title: Identification of Tissue Specific DNA Methylation Signatures for Thyroid Nodule Diagnostics

city: Duarte

first: Xiwei

last: Wu

address: 1500 E Duarte Rd

department: Molecular and Cellular Biology

institute: Beckman Research Institute

type: Methylation profiling by high throughput sequencing

summary:
PURPOSE: Thyroid cancer is frequently difficult to diagnose due to an overlap of cytological features between malignant and benign nodules. This overlap leads to unnecessary removal of the thyroid in patients without cancer. While providing some improvement over cytopathologic diagnostics, molecular methods frequently fail to provide a correct diagnosis for thyroid nodules. These approaches are based on the difference between malignant nodules and normal adjacent thyroid tissue and assume that normal thyroid tissues are the same as benign nodules. However, in contrast to normal thyroid tissues, benign thyroid nodules can contain genetic alterations that can be found in cancerous nodules.
PATIENTS AND METHODS: For the development of a new molecular diagnostic test for thyroid cancer, we evaluated DNA methylation in 109 thyroid tissues by using genome wide single base resolution DNA methylation analysis (Reduced Representation Bisulfite Sequencing). The test was validated in the retrospective cohort containing 64 thyroid nodules.
RESULTS: By conducting Reduced Representation Bisulfite Sequencing in 109 thyroid specimens, we found significant differences between normal tissue, benign nodules, and cancer. Based on tissue-specific epigenetic signatures for benign and malignant nodules, we developed a new epigenetic approach for thyroid diagnostics. According to the validation cohort, our test has an estimated specificity of 97% (95% CI, 80 to 100), sensitivity of 100% (95% CI, 86 to 100), PPV of 97% (95% CI, 82-100), NPV of 100% (95%, 85 to 100).
CONCLUSION: These data show that epigenetic testing can provide outstanding diagnostic accuracy for thyroid nodules by evaluating tissue specific DNA methylation.

overall_design:
Bisulfite-Seq: DNA methylation analysis by RRBS in bening and malignant nodules and thyroid adjacent tissues

ChIP-Seq: Analysis of H3K27Ac pattern by using ChIP-sequencing in thyroid adjacent tissues

last: Hahn

first: John

last: Yim

first: Yuman

last: Fong

first: Gerd

last: Pfeifer

first: Audrey

last: Choi

first: Arthur

middle: X

last: Li

first: Hanjun

last: Qin

first: Sue

last: Chang

first: Sun-Wing

last: Tong

first: Peiguo

last: Chu

first: Byung-Wook

last: Kim

first: Ryan

last: Lew

first: Yasmine

last: Ibrahim

first: Valeriy

last: Poroyko

first: Jinhui


title: The role of Gch1 in the macrophage response to LPS

status: Public on Nov 30 2018

city: Oxford

first: Eileen

last: McNeill

address: Wellcome Trust Centre for Human Genetics, Roosevelt Drive

department: Radcliffe Department of Medicine

country: United Kingdom

institute: University of Oxford

summary: We used microarrays to detail the global program of gene expression induced in LPS/IFNgamma stimulated macrophages that were deficient in BH4/Gch1 (GCHfl/flTie2cre vs. GCHfl/fl) over a 24 hour timecourse.

overall_design: Bone Marrow-Derived Macrophages (BMDM) were cultured from GCHfl/fl and GCHfl/flTie2cre bone marrow. Differentiated macrophages from 3 animals per genotype were stimulated with LPS in the presence of interferon-gamma and samples were taken for analysis at t=0, 2hrs, 8hr and 24hrs post stimulation.

first: Keith

middle: M

last: Channon


title: DNA methylation fingerprint of a KDM5C loss-of-function mutation in patients with intellectual disability

city: Campinas

first: Mariana

last: Maschietto

state: São Paulo

address: Rua Márcia Mendes, 619

department: Research Center

country: Brazil

institute: Boldrini Children's Hospital

type: Methylation profiling by genome tiling array

summary:
Characterization of methylation changes related do KDM5C c.807delC (p.V271X)
Mutations in KDM5C (lysine (K)-specific demethylase 5C) were associated to up to 3% of the X-linked ID cases. Males carrying KDM5C mutations present reduced activity of the KDM5C protein, increasing H3K4 tri-methylation, which ultimately leads to up-regulation of KDM5C target genes. Whole-exome sequencingi n one of three brothers with intellectual disability  followed by confirmation by Sanger sequencing in the family identified a frameshift mutation in the KDM5C exon 7 that devoid of the JmjC catalytic domain  from the protein. Two other brothers also presenting intellectual disability as well as in their clinically normal mother, who had completely skewed X-inactivation also had the mutation. An empirical Bayesian analysis of methylation levels between cases and controls identified 399 differentially methylated positions, 288 hypomethylated and 111 hypermethylated.Among the genes associated with the hypomethylated CpGs were FBXL5 and CACYBP, both involved in the ubiquitination pathway.

overall_design: Subjects (3 affected brothers and non-affected mother carrying KDM5C mutation) and controls (2 female relatives and 12 male controls) were subjected to sequencing and genome-wide methylation analyses (450K, Illumina)

first: Joao

middle: V

last: da Silva Guerra

first: Angela

last: Morgante


title: Glatiramer Acetate modulates ion channel expression and calcium homeostasis in B-cell of patients with relapsing-remitting multiple sclerosis

city: Pozzuoli

first: Rossella

last: De Cegli

address: Via Campi Flegrei 34

country: Italy

institute: Tigem

summary:
In order to investigate the effects of Glatiramer acetate (GA) in treatment-naïve RR-MS female patients’ B cells we performed Affymetrix Gene-Chip Human Genome HG-U133A_2  hybridization experiments
Transcriptome analysis before and after acute (six hours in vitro) and chronic (six months in vivo) treatment with GA. We compared the transcriptional profiles of B cells, from the above-mentioned patients, treated or not with GA for 6 hours in vitro, and before and after six months of GA treatment in vivo.

overall_design:
For the analysis, total RNA was extracted from B cells from all patients, treated or not with GA for 6 hours in vitro, and before and after six months of GA treatment in vivo.

Patient ID is represented as "Xn" in the sample titles. Technical replicates were performed for most samples. Technical replicates were produced by splitting cells from the same donor/treatment into two sets which were each used for independent RNA extraction.

first: Chiara

last: Criscuolo


title: Patients with Congenital Ichthyosis and TGM1 Mutations Overexpress Other ARCI Genes in the Skin: Part of a Barrier Repair Response?

city: Uppsala

first: Hans

last: Törmä

address: Dag Hammarskjölds väg 20

department: Dept of Medical Sciences

country: Sweden

institute: Uppsala University

summary: Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of monogenic skin disorders caused by mutations in any of >10 different genes, many of which are involved in epidermal synthesis of ω-O-acylceramides (acylCer), an essential precursor of the corneocyte lipid envelope that is also dependent on transglutaminase-1 for normal skin barrier formation.  We hypothesized that inactivating TGM1 mutations, the most common cause of ARCI, might lead to a compensatory overexpression of transcripts involved in barrier repair, including ARCI-causing genes. Using microarray we examined the global mRNA expression profile in skin biopsies from five ARCI-patients with TGM1 mutations and four healthy controls. There were a total of 602 differentially expressed genes (adjusted P<0.05). Gene ontology analysis showed enrichment of mRNA encoding proteins associated with biological pathways mainly involved in keratinocyte differentiation and adaptive/innate immune response. Moreover, among non-syndromic ARCI-causing genes, seven out of twelve were significantly increased (log2fold -change=0.98-2.05). Four genes causing syndromic ichthyosis and seven other genes involved in biosynthesis of fatty acyl-CoA and ceramides were also significantly affected. This study reveals upregulation of several ichthyosis-causing genes in the skin of patients with TGM1 mutations, indicating a compensatory induction of acylCer biosynthesis as a part of a barrier repair mechanism.

overall_design: Skin biopsies were obtained at the buttocks of ichthyosis patients with TGM1 mutations (n=5) and healthy controls (n=4).

first: Hanqian

last: Zhang

first: Maja

last: Ericsson

first: Simone

last: Weström

first: Anders

last: Vahlquist

first: Marie

last: Virtanen


title: Mechanistic Model-Guided Study of Embryonic Morphogenesis

status: Public on Nov 29 2018

city: Jerusalem

first: Avner

last: Ehrlich

address: Silberman 3-512, Edmond J. Safra Campus (Givat Ram)

department: Bioengineering

country: Israel

institute: The Hebrew University of Jerusalem

type: Expression profiling by high throughput sequencing

summary: We utilize a finite element model of implantation mechanics to guide the in vitro micropatterning human embryonic stem cells to simulate in vivo stress gradients that form during development. Micropatterned stem cells showed early formation of GATA4+/SOX17+ primitive endoderm due to migration toward regions of high stress, while NANOG+/OCT4+ epiblast was retained in regions of low stress under spontaneous differentiation.

overall_design: Recently, Blakeley and colleagues used single cell RNA-Seq of human blastocysts to identify genes that are differentially expressed between primitive endoderm and epiblast cells. RNA-Seq analysis was used to show that our NANOG+/OCT4+ cells (stress-low region) cluster with epiblast markers. while GATA6+/SOX17+ cells (stress-high region) cluster with primitive endoderm markers. We also utalized these results to compare the GSEA to GSEA of late blastocyst data.

first: Yaakov

last: Nahmias

first: Sabina

last: Tsytkin-Kirschenzweig

first: Yoel

last: Goldstein

first: Matan

last: Hofree


city: Guangzhou

first: Shanzhou

last: Huang

address: No. 58 Zhongshan Second Road

country: China

institute: First Affiliated Hospital of Sun Yat-sen University

summary: To study the mRNA modulating network and different expressed mRNA lists in MHCC-97H-SH-UCK2 cells, we use high-throughout method to genomically detect the expression profile of mRNA

overall_design: mRNA expression profile in MHCC-97H-SH-NC And MHCC-97H-SH-UCK2 liver cancer cells.  Illumina PE150 for mRNA expression profile were chosen. The high-throughout experiment service are supplied by novogene (Beijing, China).


