute: University of Oxford

summary: The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterise unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

overall_design: Targeted amplicon sequencing at two sites targeted using CRISPR/Cas9

middle: D

first: Marella

last: de Bruijn


title: RNA sequencing of Rnaseh2c knockdown Mvt1 mammary cancer cells

status: Public on May 14 2019

city: Bethesda

first: Maxwell

last: Lee

state: MD

address: Building 37, Room 5120

department: CCR

institute: National Cancer Institute, NIH

type: Expression profiling by high throughput sequencing

summary: To better understand the etiology of metastatic disease, we investigated the impact of genetic polymorphism on metastatic progression. Using RNAseq and exome data analysis, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. Mechanistic studies demonstrated that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity and it functions through engagement of the T cell-mediated adaptive immune response.

overall_design: Mvt1 mouse mammary carcinoma cells with an shRNA-mediated knockdown of Rnaseh2c were generated along with Mvt1 cells expressing a scramble control shRNA. Cell lines or primary mammary tumors generated from syngeneic orthotopic implantations of the control and knockdown cells were anlayzed. mRNA seq samples included RNA from 3 tumors, 1 from each of 3 experiments, for both scramble and knockdown (sh4). Total RNA seq samples included RNA from 3 biological replicates of cultured control or knockdown Mvt1 cells. Exome seq samples included DNA from 4 pulmonary metastases from 3 mice each scramble and knockdown.

first: Deasy

last: Sarah

first: Hunter

last: Kent


title: Serum Reponse of CF1 primary MEFs , E13

status: Public on May 10 2019

city: La Jolla

first: Shankar

last: Subramaniam

state: CA

address: 9500 Gilman Dr MC 0412

department: Bioengineering

institute: University of California, San Diego

summary: The goal of this study was to find the longitudinal transcriptional response of Mouse Embryonic Fibroblast (MEF) primary cells during the progression of a cell cycle. Using RNASeq method (single-end 50-bp chemistry on Illumina Hi-seq 2500 instrument in high-throughput mode), we measured the transcriptional response for around two cell-cycles. Using the fold-change (with respect to average response before serum addition at t = 0) time-series data, we first identified, without using a priori knowledge, the duration and timing of cell cycle phases using a change-point detection algorithm. Next, using Least Absolute Shrinkage and Selection Operator (LASSO) and Estimation Stability with Cross Validation (ES-CV), we were able to, without any prior biological knowledge, extract information on the phase-specific causal interaction of cell cycle genes, as well as temporal interdependencies of biological mechanisms through a complete cell cycle.

overall_design: MEF cells were serum starved for 36 hours (Serum Starved Conditions - 0.5% FCS, DMEM High Glucose, L-glutamine, Penicillin Streptomycin, fungizone, non-essential amino acids) prior to adding serum at t = 0 hr. At t = 0, FCS was added to 20% level by volume to stimulate cell growth. Cells were extracted using Trizol RNA isolation protocol with scraping. Total RNA was isolated using Zymo Research Direct-zol 96 RNA (R2054) kit. TruSeq Stranded mRNA HT Kit (RS-122-2101) was used for library preparation. mRNA was sequenced (single-end 50-bp) using Illumina HiSeq 2500 in high throughput mode, with 12 samples per lane. Data processing steps are: 1) Demultiplexing using Casava [configureBclToFastq.pl --mismatches 1], 2) Mapping/Alignment using RNA-STAR [STAR --genomeDir mm9 --sjdbGTFfile mouse_splice.sjdb --outSammode FULL --outFilterType BySJout], and 3) Normalization and Quantification using Homer [analyzeRepeats.pl rna mm9 -count exons -strand both].

first: Gerald

last: Pao

first: Eugene

last: Ke

first: Junko

last: Ogawa

first: Nina

last: Tonnu

first: Inder

middle: M

last: Verma

first: Maryam

last: Masnadi-Shirazi

first: Mano

last: Maurya


title: In vivo nuclear capture and molecular profiling identifies Gmeb1 as a transcriptional regulator essential for dopamine neuron function

city: Doha

first: Mohamed Nadhir

last: Djekidel

state: Doha

address: Gharafa

department: Translational Medicine Department

country: Qatar

institute: Sidra Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Midbrain dopamine (mDA) neurons play a central role in reward signaling and are widely implicated in psychiatric and neurodegenerative disorders. To understand how mDA neurons perform these functions, it is important to understand how mDA-specific genes are regulated. However, cellular heterogeneity in the mammalian brain presents a major challenge to obtaining this understanding. To this end, we developed a virus-based approach to label and capture mDA nuclei for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling, followed by predictive modeling to identify putative transcriptional regulators of mDA neurons. Using this method, we identified Gmeb1, a transcription factor predicted to regulate expression of Th and Dat, genes critical for dopamine synthesis and reuptake, respectively. Gmeb1 knockdown in mDA neurons resulted in downregulation of Th and Dat, as well as in severe motor deficits. This study thus identifies Gmeb1 as a master regulator of mDA gene expression and function, and provides a general method for identifying cell type-specific transcriptional regulators..

overall_design: mDA neurons were targeted by injection of an AAV-DIO-KASH-HA (KASH-HA) virus into the midbrain of dopamine transporter (DAT)-Cre heterozygous (het) mice. mDA KASH-HA tagged nuclei were FANS sotred for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling. To study the transcriptional effects of Gmeb1 knockdown in mDA neurons, we generated RNA-Seq profiles of mDA neurons  from (DAT)-Cre het mice injected with shGmeb1 or shScrambled virus.

first: Luis

last: Tuesta

first: Mohamed

middle: N

first: Renchao

last: Chen

first: Falong

last: Lu

first: Wengang

last: Wang

first: Bernardo

middle: L

last: Sabatini

first: Yi

last: Zhang


title: Identification of immediate early genes in the nervous system of snail Helix lucorum

city: Moscow

first: Philipp

last: Khaitovich

address: Nobelya Ulitsa 3

country: Russia

institute: Skolkovo Institute of Science and Technology

summary: Immediate early genes (IEGs) are useful markers of neuronal activation and essential components of neuronal response. While studies of gastropods have provided many insights into the basic learning and memory mechanisms, the genome-wide assessment of IEGs has been mainly restricted to vertebrates. In this study we identified IEGs in the terrestrial snail Helix lucorum. In the absence of the genome, we conducted de novo transcriptome assembly using reads with short and intermediate lengths cumulatively covering more than 98 billion nucleotides. Based on this assembly, we identified 37 proteins corresponding to contigs differentially expressed in either the parietal ganglia or two giant interneurons located within the parietal ganglia of the snail in response to the neuronal stimulation. These proteins included homologues of well-known mammalian IEGs, such as c-jun/jund, C/EBP, c-fos/fosl2 and Egr1, as well as homologues of genes not yet implicated in the neuronal response.

overall_design:
RNA-Sequencing was conducted to assemble the neuronal transcriptome and identify immediate early genes in the parietal ganglia or two giant interneurons located within the parietal ganglia of snail Helix lucorum
Please note that the Samples 25-35 were used for transcriptome assembly (i.e. no quantitative processed data).

first: Chuan

last: Xu

first: Qian

last: Li

first: Xi

last: Jiang

first: Olga

last: Efimova

first: Marina

last: Petrova

first: Alia

middle: K

last: Vinarskaya

first: Peter

last: Kolosov

first: Nikolay

last: Aseyev

first: Victor

last: Ierusalimsky

first: Pavel

last: Balaban


title: Mapping chromatin accessibility in stable cell lines expressing Drosophila Hox proteins, Hox cofactors Extradenticle and Homothorax, and the pioneer factor Gcm [ATAC-seq]

status: Public on May 15 2019

city: Cambridge

first: Rob

last: White

address: Downing Street

department: Physiology, Development and Neuroscience

institute: University of Cambridge

summary: We have used stable cell lines expressing selected Hox proteins, Hox cofactors and the pioneer factor Glial cells missing, singly or in combination, to investigate the effects on chromatin accessibility genome-wide using ATAC-Seq.

overall_design: We have examined chromatin accessibility in stable cell lines expressing selected Drosophila Hox proteins in the absence and presence of the cofactor Homothorax, in the presence of the pioneer factor Glial cells missing and in the presence of both Homothorax and Glial cells missing. We also examine chromatin accessibility in stable cell lines expressing either Homothorax or Glial cells missing. For each sample type there are two biological replicates for both induced (i) and non-induced (n) samples. For the analysis of chromatin accessibility in the basal Kc167 cells there are three biological replicates.

first: Damiano

last: Porcelli

first: Bettina

last: Fischer

first: Steven

last: Russell

first: Robert


title: RNAseq of oculomotor (OMN) and spinal motor neurons (SC) derived from mouse embryonic stem cells and purified by FACS

status: Public on May 09 2019

city: Stockholm

first: Eva

last: Hedlund

address: Svante Arrhenius v. 16C

department: Department of Biochemistry and Biophysics

country: Sweden

institute: Stockholm University

summary: Oculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo.

overall_design: A total of 24 samples were analyzed here. Of these, 12 samples are oculomotor neurons derived from mouse embryonic stem cells (mESCs)  facilitated by Phox2a over-expression. The remaining 12 samples are spinal motor neurons also mESCs-derived. mESCs were cultured and patterned with appropriate morphogens as embriod bodies for 5 days and neurons purified using FACS and collected as bulk for RNAseq or re-plated for characterization.  This was performed for 2 mESC lines (Islet1-GFP/NesEPhox2A for OMN and Hb9-GFP for SC differentiations), resulting in the following 4 groups: OMN GFP positive fraction (n=6), OMN GFP negative fraction (n=6), SC GFP positive fraction (n=6) and SC GFP negative fraction (n=6).