title: The HDAC3 Enzymatic Activity Regulates Skeletal Muscle Fuel Metabolism

city: Houston

first: Shiyang

last: Song

state: Texas

address: 2111 Holly Hall ,Apt.2206

department: Medicine

country: USA

institute: Baylor College of Medicine

summary: We address whether the functions of HDAC3 in skeletal muscle require its enzyme activity. By mutating the NCoR/SMRT corepressors in a knock-in mouse model named NS-DADm, we ablated the enzymatic activity of HDAC3 without affecting its protein levels. Compared to the control mice, skeletal muscles from NS-DADm mice showed lower force generation, enhanced fatigue resistance, enhanced fatty acid oxidation, reduced glucose uptake during exercise, upregulated expression of metabolic genes involved in branched-chain amino acids (BCAAs) catabolism, and aging-associated reduction in muscle mass, without changes in the muscle fiber type composition or mitochondrial protein content. These findings demonstrate that the metabolic function of HDAC3 in skeletal muscles requires its enzymatic activity.

overall_design: examination of 3 different samples in each genotype (WT and NS-DADm)

first: Zheng

last: Sun


title: A chemical probe for Tudor domain protein SPIN1 to investigate chromatin functions

first: Reshma

last: Nibhani

address: Windmill Road

department: NDORMS

summary: Lysine and arginine methylation are amongst the most frequent modifications on unstructured histone tails and in combination with other modifications provide the basis for a combinatorial 'chromatin or histone code'. Recognition of modified histone residues is accomplished in a specific manner by 'reader' domains that recognize chromatin modifications allowing to associate with specific effector complexes mediating chromatin functions. The methyl-lysine and methyl-arginine reader domain protein SPINDLIN1 (SPIN1) belongs to a family of 5 human genes, and has been identified as a putative oncogene and  transcriptional co-activator containing three Tudor domains, able to mediate chromatin binding. Here we report on the discovery of the potent and selective bivalent Tudor domain inhibitor VinSPINIn, which simultaneously engages Tudor domains 1 and 2 and effectively competes with chromatin binding. Inhibitor, chemoproteomic and knockdown studies in squamous cell carcinoma suggest an un-anticipated complexity of SPIN isoform mediated interactions in regulating cellular phenotypes.

overall_design: Triplicates for 2 compounds, 1 siRNA, DMSO and scrambled siRNA control at 2 time points are examined by RNA-Seq

first: Martin

last: Philpott

first: Adam

last: Cribbs

first: Udo

last: Oppermann


title: Identification of gene expression and splicing changes upon eye-specific downregulation of tri-snRNP components in Drosophila

city: Philadelphia

first: Iryna

last: Shakhmantsir

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: The spliceosome is a dynamic RNA-protein complex that executes pre-mRNA splicing and is composed of five core small nuclear ribonucleoprotein particles (U1, U2, U4/5/6 snRNP) and >150 additional proteins specific for each snRNP. We report a circadian role for Pre-mRNA Processing factor 4 (PRP4), a conserved component of the spliceosomal U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex. We broadly hypothesized that downregulation of prp4 led to the aberrant splicing of one or many of the core clock transcripts. To identify these splicing events in an unbiased way, we performed RNA-Sequencing (RNA-Seq) analysis. We reasoned that we could have a more targeted approach if we could zoom in on the overlapping splicing changes that would be driven by the knockdown of at least two different tri-snRNP components. Because the pan-neuronal knockdown of all tri-snRNP components tested in our study led to lethality, we decided to utilize an alternative broad driver. For that purpose, we selected a strong eye-specific Glass Multiple Promoter driver (GMR-Gal4). Because most of the signal from head lysates comes directly from the eye tissue and because the core splicing factors are ubiquitously expressed, GMR-specific downregulation of prp4 and prp8 promised to be a viable alternative to the pan-neuronal knockdown. We examined changes in both the total transcript levels and splicing events upon prp4 knockdown in the eye. The overall gene expression seemed to be dramatically influenced by prp4 downregulation (433 DOWN, 310 UP at FDR < 0.05). Despite the fact that PRP4 is a component of the core spliceosome that is required for constitutive exon splicing, we did not detect dramatic effects on global splicing. Only 45 genes exhibited differential alternate splicing upon prp4 downregulation at FDR < 0.05).

overall_design: 3 samples with 5 replicates each were analyzed using Illumina Next-Generation Sequencing (NextSeq 500).

first: Soumyashant

last: Nayak

first: Amita

last: Sehgal


title: 2-5A-Mediated mRNA Decay and Transcription Act in Concert to Reprogram Protein Synthesis during dsRNA Response

city: Princeton

first: Alexei

last: Korennykh

state: New Jersey

address: 216 Shultz Laboratory

department: Molecular Biology

institute: Princeton University

summary: RNA degradation by RNase L during 2-5A-mediated decay (2-5AMD) is a conserved mammalian stress response to viral and endogenous double-stranded RNA (dsRNA). To understand this mechanism we examined 2-5AMD in human cells by spike-in RNA-seq.

overall_design: Poly-IC transfection for 0-4 h followed by poly-A pulldown and RNAseq

first: Rath

last: Sneha

first: Prangley

last: Eliza

first: Korennykh

last: Alexei


title: IDH3a KO RNA-seq

city: Chicago

first: Yanrong

last: Ji

state: IL

address: Arthur J. Rubloff Building, 750 North Lake Shore Drive, 11th Floor

department: Feinberg School of Medicine

institute: Northwestern University

summary: RNA-seq of NHAs with either wild-type IDH3a expression (control) or IDH3a KO via CRISPR/Cas9

overall_design: 4 biological replicates were collected for both the wild type and KO cell lines. RNA was then extracted from these samples using the Qiagen RNeasy kit and sent to the University of Chicago for analysis

first: Jasmine

middle: L

last: May

first: Alexander

middle: H

last: Stegh


title: Analysis in melanomas and melanocytes from the same patient

city: Kobe

first: Susumu

last: Fujiwara

state: Hyogo

address: 7-5-1, Kusunoki-cho, Chuo-ku

department: dermatology

country: Japan

institute: Kobe univ

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

overall_design: Refer to individual Series


title: A microarray analysis of human epidermal keratinocytes upon depletion of the long non-coding RNA LOC100130476

city: Stockholm

first: Ning

last: Xu

address: CMM L8:02

institute: Karolinska Institutet

summary:
The lncRNA LOC100130476 (named as WAKMAR2) was found to be down-regulated in epidermal keratinocytes in human chronic non-healing wounds compared to normal acute wounds and the intact skin. However, its biological role in keratinocytes during wound repair has not been studied.
To study the genes regulated by WAKMAR2, we transfected Antisense LNA GapmeR against WAKMAR2  into human primary epidermal keratinocytes to deplete it.
We performed a transcriptome-wide gene expression level profiling of keratinocytes upon depletion of WAKMAR2 using Affymetrix arrays.

overall_design: Expression profiling of primary human epidermal keratinocytes transfected with 20nM WAKMAR2-specific GapmeR or 20nM GapmeR negative control for 24 hours (biological triplicates in each group) was performed using Affymetrix GeneTitan system.

first: Eva

middle: K

last: Herter


title: NEUROD1 dependent gene regulation in murine pancreatic endocrine cells and human stem cell derived insulin producing cells

city: New York

first: Anthony

last: Romer

state: NY

address: 1150 St. Nicholas Ave

institute: Columbia University

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: methylation analysis in melanomas and melanocytes from the same patient

summary:
DNA methylation is considered the primary epigenetic mechanism underlying the development of malignant melanoma. Since DNA methylation can be influenced by environmental factors, it is preferable to compare cancer and normal cells from the same patient. Here, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients and compared the methylation status with melanoma tissues from the same individuals. We also analyzed primary and metastatic melanoma samples, three commercially available melanocytes, and four melanoma cell lines. Cluster analysis of DNA methylation data classified freshly isolated melanomas and melanocytes into the same group, whereas the four melanoma cell lines were clustered together in a distant clade. Moreover, our analysis discovered methylation at several novel loci (KRTCAP3, AGAP2, ZNF490), in addition to those identified in previous studies (COL1A2, GPX3); however, the latter two were not observed in fresh melanoma samples. Subsequent studies revealed that NPM2 was hypermethylated and downregulated in melanomas, which was consistent with previous reports and indicated that NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease. Our results highlight the importance of using matched fresh melanoma and melanocyte samples in epigenetic studies.
Illumina Infinium 450k Human Methylation Beadchip

overall_design: total 19 samples, 8 melanocytes and 11 melanomas.methylation were performed on 5 cultured melanocytes, 7 melanomas (giving 4 matched donor pairs), 4 commercial melanoma cell lines and 3 commercial melanocyte cell lines.

first: Hiroshi

last: Nagai

first: Haruki

last: Jimbo

first: Naoe

first: Tomoyo

last: Tanaka

first: Masukazu

last: Inoie

first: Chikako

last: Nishigori


title: The soil organic matter decomposition mechanisms in ectomycorrhizal fungi are tuned for liberating soil organic nitrogen

city: Lund

first: Anders

last: Tunlid

address: Sölvegatan 37

department: Biology

institute: Lund University

summary: Many trees form ectomycorrhizal symbiosis with fungi. During symbiosis, the tree roots supply sugar to the fungi in exchange for nitrogen, and this process is critical for the nitrogen and carbon cycles in forest ecosystems. However, the extents to which ectomycorrhizal fungi can liberate nitrogen and modify the soil organic matter and the mechanisms by which they do so remain unclear since they have lost many enzymes for litter decomposition that were present in their free-living, saprotrophic ancestors. Using time-series spectroscopy and transcriptomics, we examined the ability of two ectomycorrhizal fungi from two independently evolved ectomycorrhizal lineages to mobilize soil organic nitrogen. Both species oxidized the organic matter and accessed the organic nitrogen. The expression of those events was controlled by the availability of glucose and inorganic nitrogen. Despite those similarities, the decomposition mechanisms, including the type of genes involved as well as the patterns of their expression, differed markedly between the two species. Our results suggest that in agreement with their diverse evolutionary origins, ectomycorrhizal fungi use different decomposition mechanisms to access organic nitrogen entrapped in soil organic matter. The timing and magnitude of the expression of the decomposition activity can be controlled by the below-ground nitrogen quality and the above-ground carbon supply.