first: Jik

last: Nijssen

first: Julio

last: Aguila

first: Ilary

last: Allodi


title: Expression data from leukemic patients with complex structural variants

city: Brno

first: Karla

last: Plevova

address: Kamenice 5

department: Central European Institute of Technology

country: Czech Republic

institute: Masaryk University

summary:
Structural variants can lead to an alteration of gene expression which may be associated with disease worsening. In our study we attempted to describe expression changes associated with the presence of extensive genomic rearrangements in chronic lymphocytic leukemia.
We used microarrays for establishing an algorithm for identification of unique expression profiles associated with extensive genomic rearrangements.

overall_design: Peripheral blood samples from leukemic patients were used for RNA extraction. Gene expression profiles were correlated to chromosomal abnormalities identified in the cohort.


title: Expression data from from NOS2-deficient (NOS2-/-) and wild-type mice subjected to endotoxin or saline challenge

city: Heidelberg

first: Dominik

last: Schaack

address: Im Neuenheimer Feld 420

country: Germany

institute: Heidelberg University Hospital

summary:
Hypoxic pulmonary vasoconstriction (HPV) optimizes the match between ventilation and perfusion in the lung by reducing blood flow to poorly ventilated regions. Sepsis and endotoxemia impair HPV. We previously showed that nitric oxide synthase 2 (NOS2) is required, but not sufficient, for the effect of endotoxin on HPV. The aim of the current study was to identify additional factors that might contribute to the impairment of HPV during endotoxemia.
Microarray gene expression profiling was determined using pulmonary tissues from NOS2-deficient (NOS2-/-) and wild-type mice subjected to endotoxin (LPS) or saline challenge (control).

overall_design: For gene expression profiling lungs were harvested from wild-type mice after challenge with either saline (N=4, 22h) or LPS (N=3, 7h and 22h respectively) or from NOS2-/- mice after saline (N=1, 22h) or LPS challenge (N=3, 7h and 22h respectively).

first: Cornelius

middle: J

last: Busch


title: Copy number (SNP) analysis from leukemic patients with complex structural variants

type: Genome variation profiling by genome tiling array

summary: Structural variants can lead to an alteration of gene expression which may be associated with disease worsening. In our study we attempted to describe expression changes associated with the presence of extensive genomic rearrangements in chronic lymphocytic leukemia.

overall_design:
We used Affymetrix microarrays to identify genomic rearrangements in chronic lymphocytic leukemia.
Peripheral blood samples from 10 leukemic patients were used for genomic DNA extraction. Copy number analysis was performed and obtained data were correlated with expression profiles of these cases.


title: HDAC1-mediated repression of the retinoic acid-responsive gene ripply3 promotes second heart field development

status: Public on May 11 2019

city: Cincinnati

first: Joshua

last: Waxman

state: OH

address: 240 Albert Sabin Way

department: Molecular Cardiovascular Biology Division

institute: Cincinnati Children's Hospital Medical Center

summary: Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular CMs and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5+ progenitors from hdac1 and cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5+ progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that in crg mutants the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors.

overall_design: mRNA sequencing of whole zebrafish and sorted nkx2.5:ZsYellow cells using Illumina

first: Yuntao

middle: C

last: Song

first: Tracy

last: Dohn

first: Ariel

middle: B

last: Rydeen

first: Alex

middle: V

last: Nechiporuk

middle: S


title: Methylome Dynamics of Bovine Gametes and In Vivo Early Embryos

city: Storrs

first: Xiuchun

last: Tian

state: CT

address: 1390 Storrs Road, ABL220D

department: Animal Science

institute: University of connecticut

type: Methylation profiling by high throughput sequencing

summary: Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole genome bisulfite sequencing (scWGBS) method to characterize stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived in vivo and in vitro, as well as in vivo developed embryos at the 2-, 4-, 8- and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using single oocytes or embryos. We found that the major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes were differentially methylated in numerous regions (DMRs) which were enriched in intergenic regions, indicating these noncoding regions play important roles in gamete specification. DMRs were also identified between in vivo and in vitro matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA-seq data generated from the same developmental stages, we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using scWGBS. These data provide insights into the critical features of the methylome of bovine embryos, and serve as an important reference for embryos produced by assisted reproduction, such as in vitro fertilization and cloning, and a model for human early embryo epigenetic regulation.

overall_design: Whole genome bisulfite sequencing was used to determined genome-scale DNA methylation patterns of bovine sperm and individual in vivo developed oocytes and preimplantation embryos.

first: J

last: Duan

first: Z

last: Jiang

first: F

last: Alqahtani

first: I

last: Mandoiu

first: H

last: Dong

first: X

last: Zheng

first: S

last: Marjani

last: Chen


title: Gene Expression data from human mesenchymal stromal cells

city: Warsaw

first: Anna

last: Burdzińska

address: Nowogrodzka 59

department: Department of Immunology, Transplantology and Internal Medicine

country: Poland

institute: Medical University of Warsaw

summary:
Cellular therapy is proposed for tendinopathy treatment. Bone marrow- (BM-MSC) and adipose tissue- (ASC-) derived mesenchymal stromal cells are candidate populations for such a therapy.
We used microarrays to evaluate the basal gene expression in human BM-MSCs and ASCs and to create list of differentially expressed genes between analyzed groups.

overall_design: The microarray expression analysis was performed on RNA isolated from hASC (n=5) and hBM-MSC cells (n=6) using the Affymetrix Gene Atlas system.

first: Marta

last: Koblowska


title: MicroRNA-guided regulation of heat stress response in wheat

status: Public on May 08 2019

city: Ottawa

first: Sylvie

last: Cloutier

state: Ontario

address: 960 Carling Avenue

country: Canada

institute: Agriculture and Agri-Food Canada

summary: Sequencing of 24 small RNA libraries produced 55.2M reads while 404M reads were obtained from the corresponding 24 PARE libraries. From these, 202 miRNAs were ascertained, of which mature miRNA evidence was obtained for 104 and 36 were found to be differentially expressed after heat stress. The PARE analysis identified 589 transcripts targeted by 84 of the ascertained miRNAs. PARE sequencing validated the targets of the conserved members of miRNA156, miR166 and miR393 families as squamosa promoter-binding-like, homeobox leucine-zipper and transport inhibitor responsive proteins, respectively. Heat stress responsive miRNA targeted superoxide dismutases and an array of homeobox leucine-zipper proteins, F-box proteins and protein kinases. Query of miRNA targets to interactome databases revealed a predominant association of stress responses such as signalling, antioxidant activity and ubiquitination to  superoxide dismutases, F-box proteins, pentatricopeptide repeat-containing proteins and  mitochondrial transcription termination factor-like proteins.

overall_design: A total of 144 Triticum aestivum cv Chinese Spring wheat plants were grown in a PG-40 growth cabinet under long-day conditions of 16 hours of light (300 μmol m−2 s−1) at 18°C and 8 hours of darkness at 16°C. At the boot stage, a set of 72 plants were exposed to a 37°C heat stress for 5 days while the other 72 plants remained under the control conditions mentioned above. The heat stressed plants were amply watered twice per day during the 5-day stress period to avoid a confounding effect with drought stress. Leaf tissue was collected from all heat-stressed and control individual plants immediately at the end of the stress period, i.e., time point zero day after treatment (0 DAT), and at one and four days after treatment (1 and 4 DAT). Equimolar amounts of RNA from the 18 samples representing a replicate, treatment and time point were pooled to create the 24 total RNA samples representing the four biological replicates, two treatments and three time points. These 24 total RNA samples were used to construct sRNA libraries.

first: Sridhar

last: Ravichandran

first: Raja

last: Ragupathy

first: Tara

last: Edwards

first: Michael

last: Domaratzki


title: Aberrant expression of select piRNA-pathway genes does not reactivate piRNA silencing in cancer cells

first: Pavol

last: Genzor

address: 6720A Rockledge Drive, Suite 100

department: ACESO

institute: Henry M Jackson Foundation

type: Non-coding RNA profiling by high throughput sequencing

summary: Analysis of PIWIL1 function in COLO205 [ATCC® CCL-222] colon adenocarcinoma cancer cell culture model.

overall_design: Total Small RNAs and small RNAs co-immunoprecipitated with PIWIL1 from COLO205 cells were analyzed for piRNA signatures. Total RNA sequencing from PIWIL1 short hairpin knock-downs and CRISPR-generated knockouts were used for differential gene and transposon expression.

first: Seth

last: Cordts

first: Neha

last: Bokil

first: Astrid

last: Haase


title: Longitudinal Study of Leukocyte DNA Methylation and Biomarkers for Cancer Risk in Older Adults

status: Public on May 07 2019

city: Memphis

first: Khyobeni

last: Mozhui

state: TN

address: 66 N. Pauline Street, #631

department: Preventive Medicine

institute: University of Tennessee Health Science Center

summary:
Peripheral blood samples were obtained at baseline and at follow-up visit from 20 participants in the Health, Aging and Body Composition prospective cohort study. Genome-wide CpG methylation was assayed using the Illumina Infinium Human MethylationEPIC (HM850K) microarray.
We explored longitudinal changes in CpG methylation from blood leukocytes, and likelihood of a future cancer diagnosis.

overall_design: We examined both cross-sectional differences and longitudinal changes


title: Expression levels of inositol phosphorylceramide synthase modulate plant responses to biotic and abiotic stress in Arabidopsis thaliana

city: Durham

first: Paul

middle: William

last: Denny

state: Durham

address: Lower Mountjoy

department: Biosciences

institute: Durham University

summary: This research was undertaken to investigate the global role of inositol phosphorylceramide synthase (IPCS) activity in plants and reveal its potential as a herbicide target. The non-mammalian enzyme is a key component in the plant sphingolipid biosynthetic pathway and is shown here to be a possible herbicide target.  RNA-Seq analyses demonstrated that over-expression of inositol phosphorylceramide synthase isoforms AtIPCS1, 2 or 3 in Arabidopsis thaliana resulted in the down-regulation of genes involved in plant response to pathogens. In addition, genes associated with the abiotic stress response to salinity, cold and drought were found to be similarly down-regulated. Detailed analyses of transgenic lines over-expressing AtIPCS1-3 at various levels revealed that the degree of down-regulation is specifically correlated with the level of IPCS expression. Singular enrichment analysis of these down-regulated genes showed that AtIPCS1-3 expression affects biological signaling pathways involved in plant response to biotic and abiotic stress. The up-regulation of genes involved in photosynthesis and lipid localization was also observed in the over-expressing lines.