sample_organism:
Laccaria bicolor
Paxillus involutus

platform_organism:
Laccaria bicolor
Paxillus involutus

overall_design: A time-course experiment comparing 2 different fungal species at 4 different time points under a defined growth condition. For each species and each time point, there were 3 biological replicates. In total 24 samples.

first: Cesár

last: Nicolás

first: Tomas

last: Martin-Bertelsen

first: Dimitrios

last: Floudas

first: Johan

last: Bentzer

first: Mark

last: Smits

last: Johansson

first: Carl

last: Troein

first: Per

last: Persson


title: Small and large ribosomal subunit deficiencies lead to distinct gene expression signatures that reflect cellular growth rate

city: Berkeley

first: Gloria

last: Brar

address: 626 Barker Hall

department: MCB

institute: UC Berkeley

summary: Levels of the ribosome, the conserved molecular machine that mediates translation, are tightly linked to cellular growth rate. In humans, ribosomopathies are diseases associated with cell-type-specific pathologies and reduced ribosomal protein (RP) levels. Because gene expression defects resulting from ribosome deficiency have not yet been experimentally defined, we systematically probed mRNA, translation, and protein signatures that were either unlinked or linked to cellular growth rate in RP-deficient yeast cells. Ribosome concentration was seen to be associated with translation of gene sub-classes, and profound general secondary effects of RP loss on the spectrum of cellular mRNAs were seen. Among these effects, growth-defective 60S mutants increased synthesis of proteins involved in proteasome-mediated degradation, whereas 40S mutants accumulated mature 60S subunits and increased translation of ribosome biogenesis genes. These distinct signatures of protein synthesis suggest intriguing and currently mysterious differences in the cellular consequences of deficiency for small and large ribosomal subunits.

overall_design: Sequencing of mRNA molecules and ribosome footprints in strains deleted for different ribosomal protein-encoding genes to identify changes in gene expression that are linked to total translation level

middle: A

first: Ze

last: Cheng


title: Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional program [RNA-seq]

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

institute: Altos Labs

summary: The molecular regulation of zygotic genome activation (ZGA) in mammals remains poorly understood. Primed mouse embryonic stem cells contain a rare subset of “2C-like” cells that are epigenetically and transcriptionally similar to the two cell embryo and thus represent an ideal system for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed exclusively in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA either in a Dux dependent or independent manner. Here we performed a candidate-based overexpression screen, identifying, amongst others, Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) as positive regulators of 2C-like cells and ZGA transcription. In the germ line, promoter DNA demethylation coincides with upregulation of Dppa2 and Dppa4 which remain expressed until E7.5 when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during iPSC reprogramming at the time 2C-like ZGA transcription transiently peaks. Through a combination of overexpression, knockdown, knockout and rescue experiments, together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we tease apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilised. Dppa2 and Dppa4 require Dux to initiate 2C-like ZGA transcription, suggesting they act upstream by directly regulating Dux. Supporting this, ChIP-seq analysis revealed Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild type cells, but, in contrast to Dux, can no longer do so in Dppa2/4 double knockout cells, suggesting it may act to stabilise rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux upregulation and activation of the 2C-like transcriptional program which is subsequently reinforced by Zscan4c.

overall_design: RNA sequencing of screen hits (3 biological replicates of GFP+ and GFP- sorted cells for each of 12 candidates).

first: Mélanie

last: Eckersley-Maslin

first: Cela

last: Alda-Catalinas

first: Marloes

last: Blotenburg

first: Elisa

last: Kreibich

first: Christel

first: Wolf

last: Reik


title: Skeletal muscle response to high-intensity intermittent exercise training in young men

city: Kanoya

first: Eri

last: Miyamoto-Mikami

state: Kagoshima

address: 1 Shiromizu

department: Department of Sports and Life Science

institute: National Institute of Fitness and Sports in Kanoya

summary:
High-intensity intermittent exercise training (HIIT) has been proposed as an effective approach for improving both anaerobic and aerobic capacities. However, the molecular response of muscles to HIIT remains unknown.
We used microarray to examine the effects of HIIT on global gene expression in human skeletal muscle.

overall_design: Eleven young healthy men underwent a 6-week high-intensity intermittent exercise training (HIIT). Muscle biopsy samples were collected from the vastus lateralis before (PRE) and after (POST) the 6-week HIIT. Gene expression values were compared between PRE and POST samples.

first: Katsunori

last: Tsuji

first: Naoki

last: Horii

first: Natsuki

last: Hasegawa

first: Shumpei

last: Fujie

first: Toshiyuki

last: Homma

first: Masataka

last: Uchida

first: Takafumi

last: Hamaoka

first: Hiroaki

last: Kanehisa

first: Izumi

last: Tabata

first: Motoyuki

last: Iemitsu


title: Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional program [siRNA]

first: Felix

last: Krueger

overall_design: RNA-sequencing of ESCs treated with control, Dppa2 or Dppa4 siRNAs for 4 days.

first: Mélanie


title: Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional program


title: Mesenchymal precursor cells in adult nerves contribute to mammalian tissue repair and regeneration

city: Toronto

first: Freda

last: Miller

state: ON

address: 686 Bay St

country: Canada

institute: Hospital for Sick Children

summary: Peripheral innervation plays an important role in regulating tissue repair and   regeneration. Here, we provide evidence that injured peripheral nerves provide a   reservoir of mesenchymal precursor cells that can directly contribute to murine digit tip   regeneration and skin repair. In particular, using single-cell RNA sequencing and   lineage tracing we identify transcriptionally-distinct mesenchymal cell populations   within the control and injured adult nerve, including neural crest-derived cells in the   endoneurium with characteristics of mesenchymal precursor cells. Culture and   transplantation studies show that these nerve-derived mesenchymal cells have the   potential to differentiate into non-nerve lineages. Moreover, following digit tip   amputation, the neural crest-derived nerve mesenchymal cells contribute to the   regenerative blastema and ultimately to the regenerated bone. Similarly, neural crest derived   nerve mesenchymal cells contribute to the dermis during skin wound healing.   These findings support a model where peripheral nerves directly contribute   mesenchymal precursor cells to promote repair and regeneration of injured mammalian   tissues.

overall_design: We have applied the high-throughput single-cell mRNA sequencing technique, Drop seq, to the sciatic nerves of adult CD1 and C57BL/6 mice. Processed data for 3 samples are included: 2 injured nerve and one uninjured nerve sample (predicted mesenchymal cells only).

first: Matthew

middle: J

last: Carr

first: Jeremy

middle: S

last: Toma

first: Adam

middle: P

last: Johnston

first: Patrick

middle: E

last: Steadman

first: Scott

last: Yuzwa

first: Neemat

last: Mahmud

first: Paul

middle: W

last: Frankland

first: David

middle: R

last: Kaplan

middle: D


title: Comparison Between Sost-CreERT2 and wild type Cre- Gastrocnemius Skeletal Muscle Transcriptomes

city: Indianapolis

first: Lynda

last: Bonewald

state: IN

address: 635 Barnhill Drive, VanNuys Medical Science Building

institute: Indiana University

summary:
Purpose: The purpose of this study is to identify the age when the skeletal muscle phenotype in Sost-CreERT2 mice occurs.
Methods: Gastrocnemius skeletal muscle mRNA profiles of 1-month-old Sost-CreERT2 and wild type Cre- male mice and 3-month-old Sost-CreERT2 and wild type Cre- female mice were generated by deep sequencing, in triplicate, using HiSeq4000. The sequencing data were first assessed using FastQC (Babraham Bioinformatics, Cambridge, UK) for quality control. Then all sequenced libraries were mapped to the mm10 mouse genome using STAR RNA-seq aligner with the following parameter: “--outSAMmapqUnique 60”. The reads distribution across the genome was assessed using bamutils (from ngsutils). Uniquely mapped sequencing reads were assigned to mm10 refSeq genes using featureCounts (from subread) with the following parameters: “-s 2 -p –Q 10”. Quality control of sequencing and mapping results was summarized using MultiQC. Genes with read count per million (CPM) > 0.5 in more than 2 of the samples were kept. The data was normalized using TMM (trimmed mean of M values) method. Differential expression analysis was performed using edgeR. False discovery rate (FDR) was computed from p-values using the Benjamini-Hochberg procedure.
Results: In the male mice there were 1,857 2fold upregulated genes and 1,959 2fold downregulated genes, whereas in females there were 37 upregulated and 34 downregulated genes.  Only 14 upregulated and 21 downregulated genes overlapped between males and females. A pathway analysis of the male gastrocnemius was performed showing that inflammation mediated by chemokine and cytokine signaling pathways was the major upregulated pathway in Sost-CreERT2 as compared to wild type Cre– male mice.  The Wnt signaling pathway was the major downregulated pathway in the Sost-CreERT2 male mice.