overall_design: Two biological replicates were analyzed for each AtPCS isoform (three IPCS isoforms; two levels of AtIPCS expression per isoform)

first: Elizabeth

last: Pinneh


title: Comprehensive analysis of RNA-seq kits for standard, low and ultra-low quantity samples

city: Evry

first: Eric

last: Bonnet

address: 2 rue Gaston Crémieux

country: France

institute: CNRGH

summary: High-throughput RNA-sequencing has now become the gold standard method for whole-transcriptome gene expression analysis. It is widely used in a number of applications studying various transcriptomes of cells and tissues. It is also being increasingly considered for a number of clinical applications, including expression profiling for diagnostics or alternative transcripts detection. However, RNA sequencing can be challenging in some situations, for instance due to low input quantities or degraded RNA samples. Several protocols have been proposed to overcome some of these challenges, and many are available as commercial kits. Here we perform a comprehensive testing of three recent commercial technologies for RNA-seq library preparation (Truseq, Smarter and Smarter Ultra-Low) on human reference tissue preparations, for standard (1ug), low (100 and 10 ng) and ultra-low (< 1 ng) input quantities, and for mRNA and total RNA, stranded or unstranded. We analyze the results using read quality and alignments metrics, gene detection and differential gene expression metrics. Overall, we show that the Truseq kit performs well at 100 ng input quantity, while the Smarter kit shows degraded performances for 100 and 10 ng input quantities, and that the Smarter Ultra-Low kit performs quite well for input quantities < 1 ng. All the results are discussed in details, and we provide guidelines for the selection of a RNA-seq library preparation kits by biologists.

overall_design: RNA-seq profiles of Human Universal Reference Total RNA (Clontech) and Human Brain reference RNA (Ambion) were generated by deep sequencing, in duplicate, using Illumina HiSeq2000 or 2500 sequencers.

first: Marie-Ange

last: Palomares

first: Cyril

last: Dalmasso


title: Transcriptome of Trichophyton rubrum grown on protein substrates and exposed to trans-chalcone

city: RIBEIRAO PRETO

first: Tamires

middle: Aparecida

last: Bitencourt

state: SP

address: AV COSTABILE ROMANO 2221

department: BIOTECNOLOGY

country: Brazil

institute: UNAERP

summary: We assessd the transcriptional profile of T. rubrum during grown on different protein sources to mimic the natural infection sites (keratin or elastin) and exposed to trans-chalcone in order to clarify the mechanisms involved in trans-chalcone antifungal activity

overall_design: About 1.6 10^6 conidia was added in 20mL of liquid Sabouraud followed by an incubation  for 72h at 28ºC under shaking at 150 rpm. Then the resulting mycelia was transferred to six different conditions:i) Minimal medium Cove's with nitrate and glucose, ii) Keratin medium: Cove’s minimal medium with 0.5% bovine keratin powder;  iii) Elastin medium: Cove’s medium  with 0.25% elastin; iv) Minimal medium added of 0.24µg/mL of trans-chalcone and nitrate and glucose; v) Minimal medium  with 0.5 % of keratin, added of 0.24µg/mL of trans-chalcone  and vi) Minimal medium with 0.25% of elastin,  added of 0.24µg/mL of trans-chalcone. At least two independent replicates were performed for each condition

middle: A

first: Claudia

last: Macedo


title: Zfp281 shapes the transcriptome of trophoblast stem cells and is essential for placental development

city: Fukuoka

first: Takashi

last: Ishiuchi

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

department: Medical Institute of Bioregulation

country: Japan

institute: Kyushu University

summary: Placental development is a key event in mammalian reproduction and embryogenesis. However, the molecular basis underlying extraembryonic lineage specification and subsequent placental development is not fully understood. Through a genetic screen, we identified Zfp281 as a key factor for extraembryonic development. Disruption of Zfp281 in mice caused severe defects in extraembryonic as well as embryonic tissues. Importantly, Zfp281 was preferentially expressed in the trophoblast stem cell population in an FGF-dependent manner and ensured the expression of genes necessary for placental development. Through the analysis of transcriptome and epigenome, we identified Zfp281 as an important factor to shape the transcriptome of mammalian trophoblast stem cells.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: To study the role of Zfp281 in transcriptional regulation, we performed RNA-seq using mouse and human TS cells. Furthermore, we performed H3K4me3 ChIP-seq and ATAC-seq to reveal the roles of Zfp281 in chromatin regulation.

first: Tetsuya

last: Sato

first: Hiroaki

last: Ohishi


title: Nfat5 is involved in the hyperosmotic regulation of Tmem184b, which could be involved in ibuprofen transport in renal MDCK I cells

city: Karise

first: Rune

middle: Nørgaard

last: Rasmussen

address: Vesterled 4

department: Drug transporters in ADME

country: Denmark

institute: Southern University of Denmark

summary:
Purpose: To investigate the involvement of Nfat5 in the regulation of an ibuprofen transporter
Method: Reverse transfection with siRNA against Nfat5 was used to knockdown Nfat5 in MDCK I cells. The uptake of both radiolabelled taurine and ibuprofen was measured in MDCK I cells, first treated with siRNA against Nfat5 for 24 h and afterwards cultivated with raffinose-supplemented normal growth medium (500 mOsm) for 24 h. The knock down of NFat5 was investigated by qpCR and western blotting. A transcriptome analysis of MDCK I cells treated with siRNA against Nfat5  was performed. Validation of the transcriptome analysis was perform with qPCR and radiolabelled uptake of ibuprofen.
Results: The siRNA transfection resulted in a knock down of Nfat5, and the uptake of both taurine and ibuprofen was significant decreased in the transfected MDCK I cells. The transcriptome analysis of MDCK I cells treated with siRNA against Nfat5 revealed genes regulated by Nfat5 during hyperosmotic exposure. qPCR and the transciptome analysis showed an involvement of Nfat5 in the hyperosmotic regulation of Tmem184 and uptake in Tmem184b-siRNA treated MDCK I cells showed that Tmem184b might be involved in uptake of ibuprofen.
Conclusion: The regulation of Tmem184b was found regulated by Nfat5 and could be a potential possible candidate for the gene product responsible for the uptake of ibuprofen in MDCK I cells

overall_design: The transcriptome analysis was performed in duplicate (all other experiments were performed in triplicate or more) with both control for the osmotic treatement, the off-target effect of siRNA, and controls for the reagens

first: Kenneth

last: Christensen

first: René

last: Holm

first: Carsten

middle: U

last: Nielsen


title: Giardia lamblia miRNAs as a new diagnostic tool for human giardiasis

city: Jerusalem

first: Iddo

middle: Z.

last: Ben-Dov

address: Ein Kerem

department: Nephrology and Hypertension

country: Israel

institute: Hadassah Medical Center

summary: Giardia lamblia is one of most common agents causing persistent abdominal symptoms in developed and developing countries. There are several diagnostic methods for Giardia infection, but none are optimal. In this study our aim was to find a new method based on Giardia microRNA (miRNA) that would contribute to the currently available diagnostic methods of giardiasis. Profiling Giardia small RNAs by deep sequencing revealed that the previously reported putative miR5 and miR6 are expressed in several G. lamblia isolates. These miRNAs were later tested by PCR in duodenal biopsies from 8 patients with positive pathology for giardiasis, while gastric biopsies served as matched negative controls. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. All 8 duodenal samples of patients with histologically proven G. lamblia infection were positive for Giardia miR5 with a mean Ct of 23.7. These results were superior to Ct levels of G. lamblia DNA, which were 26.3 (p=0.004). The miR6 results were close to negative. All 10 gastric biopsies were negative for miR5. Stool studies showed 90% specificity but only 50% sensitivity in diagnosing giardiasis using miR6. The results of miR5 in stool were even less accurate. In conclusion, miR5 testing for Giardia infection in duodenal biopsies, may be a breakthrough method for diagnosis of giardiasis. It seems to be superior to G. lamblia DNA in duodenal biopsies. It would be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies and duodenal aspirates from patients with persistent abdominal symptoms.

overall_design: For this part of the project, we profiled small RNA from Giardia lamblia trophozoites of 5 strains obtained from the BEI Resources

first: Tal

last: Meningher

first: Dror

last: Avni

first: Devorah

last: Gur-Wahnon

first: Eli

last: Schwartz

middle: Z

first: Refael

last: Kohen


title: Early genome activation in Drosophila is extensive with an initial tendency for aborted transcripts and retained introns

status: Public on May 06 2019

first: Jamie

last: Kwasnieski

state: Massachusetts

address: 455 Main Street

department: Biology

institute: Whitehead Institute/MIT

summary: Control of metazoan embryogenesis shifts from maternal to zygotic gene products as the zygotic genome becomes transcriptionally activated. In Drosophila, zygotic genome activation (ZGA) begins with a minor wave, but technical challenges have hampered the identification of early transcripts or obscured the onset of their transcription. Here, we develop an approach to isolate transcribed mRNAs and apply it over the course of the minor wave and the start of the major wave of Drosophila ZGA. Our results increase known genes of the minor wave by 10 fold and show that this wave is continuous and gradual. Transposable-element mRNAs are also produced, but discontinuously. Genes in the early and middle part of the minor wave are short with few if any introns, and their transcripts are frequently aborted and tend to have retained introns, suggesting that inefficient splicing as well as rapid cell divisions constrain the lengths of early transcripts.

overall_design: The goal of this study is to use NGS to identify zygotic transcripts produced during early zygotic genome activation in Drosophila.

first: Terry

last: Orr-Weaver

first: David

middle: P

last: Bartel


first: Sylvie

last: Cloutier

first: Sridhar

last: Ravichandran

first: Raja

last: Ragupathy

first: Tara

last: Edwards

first: Michael

last: Domaratzki


title: Cell-based therapy restores olfactory function in an inducible model of anosmia

city: Miami

first: Anthony

last: Griswold

state: FL

address: 1501 NW 10th Ave, BRB 318

institute: University of Miami

summary: We explore culture coniditions that promote the expansion of engraftment-compentent olfactory basal progenitor cells in vitro.