first: Delphine

last: Maurel

first: Tsutomu

last: Matsumoto

first: Julian

last: Vallejo

first: Mark

last: Johnson

first: Sarah

last: Dallas

first: Yukiko

last: Kitase

first: Marco

last: Brotto

first: Michael

last: Wacker

first: Stephen

last: Harris


title: Enhancer profiling of chronic lymphocytic leukemia cells

city: Boston

first: James

last: Bradner

state: MA

address: 450 Brookline

department: Medical Oncology

institute: Dana-Farber Cancer Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Enhancer  profiling has emerged as a powerful approach for discovering the cis-regulatory elements that define transcriptional core regulatory circuits. Characteristic biochemical and biophysical attributes of chromatin mark active enhancer elements, which can be leveraged with genome-wide assay technologies for discovery. This includes chromatin immunoprecipitation followed by sequencing (ChIP-seq) for histone H3 acetylated lysine 27 (H3K27ac). Enhancers are also characterized by regions of open chromatin, hypersensitive to digestion by transposases measurable by the assay for transposase-accessible chromatin (ATAC-seq). By integrating measures of chromatin accessibility and domains of enriched H3K27 acetylation, we have generated enhancer landscapes from chronic lymphocytic leukemia (CLL) representative cell lines including MEC1, MEC2, OSU-CLL, and CII. Targeting enhancer signaling via BET bromodomain inhibition (using the inhibitor JQ1) disrupts enhancer-dependent gene expression as measured with RNA-sequencing in CLL cell lines.

overall_design: ChIP-seq for histone H3, K27 acetylation (H3K27ac), ATAC-seq, and RNA-seq (with and without JQ1 treatment) for the CLL cell lines MEC1, MEC2, OSU-CLL, and CII

first: Christopher

last: Ott


title: Haemopedia: Human Haematopoietic Gene Expression

city: Parkville

first: Carolyn

last: de Graaf

address: 1G Royal Parade

country: Australia

institute: Walter and Eliza Hall Institute

summary: Database of gene expression in different haematopoietic cell types at haemosphere.org

overall_design: Comparison of gene expression in different haematopoietic cell types

first: Jarny

last: Choi

first: Tracey

last: Baldwin


title: mRNA sequencing of oropharyngeal cancer cell lines

status: Public on Nov 28 2018

city: CARDIFF

first: Peter

last: Giles

address: Heath Park

institute: Cardiff University

summary: mRNA sequencing was performed on oropharyngeal cancer cell lines including 4 HPV-positive and 4 HPV-negative lines. Two lines  (CUOP2 and CUOP3) were newly derived from HPV-positive tonsil cancers (derivation is described in "Sensitivity to inhibition of DNA repair by Olaparib in novel oropharyngeal cancer cell lines infected with Human Papillomavirus" by Pirotte et al. This work was undertaken with the aims of quantifying levels of HPV gene transcription and identifying human:viral fusion transcripts arising from integrated viral sequences. We also wished to compare expression of genes involved in DNA damage responses between HPV positive and negative cell lines. The published analyses showed expression of HPV encoded genes in all of the HPV-positive cell lines, and the presence of fusion transcripts derived from integrated virus. We did not identify any obvious correlation between expression of DNA repair genes and HPV status.

overall_design: mRNA sequencing was performed in the 8 cell lines. Cells were grown in standard conditions, mRNA was extracted, and a single sequencing run was undertaken for each line.

first: Evelyne

last: Pirotte

first: Ned

last: Powell


title: A multi-tissue full lifespan epigenetic clock for mice

city: Los Angeles

first: Steve

last: Horvath

state: CA

address: 695 Charles E. Young Drive South, Box 708822

department: Human Genetics

institute: University of California, Los Angeles


title: mRNA sequencing of the mouse and human Hep-Orgs, Chol-Orgs and primary hepatocytes

city: Utrecht

first: Huili

last: Hu

address: Upsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary:
The mammalian liver possesses a remarkable regenerative ability. 1) The 'oval cell' response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. 2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx).
We establish a long-term 3D organoid culture system from mouse and human fetal/pediatric/adult hepatocytes that retain key morphological and functional features of hepatocytes fate. We report the mRNA and single cell sequencing of Hep-Orgs from different donors in different passages. We compared Hep-Orgs with primary hepatocytes, proliferative hepatocytes or Chol-Orgs derived from Epcam+ biliary cells. By analyzing, we determine similar gene expression profile of Hep-Orgs with primary hepatocytes and make genes lists distinguished with undamaged hepatocytes as well. We find the Hep-Orgs resemble proliferative damage-response of hepatocytes after partial hepatectomy while Chol-Orgs express high cholangiocytes markers. The sequencing data constitutes a valuable resource to understand liver organoids especially Hep-Orgs.

overall_design:
We generated transciptome data [1] from hepatocytes unbiasely sorted from the labeled mice liver three days after 2/3 partial heptectomy [2] from mouse and human Hep-Orgs and compare them with primary undamaged hepatocytes, proliferative hepatocytes and Chol-Orgs

Please note that the processed *csv files contains extra data columns which do not belong to the current samples. The submitter provided the 'readme.txt' file indicating which column the corresponding data is included.

first: Helmuth

last: Gehart

first: Benedetta

last: Artegiani

first: Johan

last: van Es

first: Onur

last: Basak

first: Susana

last: Chuva de Sousa Lopes

first: Hans

last: Clevers


title: Rna-seq transcriptome data for Neurod1-homozygous null and Neurod1-heterozygous null murine E17 pancreatic endocrine cells

first: Anthony

last: Romer

summary: The purpose of this study was to identify genes regulated by Neurod1 during murine pancreatic endocrine cell differentiation.

overall_design: Pancreata from E17 Neurod1Floxed/Floxed and Neurod1Floxed/wt mice were dissected and digested into single cell suspensions.  Neurog3-Cre+ Rosa-Tomato+ pancreatic endocrine cells were sorted by flow cytometry and total Rna was extracted. Libraries of amplified cDNA were sequenced and analyzed for Rna expression.

first: Lori

last: Sussel


title: Rna-seq transcriptome data for insulin-GFP+ cells differentiated from NEUROD1 knock out and NEUROD1+/+ control human embryonic stem cells.

summary: The purpose of this study was to identify genes regulated by NEUROD1 in insulin expressing cells differentiated from human embryonic stem cells.

overall_design: Human embryonic stem cells, MEL-1 INSGFP/wt, were differentiated into pancreatic endocrine cells.  Total RNA was extracted from day 27 INS-GFP+ flow cytometry sorted cells. Libraries of cDNA were sequenced and analyzed for RNA expression.

first: Dietrich

last: Egli


title: Rna-seq transcriptome data for 12 week pancreatic islets  from Neurod1 beta cell specific (Neurod1Floxed/Floxed; RipCre+) and control (Neurod1Floxed/Floxed) mice.

summary: The purpose of this study was to identify genes regulated by Neurod1 in murine adult pancreatic islet beta cells.

overall_design: Pancreatic islets were isolated from 12 week Neurod1Floxed/Floxed; RipCre+ and Neurod1Floxed/Floxed mice. Total Rna was extracted.  Libraries of cDNA were sequenced and analyzed for RNA expression.


title: Single cell transcriptional profiles of control and Ikzf2-transfected mouse cochlear cells

city: Bethesda

first: Michael

middle: C

last: Kelly

state: MD

address: 41 Center Drive

department: Center for Cancer Research

institute: NCI

summary: In order to determine the transcriptional effect of Ikzf2 overexpression in mammalian auditory hair cells, mouse cochleae were transfected with either a control GFP or a Ikzf2 virus between postnatal day 1 and 3 (P1-3) and then harvested for single cell gene expression profiling at postnatal day 8 (P8) on the 10X Genomics Single Cell 3' v2 platform.

overall_design: Dataset is composed of two separate single cell RNA-Seq samples captured on the 10X Genomics Chromium platform with the Single Cell 3' Solution v2 chemistry. Viral GFP- (control) or Ikzf2-transfected cochleae were harvested and single cell suspensions prepared from transgenic mice expressing tdTomato in hair cells. Hair cells expressing tdTomato were enriched by flow sorting and then captured on the Chromium system in parellel. Library preparation and sequencing was performed as defined by the 10X Genomics Single Cell 3' Solution v2 protocol.

first: Maggie

last: Mattern


title: Analysis of 3D interactions identifies candidate host genes that transposable elements potentially regulate (4C-Seq)

city: La Jolla

first: Ramya

last: Raviram

address: 9500 Gilman Drive

institute: University of California San Diego

summary: The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation. Using adapted PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes. 4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.

overall_design: We adapted 4C-seq protocols to amplify interactions from multiple sites by designing oligos that hybridized to the consensus sequence of specific endogenous retroviruses. This was applied to different cell types (B cells, fibroblasts and mouse embryonic stem cells) obtained from mice of different inbred strains.

first: Jane

last: Skok


title: RiboTag translatome analysis of outer hair cell postnatal development

city: Baltimore

first: yang

last: song

address: HSF-3, 3rd Floor; 670 W Baltimore St.

department: Department: Institute for Genome Sciences

institute: University of Maryland in Baltimore

summary: In order to determine the regulators of outer hair cell postnatal maturation, we utilized the RiboTag mouse model to perform a detailed transcriptomic analysis of outer hair cells at five postnatal developmental time points: P8, P14, P28, 6 weeks (6wk) and 10 weeks (10wk). This analysis resulted in consistent enrichment of outer hair cell expressed genes in the immunoprecipitated RNA compared to whole cochlear input RNA from each time point. Using transcription factor binding motif prediction on a set of defined outer hair cell enriched genes, we further use this dataset to identify the helios transcription factor as a regulator of the postnatal outer hair cell transcriptome.

overall_design: Examination of the outer hair cell translatome by outer hair cell expressed HA-tagged ribosomal immunoprecipitation at 5 postnatal timepoints (P8, P14, P21, 6wk and 10wk). Immunoprecipitated samples were compared to input cochlear RNA controls in independent biological duplicates or triplicates.

last: Matern

first: Beatrice

last: Milon

first: Ronna

last: Hertzano

first: Ran

last: Elkon

first: Yang

last: Song


title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses

city: Princeton

first: Alexander

last: Ploss

address: Lewis Thomas Laboratory, Washington Road

institute: Princeton University

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

overall_design: Refer to individual Series


title: Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional program [CRISPR-KO]

first: Felix

last: Krueger

overall_design: RNA-sequencing of Dppa2 and/or Dppa4 knockout ESCs.