overall_design: Whole transcriptome sequencing was performed on cells treated with one of two Activin/TGF-beta pathway inhibitors; 3 biologic replicates per treatment.

first: Rhea

last: Choi

first: Stefania

last: Goncalves

first: Bradley

last: Goldstein


title: SAGA is important for prompt export of stress-inducible transcripts through Sgf73p-mediated mechanism

city: Daejeon

first: Minhoo

last: Kim

address: Basic Science Bld Rm418, 291 Daehak-ro, Yuseong-gu, KAIST

department: Department of Biological Sciences

country: South Korea

institute: KAIST

summary: Previous studies suggested that stress-inducible genes are SAGA (Spt-Ada-Gcn5 acetyltransferase)-dominated and characterized by distinct chromatin structures on promoter regions; however, a detailed regulatory mechanism and evolutional advantage of such specialization are not fully understood yet. Here, we newly defined SAGA-regulated genes under direct control of Sgf73p of the deubiquitylating module (DUBm). Through genetic and biochemical analyses, we observed a functional relationship between Sgf73p and the essential mRNA export factor Yra1p; Sgf73p is critical for proper chromatin localization and RNA-binding of Yra1p. Additionally, under physiological condition, Sgf73p plays a central role in quality control of messenger ribonucleoparticles (mRNPs) biogenesis and export in conjunction with the nuclear exosome. Upon environmental stress, when immediate transport of specific transcripts is imperative, Sgf73p facilitates bypass of canonical surveillance and promotes timely export of necessary transcripts. Overall, our results show Sgf73p-mediated gene expression plasticity is important for cell tolerance to stress and thus survival amidst of the uncertain environment.

overall_design: ChIP-seq samples of Flag-tagged SAGA subunits (including Sgf73p and Sgf11p), Sua7p, Yra1p, and Rpb3p in wildtype and various mutants were prepared using budding yeast; ChIP-seq samples of Flag-tagged Sgf73p in wildtype, rpt2-1 and spt20del before and after heat shock (at 42degrees for 20min).

first: Yoonjung

first: Harim

first: Daeyoup

last: Lee


title: Transcriptomic profile of several temperate Cluster A actinobacteriophages during lysogenic growth

city: Pittsburgh

first: Travis

last: Mavrich

state: Pennsylvania

address: 4249 Fifth Avenue

department: Biological Sciences

institute: University of Pittsburgh

summary: The goal of this sequencing project was to measure transcription levels of several Cluster A actinobacteriophages during lysogeny in order to study which genes contribute to lysogeny and superinfection immunity.

overall_design: Total RNA was isolated from cultures grown in log phase, depleted of rRNA and prepared for sequencing using a strand-specific library kit during lysogeny of phages Et2Brutus, Gladiator, and Trixie. A total of three different RNA samples were prepared.

first: Graham

middle: F

last: Hatfull

first: Daniel

last: Russell

first: Rebecca

last: Garlena


first: Sylvie

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

overall_design: Refer to individual Series


title: Phospho-small RNA-seq reveals circulating, extracellular mRNA/lncRNAs as potential biomarkers in human plasma: Healthy Cohort [HEALTHYPLASMA]

status: Public on May 03 2019

city: Ann Arbor

first: Muneesh

last: Tewari

state: MI

address: 109 Zina Pitcher Pl

institute: University of Michigan

summary: Extracellular RNAs (exRNAs) in blood and other biofluids have attracted great interest as potential biomarkers in liquid biopsy applications, as well as for their potential biological functions. Whereas it is well-established that extracellular microRNAs are present in human blood circulation, the degree to which messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are represented in plasma is less clear. Here we report that mRNA and lncRNA species are present as small fragments in plasma that are not detected by standard small RNA-seq methods, because they lack 5’-phosphorylation or carry 3’-phosphorylation. We developed a modified sequencing protocol (termed “phospho-sRNA-seq”) that incorporates upfront RNA treatment with T4 polynucleotide kinase (which also has 3’ phosphatase activity) and compared it to a standard small RNA-seq protocol, using as input both a pool of synthetic RNAs with diverse 5’ and 3’ end chemistries, as well exRNA isolated from human blood plasma. Using a custom, high-stringency pipeline for data analysis we identified mRNA and lncRNA transcriptome fingerprints in plasma, including multiple tissue-specific gene sets. In this cohort of healthy individuals, we demonstrate the improved capture of mRNA and lncRNA fragments using the phospho-sRNA-seq protocol. We find they primarily arise from mRNA exons, and that abundant fragments are consistently detected in multiple individuals.

overall_design: Total RNA from platelet-poor plasma was sequenced in triplicate from 5 healthy individuals.


title: Phospho-small RNA-seq reveals circulating, extracellular mRNA/lncRNAs as potential biomarkers in human plasma: PNK evaluation on synthetic, equimolar pool [SYNTHETICPNK]

summary: Extracellular RNAs (exRNAs) in blood and other biofluids have attracted great interest as potential biomarkers in liquid biopsy applications, as well as for their potential biological functions. Whereas it is well-established that extracellular microRNAs are present in human blood circulation, the degree to which messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are represented in plasma is less clear. Here we report that mRNA and lncRNA species are present as small fragments in plasma that are not detected by standard small RNA-seq methods, because they lack 5’-phosphorylation or carry 3’-phosphorylation. We developed a modified sequencing protocol (termed “phospho-sRNA-seq”) that incorporates upfront RNA treatment with T4 polynucleotide kinase (which also has 3’ phosphatase activity) and compared it to a standard small RNA-seq protocol, using as input both a pool of synthetic RNAs with diverse 5’ and 3’ end chemistries, as well exRNA isolated from human blood plasma. This series uses the synthetic pools of small RNAs to demonstrate the efficacy of phospho-sRNA-seq to enable capture of sRNAs lacking a 5’ phosphate and/or having a 3’ phosphate.

overall_design: Two TruSeq smallRNA-seq libraries were prepared from a synthetic, equimolar pool of 476 small RNAs (15 - 90 nt), containing oligos with different end chemistries: 5' P + 3'OH (n=352), 5'OH + 3'P (n=60), 3'OH + 5'OH (n=60), 5'P + 3' 2OME (n=4). Libraries were generated by 1) directly sequencing the pool or 2) pre-treating the pool with T4 Polynucleotide Kinase (PNK).


title: Epstein-Barr Virus (EBV) activates NKL homeobox gene HLX in DLBCL

city: Braunschweig

first: Claudia

last: Pommerenke

address: Inhoffenstraße 7B

institute: Leibniz Institute DSMZ

summary: NKL homeobox genes encode developmental transcription factors regulating basic processes in cell differentiation. According to their physiological expression pattern in early hematopoiesis and B-cell development, particular members of this homeobox gene subclass constitute an NKL-code. These B-cell specific genes generate a regulatory network and their deregulation is implicated in B-cell lymphomagenesis. Epstein-Barr virus (EBV) infects B-cells and influences the activity of signalling pathways including JAK/STAT and several genes encoding developmental regulators. Therefore, EBV-infection impacts the pathogenesis and the outcome of B-cell malignancies including Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL). Here, we isolated EBV-positive and EBV-negative subclones from the DLBCL derived cell line DOHH-2. These subclones served as model to investigate the role of EBV in deregulation of the B-cell specific NKL-code members HHEX, HLX, MSX1 and NKX6-3. We showed that the EBV-encoded factors LMP1 and LMP2A activated the expression of HLX via STAT3. HLX in turn repressed NKX6-3, SPIB and IL4R which normally mediate plasma cell differentiation. In addition, HLX repressed pro-apoptotic factor BCL2L11/BIM supporting cell survival. Thus, EBV aberrantly activated HLX thereby disturbing both B-cell differentiation and apoptosis in DLBCL. The results of our study contribute to better understand the pathogenic role of EBV in B-cell malignancies.

overall_design: Molecular characterization of EBV-positive and EBV-negative subclones of DLBCL cell line DOHH-2

first: Stefan

last: Nagel


title: Rhodospirillum rubrum whole transcriptome data comparison between anaerobic versus aerobic growth conditions

city: Spokane

first: Jennifer

middle: Niven

last: Shepherd

state: WA

address: 502 E Boone Avenue

department: Department of Chemistry and Biochemistry

institute: Gonzaga University

summary: RNAseq was performed on sixth generation anaerobic and aerobic R. rubrum cultures

overall_design:
Comparison of gene reads in anaerobically versus aerobically grown R. rubrum was performed to identify gene candidates in rhodoquinone biosynthesis

Raw data are not available because the UW HT Sequencing center did not provide raw data files with the analyzed data. The raw files at UW HT Sequencing center have since been deleted.

first: Kirk

last: Anders


title: microRNA in mouse peritoneal tissue

city: Guangzhou

first: Meijun

last: Si

state: Guangdong

address: 600 Tianhe Ave.

department: Nephrology

country: China

institute: The 3rd Affiliated Hospital of Sun Yat-sen University

summary: Analysis of microRNAs in peritoneal tissues from mice treated with peritoneal dialysis fluid and saline

overall_design: We collected peritoneal tissues from mice intraperitoneally-injected with peritoneal dialysis fluid for six weeks(n = 3), while mice in control group were treated with saline for six weeks(n = 3). We profiled microRNAs in peritoneal tissue by microarray.

first: Hui

last: Peng

first: Zhaoyong

last: Hu

first: Qianqian

last: Wang


title: KRASG12C inhibition produces a driver-limited state revealing collateral dependencies

city: San Francisco

first: Kevin

last: Lou

address: 600 16th Street

institute: University of California, San Francisco

summary: Inhibitors targeting KRASG12C, a mutant form of the guanosine triphosphatase (GTPase) KRAS, are a promising new class of oncogene-specific therapeutics for the treatment of tumors driven by the mutant protein. These molecules react with the mutant cysteine residue by binding covalently to the switch-II pocket (S-IIP) that is present only in the inactive guanosine diphosphate (GDP)-bound state of KRASG12C, sparing the wild-type protein. We used a genome-scale CRISPR interference (CRISPRi) functional genomics platform to systematically identify genetic interactions with the KRASG12C inhibitor in cellular models of KRASG12C mutant lung and pancreatic cancer. Our data revealed genes that were selectively essential in this oncogenic driver-limited cell state, meaning their loss enhanced cellular susceptibility to direct KRASG12C inhibition. We termed such genes “collateral dependencies” (CDs), and identified two classes of combination therapies targeting these CDs that increased KRASG12C target engagement or blocked residual survival pathways in cells and in vivo. From our findings, we also propose a new framework for assessing genetic dependencies with driver oncogenes.

overall_design: RNA sequencing of H358 cells following 24 hour treatment with DMSO, palbociclib (1 μM, CDK4/6i), ARS-1620 (1 μM), or the combination

first: Alex

middle: Y

last: Ge

first: Kevan

middle: M

last: Shokat

first: Luke

last: Gilbert


title: Unanchored Ubiquitin Chains Do Not Lead to Marked Genetic Alterations in Drosophila melanogaster

status: Public on May 01 2019

city: Detroit

first: Jessica

middle: Renee

last: Blount

address: 540 E. Canfield, 3108 Scott Hall

department: Pharmacology

institute: Wayne State School of Medicine

summary:
Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell.
We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.

overall_design: 12 samples total were analyzed, with four biological replicates for each genotype (Ub6-GG, Ub6-Stop, and Control). Controls were flies with the same genetic background as Ub6 flies, crossed to the same driver, but without the Ub6 transgene.

first: Sokol

last: Todi


title: Phospho-small RNA-seq reveals circulating, extracellular mRNA/lncRNAs as potential biomarkers in human plasma: Hematopoietic Stem Cell Transplant [HSCT]

summary: Extracellular RNAs (exRNAs) in blood and other biofluids have attracted great interest as potential biomarkers in liquid biopsy applications, as well as for their potential biological functions. Whereas it is well-established that extracellular microRNAs are present in human blood circulation, the degree to which messenger RNAs (mRNA) and long noncoding RNAs (lncRNA) are represented in plasma is less clear. Here we report that mRNA and lncRNA species are present as small fragments in plasma that are not detected by standard small RNA-seq methods, because they lack 5'-phosphorylation or carry 3'-phosphorylation. We developed a modified sequencing protocol (termed "phospho-sRNA-seq") that incorporates upfront RNA treatment with T4 polynucleotide kinase (which also has 3' phosphatase activity) and compared it to a standard small RNA-seq protocol, using as input both a pool of synthetic RNAs with diverse 5' and 3' end chemistries, as well exRNA isolated from human blood plasma. Using a custom, high-stringency pipeline for data analysis we identified mRNA and lncRNA transcriptome fingerprints in plasma, including multiple tissue-specific gene sets. In a longitudinal study of hematopoietic stem cell transplant (HSCT) patients, we found different sets corresponding to bone marrow- and liver- enriched genes, which tracked with bone marrow recovery or liver injury, providing proof-of-concept validation of this method as a biomarker approach. By accessing a previously unexplored realm of mRNA and lncRNA fragments in blood plasma, phospho-sRNA-seq opens up a new space for plasma transcriptome-based biomarker development in diverse clinical settings.

overall_design: ExRNA-seq libraries were prepared from platelet-poor plasma obtained from serial blood draws collected from two individuals undergoing bone marrow transplantation. A total of 11 samples were collected from each individual, starting prior to chemotherapy/ratiation treatment (approximately 7 days pre-HSCT) the day of transplant, and then weekly up to approximately Day 63.


title: Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks

status: Public on May 02 2019

city: Cleveland

first: Aura

last: Perez

address: 10900 Euclid Avenue

department: Pediatrics

institute: CWRU

summary: Individuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear. Using CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells.  Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR-) to directly address the role of CFTR in inflammatory gene regulation. All lines maintained CFTR mRNA production and formation of tight junctions.  CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR- lines did not.  Baseline expression of both cytokines was not different between the lines regardless of CFTR genotype.  All lines responded to TNFa and IL1b by increasing IL6 and CXCL8 (IL8) mRNA levels, but the CFTR- lines produced more CXCL8 mRNA than the CFTR+ lines.  Transcriptomes of 6 CFTR- and 6 CFTR+ lines, before and after stimulation by TNFa, were compared for differential expression as a function of CFTR genotype.  While some genes appeared to be differentially expressed simply because of CFTR’s absence, others required stimulation for differences to be apparent. Together, these data suggest cells respond to CFTR’s absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation.  With regards to inflammation, these data suggest a model in which CFTR’s absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation.

overall_design: Compare cells with intact CFTR to cells lacking CFTR for overall gene expression under basal and TNFa-stimulated conditions

first: Shuyu

last: Hao

first: Mitchell

middle: L

last: Drumm


title: Common functional alterations identified in blood transcriptome of autoimmune cholestatic liver and inflammatory bowel diseases

city: Warszawa

first: Krzysztof

last: Goryca

address: Banacha 2c

department: CeNT

institute: Uniwersytet Warszawski

summary: Primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), are heterogeneous chronic autoimmune diseases that may share underlying pathogenic mechanisms. Herein, we compared simultaneously analyzed blood transcriptomes from patients with PBC, PSC, and IBD.

overall_design: Microarray-based measurements were conducted using RNA isolated from whole blood samples from 90, 45, 95 and 93 patients with PBC, PSC, CD, and UC, respectively, and 47 healthy controls.

first: Jerzy

last: Ostrowski

first: Izabella

last: Lazowska

first: Agnieszka

last: Rogowska

last: Paziewska

first: Michalina

last: Dabrowska

first: Filip

last: Ambrozkiewicz

first: Jakub

last: Karczmarski

first: Aneta

last: Balabas

first: Anna

last: Kluska

first: Magdalena

last: Piatkowska

first: Natalia

last: Zeber-Lubecka

first: Maria

last: Kulecka

first: Andrzej

last: Habior

first: Michal

last: Mikula


title: Phospho-small RNA-seq reveals circulating, extracellular mRNA/lncRNAs as potential biomarkers in human plasma:

first: Muneesh

last: Tewari

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct


title: Asthma IV

first: Jadranka

last: Milosevic

state: PA - Pennsylvania

address: 2330 Tilbury Avenue

summary: Bronchial Epithelial Cells were isolated processed as described (Chu et al., 2002 and Zhao et al., 2011). The objective of the study was to identify differentially expressed genes between normal control (NC), mild-moderate asmathic (notSA) and severe asthmatic (SA) patients.   For demographics data, contact Dr.Sally Wenzel (wenzelse@upmc.edu)

overall_design: Bronchoscopy with endobronchial epithelial brushing was performed as previously described (Chu et al., 2002; Zhao et al., 2011).  Bronchial alveolar lavage fluids was spin down on 4000 g for 10 minutes. 0.5- 1X10^6 cells were stored in Trizol for RNA extraction. RNA in Trizol solutionwas extracted using the QIACube system (Qiagen, Valencia, CA). RNA quality was determined using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA), and only samples with an RIN higher than 7 used for microarray experiments.

first: Sally

last: Wenzel

first: Naftali

last: Kaminski


title: Genome wide gene expression profiles of Drosophila metastatic cultured tumors

status: Public on Apr 30 2019

city: Sabadell

first: Antoni

last: Berenguer-LLergo

state: Barcelona

address: Parc del Taulí, 1

department: A8G2 Group - Rheumatology Service

country: Spain

institute: Parc Taulí Research and Innovation Institute (I3PT)

summary: Clones in the intestinal adult stem cell population mutant for Apc and Apc2, and over-expressing RasV12 and Snail are able to disseminate outside the gut and form macrometastases. These macrometastases can be cultured in allografts. This data shows the changes in gene expression profile of culturde tumors after 1, 2 and 10 (T1, T2, T10) rounds of allograft transplantation

overall_design: We induced clones in adult Drosophila guts mutant for Apc and Apc2 and over-expressing UASRasV12, UASSnail and UASGFP under the Esg-Gal4 driver. We selected 3 macrometastases located at the abodmen, outside the gut, from 3 different flies, and performed an allograft culture in adult flies. We obtained a sample for each allograft at the first (T1), secod (T2) and tenth (T10) round of allograft cultire.

first: Kyra

last: Campbell

first: Andreu

last: Casali


title: A microsporidia possesses robust transcriptional response to heat shock impacting diverse cellular processes despite lack of HSF

city: New York

first: Jonathan

middle: W.

last: Snow

state: New York

address: 3009 Broadway

country: USA

institute: Barnard College

summary: The majority of fungal species prefer the 12°–30° C range and relatively few species tolerate temperatures higher than 35° C . Our understanding of the mechanisms underpinning the ability of some species to grow at higher temperatures is incomplete. Nosema ceranae is an obligate intracellular fungal parasite that infects honey bees and can cause individual mortality and contribute to colony collapse. Despite a reduced genome, this species is strikingly thermotolerant, growing optimally at the colony temperature of 35° C. In characterizing the HSR in N. ceranae, we found that this and other microsporidian species have lost the transcriptional regulator HSF and possess a reduced set of putative core HSF1-dependent HSR target genes. Despite these losses, N. ceranae demonstrates robust upregulation of the remaining HSR target genes after heat shock. In addition, thermal stress leads to alterations in genes involved in various metabolic pathways, ribosome biogenesis and translation, and DNA repair. These results provide important insight into the stress responses of microsporidia. Such new understanding will allow new comparisons with other pathogenic fungi and potentially enable discovery of novel treatment strategies for microsporidia infections affecting food production and human health.