first: Mélanie

last: Eckersley-Maslin

first: Cela

last: Alda-Catalinas

first: Marloes

last: Blotenburg

first: Elisa

last: Kreibich

first: Christel

first: Wolf

last: Reik


title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses (Experiment 2: scRNA-seq)

summary: We performed an in-depth characterization and comparison of the immune system diversity and complexity in the spleen of conventional NRG-HIS mice and NFA2-HIS/Ftl3LG mice upon YFV-17D infection, a live-attenuated virus that induce that induce potent protective immunity in human. To do so, we employed Seq-Well, a recently developed platform for massively parallel single-cell RNA-Seq (scRNA-Seq), on splenocytes from NRG-HIS mice and NFA2-HIS/Flt3LG mice at six-weeks post YFV-17D infection. Our data provide an in-depth view of the cellular composition of the HIS in conventional and second-generation humanized mice. They also highlight the enhanced engraftment and functionality of the critical role of the myeloid and NK cell compartment in NFA2-HIS/Flt3LG mice, which is likely critical in promoting an enhanced transcriptomic, cellular and humoral response to YFV-17D.

overall_design: We isolated splenocytes from two NRG-HIS mice and two NFA2-HIS/Flt3LG mice at six-weeks post YFV-17D infection and sorted these cells by human CD45 (hCD45) or human CD33 (hCD33) expression. We ran parallel Seq-Well arrays for each sorted population, enabling both unbiased characterization of the relative abundances of all lymphocytes, as well as a deeper examination of the cellular diversity within the myeloid compartment.

first: Florian

last: Douam

first: Carly

middle: G

last: Ziegler

first: Gabriela

last: Hrebikova

first: Bruno

last: Fant

first: Robert

last: Leach

first: Lance

last: Parson

first: Wei

last: Wang

first: Jenna

last: Gaska

first: Benjamin

middle: Y

last: Winer

first: Brigitte

last: Heller

first: Alex

last: Shalek


title: Ikzf2/helios mutant cochlear RNA-seq analysis

first: yang

last: song

summary: Given the potential role of the helios transcription factor in postnatal outer hair cell gene regulation indicated by our RiboTag translatome analyses, our next goal was to identify and validate genes that are regulated by helios in outer hair cells by performing RNA-seq on P8 cello mutant animals compared to wild-type littermates.

overall_design: Difference in cochlear gene expression between wild-type Ikzf2+/+ animals and mutant Ikzf2cello/cello littermates was analyzed by RNA-seq in biological duplicates.

first: Lauren

last: Chessum

first: Maggie

last: Matern

first: Beatrice

last: Milon

first: Michael

last: Bowl

first: Ronna

last: Hertzano

first: Rani

last: Elkon

first: Yang

last: Song


title: Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses (Experiment 1: RNA-seq)

first: Alexander

last: Ploss

summary: By taking advantage of the highly immunogenic live-attenuated yellow fever virus vaccine YFV-17D, we performed an in-depth comparison of transcriptomic responses in human vaccinees, conventional humanized mice, and second generation humanized mice. We demonstrate that selective expansion of human myeloid and natural killer cells in humanized mice promotes transcriptomic responses akin to those of human vaccinees

overall_design: The transcriptome of human PBMCs from different humanized mouse models (conventional model: 3 cohorts/samples of 5 mice each; NFA2-HIS/Fluc model: 2 cohorts/samples of 4 mice each; NFA2-HIS/Flt3LG model: 3 cohorts/samples of 4 mouse each) was determined by RNA-Seq prior (day 0 post infection; control) and after YFV-17D infection (day 11 post infection; post infection). Resulting data sets of differentially expressed genes were compared to data sets from human vaccinees (GSE13485 and GSE13699). Total number of samples processed for RNA-seq: 16

first: Florian

last: Douam

first: Carly

last: Ziegler

first: Gabriela

last: Hrebikova


title: Single-cell ID-seq identifies BMP signaling as a driver of a late stage epidermal differentiation program

city: Nijmegen

first: Jessie

middle: van

last: Buggenum

address: Heyendaalseweg 135

institute: Radboud University

summary: We developed a single cell application of the sequencing-based immunodetection method ID-seq which makes it possible to multiplex measurements of many different epitopes from proteins and phosphoproteins in a high throughput setting. A FACS sort step was implemented to allow sorting of single cells in 96 well plates. With these DNA-tagged antibodies and dedicated sample preparation protocol adapted for single cells, we studied the studied the changes of 70 (phospho-)proteins in primary human skin cells compared to a subpopulation with low ITGB1 protein expression. Given that ITGB1 is a skin stem cell marker, we indeed found that the ITGB1 low cells are differentiated and we found a role for BMP signaling in this process.

overall_design:
We present an single cell immuno-detection sequencing assay for multiplexed protein detection, based on next-generation sequencing (NGS) of barcoded DNA-tags coupled to antibodies.
Sequencing samples contain reads with antibody specific DNA-tag  sequences. Using the antibody and sample index files and the indicated processing pipeline from github.com/jessievb/IDseq, one will obtain a count table of tags per sample.

first: Roderick

last: van Eijl

first: Klaas

last: Mulder


title: CDK12 Regulates DNA Repair Genes by Suppressing Intronic Polyadenylation

first: Sara

middle: Jane

last: Dubbury

address: 500 Main Street, 76-417

department: Koch Institute

institute: MIT

summary: Mutations that attenuate DNA repair by homologous recombination (HR) promote tumorigenesis and sensitize cells to chemotherapeutic agents that cause replication fork collapse, a phenotype known as “BRCAness.” BRCAness tumors arise from loss-of-function mutations in 22 genes. Of these genes, all but one (Cdk12) directly function in the HR repair pathway. Cdk12 phosphorylates Serine 2 of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeat, a modification that regulates transcription elongation, splicing, and cleavage/polyadenylation. Genome-wide expression studies suggest that Cdk12 depletion abrogates the expression of several HR genes relatively specifically, blunting HR repair. This observation suggests that Cdk12 mutational status may predict tumor sensitivity to targeted treatments against BRCAness, such as Parp 1 inhibitors, and that small-molecule inhibitors of Cdk12 may induce sensitization of otherwise HR-competent tumors to these treatments. Despite this growing clinical interest, the mechanism behind the apparent specificity of Cdk12 in regulating HR genes remains unknown.  Here we find that Cdk12 globally suppresses intronic polyadenylation events, enabling the production of full-length gene products. Many HR genes harbor significantly more intronic polyadenylation sites compared to all expressed genes, and the cumulative effect of these sites accounts for the increased sensitivity of HR gene expression to Cdk12 loss. Finally, we find evidence that Cdk12 loss-of-function mutations cause increased intronic polyadenylation within HR genes in human tumors, suggesting that this mechanism is conserved. This work clarifies the biological function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumor biomarker.

overall_design: V6.5 mouse embryonic stem cells with endogenous Cdk12 knock out complemented by doxycycline (Dox)-inducible Flag- HA- epitope tagged Cdk12 transgene (Cdk12△) were grown in the presence of Cdk12 (+Dox) or were depleted of Cdk12 (-Dox). RNA sequencing experimental design: PolyA-selected RNA was isolated from two independently derived Cdk12△ clones both in biological duplicate for cells mainained in Dox (+Dox), withdrawn from Dox for 24 hours (-Dox 24hrs), or withdrawn from Dox for 48 hours (-Dox 48hrs). In parallel, polyA-selected RNA was isolated in biological duplicate from parental Cdk12 Floxed cells (V6.5 mouse embryonic stem cells with homoqygous LoxP sites flanking Cdk12 exon 4 without introduction of the Dox-inducbile transgene) that were pretreated with Dox for 17 days and then either harvested in Dox or harvested after withdrawal of Dox for 24 or 48 hours to serve as a control for gene expression changes caused by long-term Dox treatment followed by short term withdrawal. A total of 18 libraries were prepared and sequenced. ChIP Sequencing Experimental Design: We performed chromatin immunoprecipitation (ChIP) sequencing on Cdk12∆ cells in +Dox or –Dox 48 hour conditions for total RNA Polymerase II (RNAPII), RNAPII phosphorylated on Serine 2 of the CTD (Ser2p), and RNAPII phosphoryalted on Serine 5 of the CTD (Ser5p). Two independent antibodies were used for each antigen as follows: 8WG16 (Abcam ab817) and Rpb3 (Bethyl A303-771A) for total RNAPII density; H5 Clone (Abcam ab24758) and 3E10 Clone (Millipore 04-1571) for Ser2p RNAPII density; and H14 clone (Abcam Ab24759) and 3E8 clone (Millipore 04-1572) for Ser5p RNAPII density. Two biological replicates were processed for each antibody and dox condition. For each dox condition, we also processed 4 negative control libraries: one whole-cell extract (WCE) sample and 3 mock IP samples with the following antibodies: goat anti-mouse IgM (Thermo Fisher 31172), goat anti-rat IgG (Thermo Fisher 31226), mouse IgG2a [MOPC-173] Isotype Control (Abcam ab18413). A total of 32 libraries were prepared and sequenced. For detailed descriptions of the experimental design, please refer to the referenced manuscript.

first: Paul

last: Boutz

first: Phillip

last: Sharp


title: H3K4me3 ChIP-seq of primary human keratinocytes after 6 hours of differentiation induction

summary: To determine as a proxy transcriptional activation after induction of differentiation, we performed H3K4me3 ChIP-seq experiment of primary human keratinocytes that were treated with EGFR inhibitor AG1478 and BMP2/7 for 6 hours.