sample_organism:
Apis mellifera
Vairimorpha ceranae

platform_organism:
Apis mellifera
Vairimorpha ceranae

overall_design: Six samples at 2 temperatures

middle: W

first: Nora

middle: K

last: McNamara-Bordewick

first: Mia

last: McKinstry


title: Transcriptomic analysis of Vibrio sp. dhg in different sugar media.

city: San Diego

first: Bernhard

middle: O.

last: Palsson

state: California

address: 9500 Gilman Dr

department: Bioengineering

institute: University of California, San Diego

summary: Brown macroalgae holds an enormous potential as a future feedstock because it rapidly forms large biomasses and has high carbohydrate content (35% of its dry weight consists of alginate and mannitol). However, utilization of brown macroalgae by conventional microbial platforms (e.g., Escherichia coli and Saccharomyces cerevisiae) has been limited due to the inability of these platforms to metabolize alginate. Although recent studies engineered them to utilize alginate, their growth rates and metabolic activities are still too low for industrial applications, likely due to the unoptimized expression of multiple xenogeneic genes. Here, we isolated Vibrio sp. dhg, a novel, fast-growing bacterium that has been naturally evolved for efficient alginate assimilation (growth rate = 0.98 h-1). Especially, both the growth rate and sugar uptake rate of V. sp. dhg are substantially higher than the rates of E. coli for most biomass-derivable sugars. Based on our systematic characterization of its metabolism and gene expression architecture, we were able to develop a genetic toolbox for its engineering. By using this microorganism, we successfully demonstrated its ability to produce a broad spectrum of chemicals from alginate-mannitol mixtures with high productivities (1.1 g ethanol/L/h, 1.3 g 2,3-butanediol and acetoin/L/h, and 0.69 mg lycopene/L/h). Collectively, the V. sp. dhg strain is a powerful platform for the conversion of brown macroalgae sugars whose usage will dramatically accelerate the production of value-added biochemicals in the future.

overall_design: Vibrio sp. dhg strain was cultivated in a minimal medium supplemented in glucose, alginate, or mannitol as a sole carbon source.

first: Hyun-Gyu

last: Lim

first: Sungwoo

last: Park

first: Sang-Woo

last: Seo

first: Gyoo-Yeol

last: Jung


title: The Hox Transcription Factor Ubx stabilizes Lineage Commitment by Suppressing Cellular Plasticity

city: Heidelberg

first: Katrin

last: Domsch

address: INF 230

department: COS - Developmental Biology

institute: Heidelberg University


title: Gene expression in three tissues from two crab-eating macaques (Macaca fascicularis, cynomolgus monkey).

city: Biberach a.d. Riss

first: Eric

last: Simon

address: Birkendorfer Str. 65

institute: Boehringer Ingelheim Pharma GmbH & Co KG

summary: We provide RNA-Seq data for cerebral cortex, liver and kidney cortex from two untreated female crab-eating macaques.

overall_design: Three tissues from two animals, without treatment

first: Julia

last: Söllner

first: Germán

last: Leparc

first: Matthias

last: Zwick

first: Tanja

last: Schönberger

first: Tobias

last: Hildebrandt

first: Kay

last: Nieselt


title: Effects of high fructose and high glucose on third instar larval fat body gene expression in Drosophila melanogaster

status: Public on Apr 26 2019

city: Binghamton

first: Laura

middle: Palanker

last: Musselman

address: 4400 Vestal Parkway East

institute: Binghamton University

summary: We characterized monosaccharide-dependent gene expression in the Drosophila fat body using fructose and glucose. Control and high-sugar diets were compared and RNA-seq was used to identify potential target genes.

overall_design: Drosophila were reared on control (0.3 M fructose or glucose) or high sugar (1.7 M fructose or glucose) diets until the wandering third instar stage.   Fat bodies were isolated and RNA was extracted to determine the effects of each sugar at different concentrations on gene expression using Illumina RNA-seq.

first: Thomas

middle: J

last: Baranski


title: Genome-wide essential gene identification for SubAB toxin-induced cell death using CRISPR/CAS9 screening system

city: Shinjuku-ku

first: Tsuyoshi

last: Sekizuka

state: Tokyo

address: 1-23-1 Toyama

department: Pathogen Genomics Center

institute: National Institute of Infectious Diseases

summary: Isolation of genes whose knockout confer resistance to SubAB-Induced cell death

overall_design: A genome-wide CRISPR/CAS9 knockout screen in HeLa cells was performed to identify host factors involved in the regulation of glycan biosynthesis by exploiting SubAB sensitivity. Lentivirus-based GeCKO v2 pooled library, which is delivered as two half-libraries (A and B), was used. Two independent sgRNAs-expressing cell libraries (A-1, A-2, B-1, B-2) were prepared by transducing the lentivirus libraries, and cells were then treated with SubAB to assess toxicity. The sgRNAs integrated into the cellular genomes of the surviving cells were amplified by PCR and analyzed with high-throughput sequencing. For untreated controls, cells in each cell library were cultured for the same period as SubAB-treated cells with passages several times. sgRNA profiles of control and SubAB-treated HeLa cell were generated by deep sequencing, in biological replicate 2, using Illumina MiSeq.

first: Toshiyuki

last: Yamaji

first: Makoto

last: Kuroda


title: Gene differential expression after cold acclimation in D. suzukii

status: Public on Apr 25 2019

city: Rennes

last: Enriquez

state: Bretagne

address: 263 av. du Général Leclerc

institute: Université de Rennes1

summary: RNA sequencing revealed differential expression of a large cluster of genes related to ion transport, signaling and oogenesis. These processes are probably key mechanisms underlying cold tolerance in D. suzukii.

overall_design: comparison of 3 replicates of ctrl (COF) x 3 replicates of cold acclimated flies (CAF). 15 females per replicate, whole body

first: Hervé

last: Colinet


title: A comprehensive single cell transcriptional landscape of human hematopoietic progenitors

city: Boston

first: Luca

last: Biasco

state: MA

address: 1 Jimmy Fund Way

institute: Dana-Farber/Boston Children’s Cancer and Blood Disorders Center

summary: Hematopoietic Stem/Progenitor cells (HSPCs) are endowed with the role of maintaining a diverse pool of blood cells throughout the human life. Despite recent efforts, the nature of the early cell fate decisions remains contentious. Using single-cell RNA-Seq, we show that existing approaches to stratify bone marrow CD34+ cells reveal a hierarchically-structured transcriptional landscape of hematopoietic differentiation. Still, this landscape misses important early fate decisions. We here provide a broader transcriptional profiling of bone marrow lineage negative hematopoietic progenitors that recovers a key missing branchpoint into basophils and expands our understanding of the underlying structure of early adult human haematopoiesis. We also show that this map has strong similarities in topology and gene expression to that found in mouse. Finally, we identify the sialomucin CD164, as a reliable marker for the earliest branches of HSPCs specification and we showed how its use can foster the design of alternative transplantation cell products.

overall_design: Single-cell mRNA sequencing of freshly isolated hematopoietic progenitors from human bone marrow. Sample HSC (Donor A) represents 1282 single cells. Sample MPP (Donor A) represents 215 single cells. Sample MLP (Donor A) represents 123 single cells. Sample PreB/NK (Donor A) represents 592 single cells. Sample MEP (Donor A) represents 1211 single cells. Sample CMP (Donor A) represents 1576 single cells. Sample GMP (Donor A) represents 1012 single cells. Sample Lin-CD34+CD164+ (Donor B) represents 6343 single cells. Sample Lin-CD34-CD164high (Donor B) represents 4434 single cells. Sample Lin-CD34lowCD164high (Donor B) represents 4266 single cells. Sample Lin-CD34-CD164low (Donor B) represents 358 single cells.

first: Danilo

last: Pellin

first: Mariana

last: Loperfido

first: Cristina

last: Baricordi

first: Samuel

last: Wolock

first: Annita

last: Montepeloso

first: Olga

last: Weinberg

first: Alessandra

last: Biffi

first: Allon

last: Klein


title: Genome-wide binding of the meiosis specific Rec8 cohesin subunit in metaphase I

city: Edinburgh

first: Daniel

last: Robertson

state: Midlothian

address: 2.28 Michael Swann Building, Kings Buildings, Mayfield Road

department: Discovery Research Platform for Hidden Cell Biology

country: United Kingdom

institute: University of Edinburgh

summary: ChIP-seq profile of Rec8 binding sites arrested in metaphase I by depletion of the APC/C activator CDC20. The aim of this experiment is to identify Rec8 binding sites in metaphase I.

overall_design: Diploid S. cerevisiae SK1 cells were constructed containing Rec8-3Ha in an pGAL1-NDT80 pGPD1-GAL4.ER pCLB2-CDC20 background. Cells were allowed to sporulate in sporulation media for 5 hours before addition of β-estradiol to release from prophase arrest. Cells were grown for 75min to arrest in metaphase I after which formaldehyde was added to the cultures and cells were fixed for 30min. We chose this double-arrest strategy (with a prophase I arrest followed by a metaphase I arrest) to ensure efficient arrest in metaphase I, which is less robust in cells without Spo13. Samples were subsequently processed according to the standard lab ChIP-Seq protocol.

first: Stefan

last: Galander

first: Adele

last: Marston


title: Genome-wide binding of the meiosis I-specific Spo13 in the presence and absence of cohesin

summary: ChIP-seq profile of Spo13 binding sites arrested in prophase I by deletion of the meiotic transcription factor NDT80. Previous ChIP-ChIP data had indicated that Spo13 binding correlates with cohesin binding sites. Therefore, in addition to wild type cells, we also investigated cells deficient of the meiosis-specific cohesin subunit Rec8 to identify cohesin-dependent binding sites of Spo13.

overall_design: Diploid S. cerevisiae SK1 cells were constructed containing Spo13-3Flag in an ndt80Δ background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 30min. Samples were then processed according to the standard lab ChIP-Seq protocol.


title: Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility

first: Caleb

last: Lareau

address: 417 E 68th St, Zuckerman - ZRC 1132

institute: Memorial Sloan Kettering

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

overall_design: Refer to individual Series


title: Characterization of induced susceptibility effects on soybean- soybean aphid interactions

city: Brookings

first: Madhav

middle: P.