overall_design: H3K4me3 data of control and 6h AG1478 treated primary human keratinocytes

last: van Buggenum


title: (2-Benzimidazolyl)acetonitrile derivative for the highly selective la-beling 5-formyluracil and its application in genome-wide mapping in tissues

city: Wuhan City

first: Fan

last: Wu

address: Bayi Road No. 299

institute: Wuhan Univwesity

summary: Natural chemical modifications to 5-formyluracil (5fU) has been identified as an important oxidative nucleobase modification, is present in the genomes of many cell types. However, its distribution in genome is poorly understood. Herein, we present a selec-tive labeling and pull-down strategy termed fU-Seq for genome-wide 5fU profiling in mouse hippocampus and human thyroid carcinoma tissues. The mainly essence is using an azido modified (2-benzimidazolyl)acetonitrile derivative that selectively labels 5fU. Surprisingly, 5fU is highly enriched at intergenic and intron, and 5fU-rich regions are positively correlated with H3K27me3 and negatively correlated with H3K27ac, indicating that 5fU is associated with transcription. Collectively, the sequencing of 5fU allow comprehensive analysis of 5fU in genome and provide effective technology for mapping and paves the way for addressing biological and chemical questions regarding this nucleobase modification and for revealing its potential roles in epigenetics.

overall_design: A small molecule was applied for pull-down of 5-formyluracil in the genome of mouse hippocampus tissues and human thyroid carcinoma tissues

first: Y

last: Wang

first: C

last: Liu

first: F

last: Zhou

first: Z

first: X


title: Understanding Early Stage Myelodysplastic Syndrome Pathobiology

city: Cincinnati

first: H. Leighton

last: Grimes

state: OH

address: 3333 Burnet Ave. MLC 7038

department: Immunobiology

institute: Cincinnati Childrens Hospital Medical Center

summary: Delineating key HSC regulators is of significant interest for informing the treatment of hematologic malignancy. While HSC activity is enhanced by overexpression of SKI, the transforming growth factor-beta (TGFβ) signaling antagonist corepressor, its requirement in HSC is unknown. Here we reveal a profound defect in Ski-/- HSC fitness but not specification. Transcriptionally, Ski-/- HSC exhibited striking upregulation of TGFb superfamily signaling and splicing alterations. As these are both common aspects of myelodysplastic-syndrome (MDS) pathobiology with prognostic value, we investigated the role of SKI in MDS. A SKI-correlated gene signature defines a subset of low-risk MDS patients with active TGFβ signaling and deregulated RNA splicing (e.g. CSF3R). The apparent paradox of Ski-/- HSC sharing molecular aspects of MDS with elevated SKI-mRNA is resolved by miR-21 targeting of SKI in MDS. We conclude that miR-21-mediated loss of SKI contributes to early stage MDS pathogenesis by activating TGFβ signaling and alternative splicing while hindering HSC fitness.

overall_design: Single cell RNA seq of transplanted fetal liver-derived hematopoietic stem cells

first: David

last: Muench

first: Nathan

last: Salomonis

first: H


title: Comprehensive transcriptome anaylsis of psoriasis in a Han Chinese population

city: Shanghai

first: Xiaochun

last: Xue

address: 168 Changhai Road

department: Department of Pharmacy

institute: Changhai Hospital, Second Military Medical University

summary: Psoriasis is a chronic inflammatory skin disease related to immune, whose complexity of molecular mechanisms is still not fully clear. RNA sequencing has been widely applied in various fields including biological medicine. According to the bioinformatics analysis of differential genes, biomarkers and drug targets have been discovered for the diagnosis and treatment of diseases. Besides, the pathological mechanisms of disease and functions of gene can be evaluated. In the present study, we report the application of RNA sequencing in skin tissues from psoriatic and healthy persons. By obtaining 2139 differential expressed genes (DEGs), 208 significantly differential GO terms and 44 significantly differential pathways were generated. We found that the functions of  DEGs were mainly related to cell cycle, inflammatory, virus, immune response and metabolic process.The major pathways included cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, chemokine signaling pathway, cell cycle, metabolic pathways, ribosome, peroxisome, steroid biosynthesis and biosynthesis of unsaturated fatty acids. Furthermore, co-expression network was constructed to identify core genes and relations between genes. we considered genes with high values of degree and k-core difference in the co-expression network as core genes, such as IFNG, IL26, TLR3, PRKCQ, TLR4, CD274, CDK1 and IL17A. We chose CD274, an important immune checkpoint, to evaluate its regulatory mechanisms. Candidate genes related to CD274 were evaluated by the co-expression network analysis, and the relations between CD274 and candidate genes were validated in epidermal keratinocytes. Finally, IFNG and CDK1 inhibitor (indirubin) were found increasing the expression levels of CD274. In addition, indirubin was confirmed to attenuate mouse psoriasis-like skin lesion with the mechanisms related to CD274. In conclusion, this study provides us a comprehensive transcriptome analysis method on psoriasis to identify core genes and explore the important regulatory functions of genes.

overall_design: Nine normal skins from healthy volunteers and 18 lesional skins from patients with psoriasis vulgaris  were obtained for RNA sequencing

first: Jinhong

last: Hu

first: Jianhua


title: Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomesof Wild Type , RIG-I-/-, MAVS -/- mice during influenza infection

city: Richmond

first: Mikhail

last: Dozmorov

state: VA

address: 830 E Main St

department: Biostatistics

institute: Virginia Commonwealth University

summary:
Purpose:   The goals of this study are to compare Next-generation sequencing (NGS)-derived transcriptome profiling (RNA-seq) in the lung of three tyeps of mice during influenza infection.
Methods: Total RNA from lung was extracted using a modified TRIzol protocol and spectrophometrically quantitated. Library preparation and sequencing were conducted using 3’ inTAG next-generation sequencing . Differential gene expression for day 6 post influenza infection was determined relative to mock inoculated mice.
Results: Differentially expressed genes were defined using p-value <0.01 and FDR-corrected p-value <0.1 cutoffs. We identified  the transcripts in the lung of RIG-I-/-, MAVS -/- mice during influenza infection
Our study represents the first detailed analysis of lung transcriptomes of Wild Type , RIG-I-/-, MAVS -/- mice during influenza infection , with biologic replicates, generated by RNA-seq technology.

overall_design: Lung tissue mRNA profiles of influenza infected wild type (WT), RIG-I-/-, MAVS -/- mice at day 6 were generated by deep sequencing, n=6.

first: Wenxin

first: Xiaoqiu

first: Wei

last: Zhang

first: Lili

last: Tian

first: J

last: Booth

first: Elizabeth

last: Duggan

first: Sunil

last: More

first: Lin

first: Jordan

last: Metcalf


title: M210B4 cells activated with anti-CD84 (clone 152-1D5)

city: Rehovot

first: Idit

last: Shachar

address: Herzl 234

department: Immunology

institute: Weizmann Institute of science

summary:
CD84 can be expressed on stromal cells in CLL, its target gene in these cells have yet to be identified.
This experiment aimed at determining the CD84 regulated genes on stroma.

overall_design: We compared gene expression profiles in M210B4 cells treated with the isotype (IgG1) or CD84 activation. Cell were lysed and total RNA was isolated, reverse-transcribed, fragmented, labeled and hybridized to Affymetrix Mouse Gene 2.0 ST.

first: Hadas

last: Lewinsky


title: Comparative Transcriptomics of STR/ort, C57BL/6 and MRL/MpJ Knee Joints

city: Livermore

first: Aimy

last: Sebastian

address: 7000 East Ave

institute: Lawrence Livermore National Laboratory

summary: Injuries to the anterior cruciate ligament (ACL) often result in post-traumatic osteoarthritis (PTOA). PTOA accounts for ~12% of all osteoarthritis (OA) cases, yet the mechanisms contributing to OA after joint injury are not well understood. To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced gene expression changes in knee joints of three mouse strains with varying susceptibility to PTOA: STR/ort (highly susceptible), C57BL/6 (moderately susceptible) and super-healer MRL/MpJ (not susceptible) and identified genes differentially expressed between these strains at 0-day [before injury], 1-day, 1-week, and 2-weeks post-injury. This study highlights many new potential therapeutic targets and OA biomarkers.

overall_design: Comparative transcriptomics to understand the molecular changes associated with early stages of PTOA development in STR/ort, C57BL/6 and MRL/MpJ mice and  to identify genes that contribute to increased OA susceptibility in STR/ort and resistance to PTOA in MRL/MpJ.

first: Gabriela

last: Loots


title: Gene expression data in mouse whole lung

city: San Francisco

first: Paul

middle: John

last: Wolters

address: 513 Parnassus Ave HSE 1355B

department: Pulmonary, Critical Care, Allergy and Sleep Medicine

institute: University of California, San Francisco

summary: Telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to spontaneous development of pulmonary fibrosis in mice (SPC-creTRF1flox/flox mice). We sought to identify gene signature in these lungs in an effort to understand molecular mechanisms of the disease.

overall_design: Tamoxifen was administered to SPC-creTRF1flox/flox mice and TRF1flox/flox littermate controls beginning at 8 weeks age. Lungs from SPC-creTRF1flox/flox mice were collected after 3 months and 9 months of tamoxifen administration whereas lungs from TRF1flox/flox controls were collected after 9 months of tamoxifen administration. This design is intended to identify gene changes at both early and late stage of the disease and compare with controls. Mouse whole lungs were harvested for RNA extraction and hybridization on one color Agilent microarrays.

first: Ram

last: Naikawadi


title: Differential expression of antiviral factors in H9c2 cells overexpressing Cacnb4 of the Cav1.2 channel

status: Public on Nov 27 2018

city: Mexico

first: Jorge

middle: Alberto

last: Sanchez

state: DF

address: Av. Politecnico 2508

department: Farmacología

country: Mexico

institute: Cinvestav

summary: L-type voltage gated Ca channels play a critical role in E-C coupling in cardiac muscle. alpha1C is associated with beta auxiliary subunits (b1-b4), which regulate cardiac Ca channel gating properties. Here we report a preliminary exploratory study suggesting a novel role of beta4 subunit in heart. We observed that overexpression of beta4 subunit increases the expression of a wide variety of endogenous genes related to antiviral activity. This includes genes in the downstream signalling of RIG-1 pathway such as RIG-1, Irf7 and Ifitm3. The increase expression of these factors may have an antiviral protective role against infection.

overall_design: Examination of an overall differential expression by the beta4 subunit

first: Eshwar

last: Tammineni

first: Elba

last: Carrillo

last: Sánchez


title: Genes regulated in HT-1080 cells after treatment with IRAK4 inhibitor

city: Kraków

first: Andrzej

last: Mazan

address: Bobrzyńskiego 14

country: Poland

institute: Selvita S.A.


title: RNA Sequencing Analysis of villus tissues from unexplained recurrent spontaneous abortion patients and controls with induced abortions

city: Nanjing

first: Zhenyao

last: Huang

address: Dongshan

institute: Nanjing Medical University

summary: Recurrent spontaneous abortion (RSA), defined as the failure of two or more consecutive clinical pregnancies before 20 weeks of gestation, affects approximately 1% of couples attempting to conceive. However, the underlying mechanisms of 50% of cases are unknown. The villus plays a crucial role during pregnancy, which can provide a delicate balance between immune tolerance and defense to maintain the pregnancy.We performed RNA-seq to identify gene expression alterations in the villus of RSA patients and controls. RNA-seq was done using TruSeq Stranded mRNA Library Preparation. Briefly, intact RNA was fragmented, end repaired, adapter ligation and PCR amplified following illumina protocol. Libraries were sequencing by illumins Hiseq 2000. After quality control, sequence data were processed with STAR to generate read alignments with hg19. Raw read counts for annotated genes were obtained with featureCounts with default settings, normalized and analyzed using DEseq2.

overall_design: Intact RNA isolated from 6 human decidua tissues was fragmented, end repaired, adapter ligation and PCR amplified following illumina protocol. Libraries were sequencing by illumins Hiseq 2000.

first: Chuncheng

last: Lu


title: Serotonin signals through a gut-liver axis to regulate hepatic steatosis

city: Daejeon

first: Byoung-Ha

last: Yoon

address: Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea

country: South Korea

institute: KRIBB

summary:
Purpose: While various functions of peripheral serotonin are known, the direct role of serotonin in regulating hepatic lipid metabolism in vivo is not well understood. We studied whether serotonin directly acts on liver to regulate lipid metabolism.
Methods: Methods: 12 weeks aged liver-specific Htr2a KO (Albumin-Cre+/-; Htr2aflox/flox, herein named Htr2a LKO) mice and wildtype (WT) littermates were fed a high-fat diet (HFD, 60% fat calories) for 8 weeks.
Results: Hepatic lipid droplet accumulation, NAFLD activity score, and hepatic triglyceride levels were dramatically reduced in HFD-fed Htr2a LKO mice compared to WT littermates.
Conclusions:  Gut-derived serotonin is a direct regulator of hepatic lipid metabolism via a gut TPH1-liver HTR2A endocrine axis. And shows promise as a novel drug target to ameliorate NAFLD with minimal systemic metabolic effects.

overall_design: Liver mRNA profiles of wild type (WT) and Htr2a LKO mice were generated by deep sequencing, in triplicate, using Illumina Hiseq.

first: Wonsuk

last: Choi

first: Jun

last: Namkung

last: Park

first: Sangkyu

first: Hail

last: Kim


title: Transcriptional profiling of hypoxia-regulated non-coding RNAs in human primary endothelial cells

city: Kuopio

first: Minna

middle: U

last: Kaikkonen

department: A.I. Virtanen Institute, Department of Biotechnology and Molecular Medicine

country: Finland

institute: University of Eastern Finland

summary: Hypoxia is human atherosclerotic lesions and has multiple adverse effects on endothelial cell metabolism. Recently, key roles of long non coding RNAs (lncRNAs) in the development of atherosclerosis have begun to emerge. In this study, we investigated the long non-coding RNA profiles of HUVECs subjected to hypoxia using GRO-Seq. We demonstrate that hypoxia regulates the nascent transcription of ~1800 lncRNAs. Interestingly, we provide evidence that promoter associated lncRNAs are more likely to be induced by hypoxia compared to enhancer associated lncRNAs which exhibit equal distribution of up- and downregulated transcripts. We also found that hypoxia leads to significant induction in the activity of superenhancers next to genes and transcription factors implicated in angiogenesis, cell survival and adhesion whereas superenhancers near regulators of peptidyl-tyrosine dephosphorylation, signal transduction and GTPase activity were repressed. Despite majority of lncRNAs exhibiting low detection in RNA-Seq, a subset of lncRNAs were expressed at comparable levels to mRNAs. Among these MALAT1, HYMAI, LOC730101, KIAA1656 and LOC339803 were found differentially expressed in human atherosclerotic and may thus serve as potential biomarkers for lesion hypoxia.

overall_design: GRO-Seq and RNA-Seq. Additional datasets for this study can be found under GEO Accession GSE98060 and GSE103945.

first: Pierre

last: Moreau

first: Maria

last: Liivrand


title: Similarities and differences in the regulation of HoxD genes during chick and mouse limb development [4C-seq]

city: Yokohama

first: Nayuta

last: Yakushiji-Kaminatsui

state: Kanagawa

address: 1-7-22 Suehiro-cho, Tsurumi-ku

institute: RIKEN Center for Integrative Medical Sciences

summary: Hoxd genes are important for limb development in all tetrapods examined thus far. In mouse, they are controlled by a complex bimodal regulation, which regulates first the proximal patterning, then the distal structure, allowing at the same time the formation of the wrist and ankle articulations. We analyzed the existence of this regulatory mechanism in chicken, i.e. in an animal where large morphological differences exist between fore- and hindlimbs. We report that while this bimodal regulation is globally conserved between mammals and avian, some important modifications evolved at least between these two model systems, in particular regarding the activity of specific enhancer and the position of the TAD boundary and the forelimb versus hindlimb comparative regulations. Some aspects of these regulations seem to be more conserved between chick and bats than with the mouse situation, which may relate to the extent to which forelimbs and hindlimbs of these various animals differ in their functions.

sample_organism:
Gallus gallus
Mus musculus

platform_organism:
Gallus gallus
Mus musculus

overall_design: 4C-seq analysis of 3D chromatin interactions in mouse or chick limb mesenchymal cells

first: N

first: G

last: Andrey

first: L

last: Lopez-Delisle

first: D

last: Duboule


title: RNA-seq data in WT, roX1, roX2, roX1roX2 mutants in D. melanogaster

city: Umeå

first: Jan

last: Larsson

address: University Hospital, byggnader 6 K och 6 L

department: Department of Molecular biology

institute: Umeå university

summary: Study of single and double mutants of the two roX RNAs in D. melanogaster

first: Marie-Line

last: Faucillion


title: Effect of BMP inhibition or stimulation of primary human keratinocytes

first: Jessie

last: Buggenum

summary: BMP treatment induces expression of late differenitation genes in primary human keratinocytes.

overall_design: RNA-seq analysis after treatment with EGFR inhibitor AG1478 with or without BMP27 or BMP inhibitor DMH1. each treatment and control was performed in triplicate

last: van Buggenum

first: Klaas

last: Mulder


title: Similarities and differences in the regulation of HoxD genes during chick and mouse limb development [RNA-Seq]

overall_design: RNA-seq analysis of limb tissues from mouse (E12.5) or chick (HH20 and HH30)

last: Beccari

first: A

last: Necsulea


title: BET Inhibition Improves NASH and Liver Fibrosis

city: Collegeville

first: Sarah

last: Middleton

address: 1250 S Collegeville Rd

department: Target Sciences - Computational Biology

institute: GSK

summary: Non-alcoholic fatty liver disease (NAFLD) is a leading form of chronic liver disease with large unmet need. Non-alcoholic steatohepatitis (NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. To identify potential new therapeutics for NASH, we used a computational approach based on Connectivity Map (CMAP) analysis, which pointed us to a potential application of bromodomain and extra-terminal motif (BET) inhibitors for treating NASH. To experimentally validate this hypothesis, we tested a small-molecule inhibitor of the BET family of proteins, GSK1210151A (I-BET151), in the STAM mouse NASH model at two different dosing timepoints (onset of NASH and onset of fibrosis) to assess its potential effectiveness for the treatment of NASH and liver fibrosis. I-BET151 decreased the non-alcoholic fatty liver disease activity score (NAS), a clinical endpoint for assessing the severity of NASH, as well as progression of liver fibrosis and interferon-γ expression. Transcriptional characterization through RNA-sequencing pointed to alterations in molecular mechanisms related to interferon signaling and cholesterol biosynthesis following treatment, as well as reversal of gene expression patterns linked to fibrotic markers. Altogether, these results suggest that inhibition of BET proteins may present a novel therapeutic opportunity in the treatment of NASH and liver fibrosis.

overall_design: RNA-sequencing of liver tissue from 4 naive vehicle-treated mice, 4 STAM vehicle-treated mice, 4 STAM I-BET151-treated mice

first: Vinod

last: Kumar


title: Single-cell ID-seq identifies BMP signaling as a driver of a late stage epidermal differentiation program.

institute: Radboud University

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

overall_design: Refer to individual Series


title: Promotion of myoblast differentiation by Fkbp5 via isomerization of Cdk4.