last: Nepal

state: South Dakota

address: Northern Plains Biostress 249D

department: Biology and Microbiology

institute: South Dakota State University

summary:
Purpose: Soybean aphid (Aphis glycines Matsumura; SBA) is major pest of soybean (Glycine max) in the United States of America. One previous study on soybean, soybean-aphid interactions showed that avirulent (biotype 1) and virulent (biotype 2) biotypes can co-occur and potentially interact on resistant and susceptible soybean resulting induced susceptibility. The main objective of this research was to employ RNA sequencing technique to characterize the induced susceptibility effect in which initial feeding by virulent aphids can increase the suitability of avirulent aphids in both susceptible and resistant cultivars.
Methods: The data in this submission come from the green house experiment using two genotypes of soybean: susceptible soybean cultivar was LD12-15838R and the resistant cultivar was LD12-15813Ra (with Rag1 gene) and two aphid populations: biotype 1 (avirulent) and biotype 2 (virulent biotype 2). RNA was extracted from the leave samples from resistant and susceptible cultivars treated with no aphids, biotype 2: biotype1 collected at day 1 and no aphids, biotype 2: biotype1 and no aphids: biotype1 at day 11 using PureLink RNA mini kit (Invitrogen, USA). RNA samples were treated with TURBOTM DNase (Invitrogen, USA) to remove any DNA contamination following the manufacturer’s instructions. Assessment of the isolated RNA integrity was performed by 1% agarose gel electrophoresis, and RNA concentration was measured by Nanodrop 2000 (Thermo Fisher Scientific, USA). Three replicates from these treatments in resistant and susceptible cultivars were pooled in equimolar concentration. RNAseq library construction was prepared using Illumina’s TruSeq Stranded mRNA Kit v1 (San Diego, CA). The libraries were quantified by QuBit dsDNA HS Assay (Life Technologies, Carlsbad, CA) and pooled in equimolar concentrations. The libraries were sequenced on an Illumina NextSeq 500 using a NextSeq 500/550 High Output Reagent Cartridge v2 (San Diego, CA) at 75 cycles.
Results: A total of 10 RNA libraries were prepared and sequenced with the sequencing depth ranging from 24,779,816 to 29,72,4913. Total reads of 266,535,654 were subjected to FastQC analysis to determine the data quality using various quality metrics such as mean quality scores, per sequence quality scores, per sequence GC content, and sequence length distribution. The phred quality scores per base for all the samples were higher than 30. The GC content ranged from 45 to 46% and followed the normal distribution. After trimming, more than 99% of the reads were retained as the clean and good quality reads. Upon mapping these reads, we obtained high mapping rate ranging from 90.4% to 92.9%. Among the mapped reads, 85.8% to 91.9% reads were uniquely mapped.
Conclusions: The objective of this study is to characterize the mechanism of induced susceptibility in soybean via transcriptional response study of soybean in presence of biotype 1 and biotype 2 soybean aphids using RNA-Seq. The data resulted from this study might provide insights into the interactions between soybean and soybean aphids and identify genes, their regulation and enriched pathways that may be associated with resistance or susceptibility to A. glycines.

first: Adam

last: Varenhorst

first: Surendra

last: Neupane


title: Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis

status: Public on Apr 24 2019

city: Aachen

first: Oliver

last: Krenkel

address: Pauwels Str. 30

institute: University Hospital Aachen

summary:
Objective: Bone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets, and dietary overnutrition may affect macrophages in liver and bone marrow. We therefore aimed at characterizing in-depth the functional adaptations of myeloid cells in fatty liver.
Design: We employed single-cell RNA-sequencing to comprehensively assess the heterogeneity of myeloid cells in liver and bone marrow during NAFLD, by analyzing C57BL/6 mice fed with a high-fat, high-sugar, high-cholesterol "Western diet" for 16 weeks. We also characterized NAFLD-driven functional adaptations of macrophages in vitro and their functional relevance during steatohepatitis in vivo.

overall_design:
Liver myeloid leukocytes and their bone marrow precursors adapt a common and functionally relevant inflammatory signature during NAFLD progression.
Bone marrow derived macrophages were cultivated for 7 days followed by 100 ng/ml LPS stimulation. Macrophages were preincubated for the first 4 days with and without 10 mM fatty acid (palmitic and oleic acid 1:3).  ND=standard chow diet and WD=Western diet.

first: Jana

last: Hundertmark

first: Ali

middle: T

last: Abdallah

first: Marlene

last: Kohlhepp

first: Tobias

last: Puengel

first: Tilman

last: Roth

first: Diogo

last: Branco

middle: C

last: Mossanen

first: Tom

last: Luedde

first: Christian

last: Trautwein

first: Ivan

middle: G

last: Costa

first: Frank

last: Tacke


title: The initiation of puberty in Atlantic salmon brings about large changes in testicular gene expression that are modulated by the energy status.

status: Public on Apr 29 2019

city: Bergen

first: Diego

last: Crespo

address: Nordnesgaten 50

department: Reproduction and Developmental Biology

country: Norway

institute: Institute of Marine Research

summary: When puberty starts before males reach harvest size, animal welfare and sustainability issues occur in Atlantic salmon (Salmo salar) aquaculture. Hallmarks of male puberty are an increased proliferation activity in the testis and elevated androgen production. Examining transcriptional changes in salmon testis during the transition from immature to maturing testes may help understanding the regulation of puberty, potentially leading to procedures to modulate its start. Since differences in body weight influence, via unknown mechanisms, the chances for entering puberty, we used two feed rations to create body weight differences. Maturing testes were characterized by an elevated proliferation activity of Sertoli cells and of single undifferentiated spermatogonia. Pituitary gene expression data suggest increased Gnrh receptor and gonadotropin gene expression, potentially responsible for the elevated circulating androgen levels in maturing fish. Transcriptional changes in maturing testes included a broad variety of signaling systems (e.g. Tgfβ, Wnt, insulin/Igf, nuclear receptors), but also, activation of metabolic pathways such as anaerobic metabolism and protection against ROS. Feed restriction lowered the incidence of puberty. In males maturing despite feed restriction, plasma androgen levels were higher than in maturing fish receiving the full ration. A group of 449 genes that was up-regulated in maturing fully fed fish, was up-regulated more prominently in testis from fish maturing under caloric restriction. Moreover, 421 genes were specifically up-regulated in testes from fish maturing under caloric restriction, including carbon metabolism genes, a pathway relevant for nucleotide biosynthesis and for placing epigenetic marks. Undifferentiated spermatogonia and Sertoli cell populations increased at the beginning of puberty, which was associated with the up-regulation of metabolic pathways (e.g. anaerobic and ROS pathways) known from other stem cell systems. The higher androgen levels in males maturing under caloric restriction may be responsible for the stronger up-regulation of a common set of (449) maturation-associated genes, and the specific up-regulation of another set of (421) genes. The latter opened regulatory and/or metabolic options for initiating puberty despite feed restriction. As a means to reduce the incidence of male puberty in salmon, however, caloric restriction seems unsuitable.

overall_design: Induced gene expression in Atlantic salmon testis tissue measured after exposure to normal (NR) and restricted ration (RR) feeding during the entry of puberty. In total, 37 samples were analyzed: 16 biological replicates from fish subjected to a NR and 21 biological replicates from fish subjected to a RR.

first: Jan

last: Bogerd

first: Elisabeth

last: Sambroni

first: Florence

last: LeGac

first: Eva

last: Andersson

first: Rolf

middle: B

last: Edvardsen

last: Bergman

first: Björn

last: Björnsson

first: Geir

last: Taranger

first: Rüdiger

last: Schulz


title: Single cell analysis of HSV-1 infection reveals anti-viral and developmental programs are activated in distinct sub-populations with opposite outcomes

city: Chicago

first: Nir

last: Drayman

state: IL

institute: University of Chicago

summary: Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, where most infected cells give rise to none or few viral progeny while some cells produce thousands. Analysis of HSV-1 infection by population averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming includes the re-localization of b-catenin into the host nucleus and viral replication compartments and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

overall_design: HDFn cells mock-infeted, infected with wt HSV-1 or dICP0 HSV-1 for 5 hours. drop-seq data for single-cell RNA-sequncing as well as bulk RNA-seq of sorted cell population (ICP4 positive or negative in each experimet, in duplicates)

first: Parthiv

last: Patel

first: Luke

last: Vistain

first: Savas

last: Tay


title: Switching from Random to Imprinted X-inactivation by Maternal PRC2

city: Ann Arbor

first: Sundeep

last: Kalantry

address: 1241 Catherine Street

department: Human Genetics

institute: University of Michigan

summary: Imprinted X-inactivation is a paradigm of transgenerational epigenetic inheritance in mammals and silences genes exclusively on the paternally-inherited X-chromosome.  Here, we test the hypothesis that oocyte-derived maternal Polycomb repressive complex 2 (PRC2) protein EED orchestrates the selective inactivation of the paternal-X in mouse embryos.  In maternal Eed null (Eedm-/-) but not zygotic Eed null female and male embryos, the maternal X-chromosome ectopically induced Xist lncRNA, which silences X-linked genes.  Subsequently, Xist was silenced from one of the two X-chromosomes in females and from the sole maternal-X in males.  As a result, later-stage female Eedm-/- embryos displayed random X-inactivation.  The kinetics of Xist RNA induction in Eedm-/- embryos resemble that of early human embryos, which lack oocyte-derived PRC2 and only undergo random X-inactivation.  Thus, expression of PRC2 genes in the oocyte and transmission of the gene products to the embryo may dictate the occurrence of imprinted X-inactivation in mammals.

sample_organism: Mus musculus domesticus x M. m. molossinus

overall_design: Allele-specific mRNA profile of Eedfl/fl, Eedfl/-, Eed-/-, Eedm-/-, and Eedmz-/- mouse E3.5 embryos by RNA-Seq

first: Clair

last: Harris

first: Marissa

last: Cloutier

first: Megan

last: Trotter

first: Michael

last: Hinten

first: Srimonta

last: Gayen


title: Stromal Fibroblasts Drive Single Cell Heterogeneity in Pancreatic Cancer

first: David

last: Ting

address: 149 E 13th street

department: Cancer Center

institute: Massachusetts General Hospital

summary: To understand the interplay between cancer and stroma, we performed single cell RNA-sequencing of PDAC cells admixed with stromal fibroblasts and defined different single cell populations with varying levels of proliferative and metastatic transcriptional states. PDAC cell behavior in vitro and in vivo on these phenotypic axes could be tuned with the proportion of stromal fibroblasts. These cell types were identified in human pancreatic tumors, and specific subpopulations were associated with worsened outcomes.

overall_design: 92 single PDAC cells and 92 single CAF cells were micromanipulated and prepared for sequencing (23 of each cell type from four culture ratios). The 24th sample from each cell type-culture condition combination is a population control obtained by micromanipulating 100 cells of the given type from the given culture condition and preparing it as if it were a single cell, giving a total of 96 PDAC samples and 96 CAF samples. During the course of library construction, 3 samples were lost, all PDAC cells from the 30:70 condition (two single cells and the population control), leaving 93 total PDAC samples and 96 total CAF samples.

first: Matteo

last: Ligorio

first: Srinjoy

last: Sil

first: Sandra

last: Misale

first: Murat

last: Karabacak

first: Nicole

middle: V

last: Jordan

first: Niyati

last: Desai

first: Kshitij

middle: S

last: Arora

first: Anupriya

last: Kulkarni

first: Mihir

last: Rajurkar

first: Mauro

last: Di Plato

first: Myriam

last: Boukhali

first: Jackson

middle: P

last: Fatherree

first: Eric

last: Tai

first: Kevin

middle: D

last: Vo

first: Leah

last: Damon

first: Kristina

last: Xega

first: Rushil

first: Melissa

last: Choz

first: Francesca

last: Bersani

first: Vishal

last: Thapar

first: Miguel

middle: N

last: Rivera

first: Vikram

last: Deshpande

first: Cyril

last: Benes

first: Shyamala

last: Maheswaran

first: Daniel

middle: A

last: Haber

first: Carlos

last: Fernandez-Del Castillo

first: Cristina

middle: R

last: Ferrone

first: Wilhelm

last: Haas

first: Martin

last: Aryee


title: Genome-wide nucleosome positioning maps from undifferentiated human iPS cells (hIPS) and iPS cells differentiated to neural progenitor cells (NPC).

city: Cardiff

first: Adrian

middle: J.

last: Harwood

address: Hadyn Ellis Building, Maindy Road,

department: Neuroscience & Mental Health Research Institute

institute: Cardiff University

summary: We report the application of MNase-seq (Kent, Adams et al. 2011) to construct genome-wide nucleosome maps from human cells. To date, genome-wide changes in chromatin structure that occur during development in human cells have not been investigated widely. We have constructed and compared genome-wide chromatin maps from undifferentiated human iPS cells and and iPS cells differentiated to neural progenitor cells (NPC).

overall_design: Examination of nucleosome positioning in human undifferentiated iPS cells and neuro-projenitor cells.

first: Janet


title: Transcriptome comparison of white adipose tissue between control and adipose specific HuR knockout mice

status: Public on Apr 23 2019

city: Jinan

first: Wencheng

last: Zhang

state: Shandong

address: Wenhua Xi Road No.44

department: cardiology

institute: Qilu hospital

summary: Human antigen R (HuR) is a member of the Hu family of RNA-binding proteins and is involved in many physiological processes. To investigate the role of adipose HuR, we generate adipose-specific HuR knockout (HuRAKO) mice. As compared with control mice, HuRAKO mice show obesity when induced with a high-fat diet, along with insulin resistance, glucose intolerance, hypercholesterolemia and increased inflammation in adipose tissue. The obesity of HuRAKO mice is attributed to adipocyte hypertrophy in white adipose tissue due to decreased expression of adipose triglyceride lipase (ATGL), a critical lipase involved in lipolysis. HuR positively regulates ATGL expression by promoting the mRNA stability and translation of ATGL. Consistently, the expression of HuR in adipose tissue is reduced in obese humans, which is associated with reduced ATGL expression. This study suggests that adipose HuR may be a critical regulator of ATGL expression and lipolysis and thereby controls obesity and metabolic syndrome.

overall_design: Adipose tissue mRNA profiles of control and adipose specific HuR knockout mice after 16 weeks high-fat diet feeding were generated by deep sequencing,in triplicate, using Illumina HiSeq 4000.

first: Jingyuan

last: Li


title: Analysis of possible mechanisms behind functional recovery following neural progenitor cell transplantation into spinal cord injury.

city: Stockholm

first: Mikael

last: Svensson

state: Stockholm

address: Solnavägen 30

department: Clinical neuroscience

institute: Karolinska Institutet

summary: Long-term survival and integration of neural progenitor cells (NPCs) transplanted following spinal cord injury (SCI) have been observed. However, questions concerning the differentiation choice of NPCs, their mechanism of action and their contribution to functional recovery remains unanswered. Therefore, we investigated global transcriptomal changes in transplanted NPCs transplanted into SCI. We found that NPCs transplanted into SCI up-regulate genes related to nerve cell function and signaling and differentiation, and down-regulate genes related to cytokine signaling and apoptosis.

overall_design: Stem cells transplantation in spinal cord injury in rodents

first: Sreenivasa

last: Sankavaram

first: Ramil

last: Hakim

first: Arvid

last: Frostell

first: Ruxandra

last: Covacu

first: Susanne

last: Neumann

first: Lou

last: Brundin


title: Genome-wide binding of the cohesin protector Sgo1 in the presence and absence of the meiosis I-specific Spo13 protein

last: Robertson

summary: ChIP-seq profile of Sgo1 binding sites arrested in metaphase I by deletion of the APC/C activator CDC20. The aim of this experiment is to identify whether Sgo1 binding differs in wild type cells and cells deficient of the meiosis I-specific protein Spo13, which is required for retention of pericentromeric cohesin after metaphase I.

overall_design: Diploid S. cerevisiae SK1 cells were constructed containing Sgo1-6Ha in an pGAL1-NDT80 pGPD1-GAL4.ER pCLB2-CDC20 background. Cells were allowed to sporulate in sporulation media for 5 hours before addition of β-estradiol to release from prophase arrest. Cells were grown for 75min to arrest in metaphase I after which formaldehyde was added to the cultures and cells were fixed for 30min. We chose this double-arrest strategy (with a prophase I arrest followed by a metaphase I arrest) to ensure efficient arrest in metaphase I, which is less robust in cells without Spo13. Samples were subsequently processed according to the standard lab ChIP-Seq protocol.

first: Stefan

last: Galander

first: Adele

middle: L

last: Marston


title: Reductional meiosis I chromosome segregation is established by coordination of key meiotic kinases

first: Daniel


title: Identification of potential biomarkers for diagnosis of pancreatic and biliary tract cancers by sequencing of serum microRNAs

city: Seoul

first: Kwondo

last: Kim

address: 1 Gwanak-ro, Gwanak-gu

institute: Seoul National University

summary: We profiled expression of serum miRNAs derived from pancreatic and biliary tract cancer patients and detected biomarker candidates using high-throughput sequencing

overall_design: 21 pancreatic cancer patients, 10 biliary tract cancer patients, and 24 healthy controls

first: Si Young

last: Song


title: Novel Genes and Metabolite Trends in Bifidobacterium longum ssp. infantis Bi-26 Involved in Metabolism of Human Milk Oligosaccharide 2’-fucosyllactose

city: Madison

first: Wesley

middle: w.

last: Morovic

state: WI

address: 3329 Agriculture Dr.

department: Genomics & Microbiome Science

institute: DuPont Nutrition & Health

summary: Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between Bifidobacterium utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2’-fucosyllactose (2’-FL) in Bifidobacterium longum ssp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600=0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2’-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2’-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded 2’-FL metabolism is a complex process involving gene clusters throughout the genome producing more metabolites compared to lactose. These results provide valuable insight on the mode-of-action of 2’-FL utilization by Bifidobacterium longum ssp. infantis Bi-26.

overall_design: Bifidobacterium longum subsp. infantis strain Bi-26 was used in this study with 2'-FL as the test carbon source and lactose as the control carbon source, Samples were done in duplicate with samples taken at early phase (A600 ~0.2) mid log (0.5-1.1) and late log (1.1+)

first: Bryan

last: Zabel

first: Paige

last: Roos


title: Gene expression profiling of neural crest progenitor cultures derived from human embryonic stem cells carrying nonsense mutations in the Polycomb gene ASXL1

city: München

first: Friederike

last: Matheus

address: Ingolstädter Landstrasse 1

department: Institute for Stem Cell Research

institute: Helmholtz Center Munich


title: Bulk RNA-sequencing of spinocerebellar tract neuron populations in cervical and thoracic sections of mouse spinal cord

status: Public on Apr 18 2019

city: New York

first: Vilas

last: Menon

state: NY

department: Neurology

institute: Columbia University Medical Center

summary: We profiled populations of mouse spinocerebellar tract neurons (SCTNs) in cervical and thoracic sections of mouse spinal cord, as defined by retrograde projection labeling. For each sample, SCTNs were labeled by injecting CTB (Alexa555 conjugated form, 1µg/µl in PBS) into  the cerebellum using NanojetII at P4 and examined at P6-P7.  Labeled SCTNs were collected manually from 150-300µm transverse spinal cords slices were generated using a razor blade. Cells were collected from each injection experiment and then profiled using standard Illumina bulk RNA-seq protocols

overall_design: We originally collected 4 biological replicates (from different mice) each of cervical and thoracic SCTN populations. For the cervical population samples, we collected 185, 182, 179, and 178 cells respectively. For thoracic population samples, we collected 310, 305, 473, and 341 cells respectively. Alignment and quantification was carried out using the STAR aligner with default parameters

first: Myungin

last: Baek