city: Minneapolis

first: Nobuaki

last: Kikyo

state: Minnesota

department: Stem Cell Institute

institute: University of Minnesota

summary: The molecular chaperons FK506-binding proteins (Fkbps) comprise one of three families of peptidyl prolyl isomerases, which promote the transition between cis- and trans-conformations of peptidyl prolyl bonds. Mouse Fkbp family is composed of at least 15 members, but the functions of the large family in cell proliferation and differentiation remain elusive. During myoblast differentiation, the cells need to exit the cell cycle before fusion and terminal differentiation to form myotubes. The clear distinction between proliferation and differentiation provides an ideal model with which to investigate the roles of Fkbps in these two cell biological events. We found that depletion of FkbpC in mouse myoblasts delayed the exit from the cell cycle and expression of myotube-specific genes, whereas its overexpression caused opposite effects. At a mechanistic level, our study revealed a crucial function of FkbpC in Cdk4 activation during myoblast proliferation. Cdk4 undergoes conformational changes in the HSP90/Cdc37/Cdk4 complex as a prerequisite for activation through binding to CyclinD1 accompanied by phosphorylation. Our results showed that FkbpC depletion released Cdk4 from the HSP90 complex, which increased the Cdk4/CyclinD1 complex in myoblasts and sustained high levels of phosphorylated Cdk4 and Rb during differentiation. These results explain the delayed cell cycle exit and differentiation in the depleted cells. In addition, after synchronizing the cell cycle of myoblasts we found dynamic changes of the amounts of FkbpC and Cdk4 in the HSP90 complex during the G1/S transition. Knockout mice of FkbpC demonstrated delayed muscle regeneration after chemical damage, providing an in vivo evidence for the essential role of FkbpC in muscle differentiation. Collectively, our study uncovered FkbpC's critical function as a novel switch regulating the transition from proliferation to differentiation through controlling one of the central regulators of proliferation, Cdk4.

overall_design: mRNA profiles of Fkbp4 knockdown, Fkbp5 knockdown and control C2C12 cells at d0, d3 and d5 were generated by using Illumina HiSeq2500.

last: Ruiz-Estevez

first: James

last: Staats

first: Ellen

last: Paatela

first: Dane

last: Munson

first: Nobuko

last: Katoku-Kikyo

first: Reilly

last: Hostager

first: Ce

last: Yuan

first: Yoko

last: Asakura

first: Lan

last: Mu

first: Atsushi


title: anti-Eomes ChIPseq of control OT-I cells and Eomes-overexpressing OT-I cells

status: Public on Nov 26 2018

city: Stanford

first: Mark

last: Davis

address: Stanford University

institute: Institute for Immunity, Transplantation and Infection

summary: High amount of Eomes might drive T cell exhaustion. In order to understand how Eomes contributes to exhaustion of CD8+ T cell in the TME as a transcription factor, we conducted anti-Eomes ChIPseq analysis of control OT-I cells and Eomes-overexpressing OT-I cells.

overall_design: Eomes was cloned into a retroviral expression vector (RVKM) which also encodes an IRES-hCD2 cassette. This vector was transfected into Pheonix to package retrovirus. The empty vector was used as a control. CD8+ T cells were isolated from spleen and lymph nodes of OT-I mice using Dynabeads Flowcomp mouse CD8 kit (Invitrogen). Then the cells were stimulated with SIINFEKL peptide (OVA257-264) at 2.5 ng/mL in the presence of 10U/mL IL-2 for 24 hr. Retroviral supernatants were harvested, filtered, and supplemented with 6μg/mL polybrene. OT-I T cell cultures were spinduced with the retroviral supernatant for 90 min at 1800 rpm, 32ºC. 48 hr later, hCD2+ cells were sorted prior to re-stimulation. hCD2+ OT-I cells were plated at 40,000 cells/well in 96-well plates and re-stimulated with 2.5ng/mL OVA with 10U/mL IL-2 for 3 days before harvested for ChIPseq analysis. Re-stimulated control or Eomes-overexpressing OT-I cells were harvested, fixed with 1% formaldehyde for 10min at room temperature and stopped by adding glycine to 125mM. ChIP experiment was done following instructions of Active Motif’s ChIP assay kit (53035) with slight modifications, in which 10 cycles of sonication (Bioruptor) were added after enzyme digestion, and dynabeads protein G (Life Technologies) were used for immunoprecipitations. 5μg anti-TBR2/Eomes antibodies (ab23345, abcam) were used for each reaction. The precipitated DNA was sent to BGI genomics for library construction and deep sequencing. To analyze the sequencing data, the sequencing reads were mapped to the Mus musculus genome (version mm10) using Bowtie2 with no more than two mismatches. After PCR duplicates removal by samtools, MACS2 was used for peak calling, compared with each input control with FDR less than 0.001.

first: Jing

last: Li

first: Yi

last: He


title: RNAseq of control OT-I cells and Eomes-overexpressing OT-I cells

summary: High amount of Eomes might drive T cell exhaustion. In order to understand how Eomes contributes to exhaustion of CD8+ T cell in the TME, we conducted transcriptional analysis of control OT-I cells and Eomes-overexpressing OT-I cells

overall_design: Eomes was cloned into a retroviral expression vector (RVKM) which also encodes an IRES-hCD2 cassette. This vector was transfected into Pheonix to package retrovirus. The empty vector was used as a control. CD8+ T cells were isolated from spleen and lymph nodes of OT-I mice using Dynabeads Flowcomp mouse CD8 kit (Invitrogen). Then the cells were stimulated with SIINFEKL peptide (OVA257-264) at 2.5 ng/mL in the presence of 10U/mL IL-2 for 24 hr. Retroviral supernatants were harvested, filtered, and supplemented with 6μg/mL polybrene. OT-I T cell cultures were spinduced with the retroviral supernatant for 90 min at 1800 rpm, 32ºC. 48 hr later, hCD2+ cells were sorted prior to re-stimulation. hCD2+ OT-I cells were plated at 40,000 cells/well in 96-well plates and re-stimulated with 2.5ng/mL OVA with 10U/mL IL-2 for 3 days before harvested for RNAseq analysis. Total RNA was extracted from re-stimulated control or Eomes-overexpressing OT-I cells and sent to BGI Genomics for library construction. The library products were sequenced via Illumina Hiseq4000 by BGI Genomics. The sequencing reads were filtered by SOAPnuke without quality problems. Genome mapping was done by HISAT. Clean reads were mapped to the mm10 reference genome using Bowtie2, and gene expression indicated by RPKM (Reads Per Kilobases per Million reads) was calculated by RSEM. Differentially expressed genes (DEG) were detected with PoissonDis by at least 1.5 fold change and FDR lower than 0.01.


title: High levels of Eomes promote exhaustion of anti-tumor CD8+ T cells


title: Effect of high-fat diet on hepatic gene expression in SM/J mice

city: Aurora

first: Madeline

last: Keleher

state: CO

department: Pediatrics

institute: University of Colorado Anschutz

summary:
We identified 4,356 genes with expression differences associated with a high-fat diet, with 184 genes exhibiting a sex-by-diet interaction. Dietary fat dysregulated several pathways, such as cytokine-cytokine receptor interaction, chemokine signaling, and oxidative phosphorylation.

Grant:
Funding source: American Heart Association
Grant number: 16PRE26420105
Title: The effect of maternal over-nutrition on obesity, epigenetics, and gene expression
Awarded to Madeline Keleher

overall_design: We performed RNA-seq in 21 total libraries, each with two mice of the same sex and diet pooled together (There were 6 low-fat-fed female libraries, 5 libraries of high-fat-fed females, 5 libraries of low-fat-fed males, and 5 libraries of high-fat-fed males). A 1x50 single read sequencing run was done on an Illumina HiSeq 2500 machine (Illumina Inc.)


title: Effect of high-fat diet on hepatic DNA methylation in SM/J mice

summary:
We identified differentially methylated regions across the genome in the liver associated with a high-fat diet.

Grant:
Funding source: American Heart Association
Grant number: 16PRE26420105
Title: The effect of maternal over-nutrition on obesity, epigenetics, and gene expression
Awarded to Madeline Keleher

overall_design: We fed mice a high-fat or low-fat diet from weaning at 3 weeks old to necropsy at 17 weeks old. We extracted DNA from liver tissue and performed MRE-seq and MeDIP-seq in 8 total libraries, each with four mice of the same sex and diet pooled together.


title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions (human fibroblast ChIP-seq)

city: Edinburgh

first: Duncan

last: Sproul

state: Mid Lothian

address: Crewe Road South

department: MRC Human Genetics Unit

institute: University of Edinburgh

summary: DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, encoding the DNA methyltransferase DNMT3A, that cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2/3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation canyons/valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3aW326R pluripotent cells in vitro, and is also evident in Dnmt3aW326R/+ dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2/3 normally limits DNA methylation of polycomb-marked regions. Our findings implicate the interplay between DNA methylation and polycomb at key developmental regulators as a determinant of organism size in mammals.

overall_design:
This series contains ChIP-seq from human fibroblast cells.
Raw sequencing reads protected as patient material, submitted to EGA. Accession: EGAS00001003232

first: Patricia

last: Heyn

first: Andrew

last: Andrew


title: Maternal Eed knockout causes loss of H3K27me3 imprinting and random X-inactivation  in the extra-embryonic cells

city: Cincinnati

first: Zhiyuan

last: Chen

state: Ohio

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

summary: Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 lysine 27 tri-methylation (H3K27me3) can mediate DNA methylation-independent genomic imprinting. However, the regulatory mechanisms and functions of this new imprinting mechanism are largely unknown. Here we demonstrate that maternal Eed, an essential component of the Polycomb group complex 2 (PRC2), is required for establishing H3K27me3 imprinting. We found that all H3K27me3 imprinted genes, including Xist, lose their imprinted expression in Eed maternal KO (matKO) embryos, resulting in male-biased lethality. Surprisingly, although maternal X chromosome inactivation (XmCI) occurs in Eed matKO embryos at preimplantation due to loss of Xist imprinting, it is resolved at peri-implantation. Ultimately, both X chromosomes are reactivated in the embryonic cell lineage prior to random XCI, and only a single X chromosome undergoes random XCI in the extra-embryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamics in the absence of Xist imprinting.

overall_design:
mRNA profiling was performed for morula embryos and E6.5 extra-embryonic ectoderm (ExE) of Eed control and maternal knockouts. For morula embryos, two replicates were generated for each group.  For E6.5 ExE samples, four replicates were produced for the control group (2 males and 2 females) and nine replicates were generated for the maternal KO group (5 males and 4 females).
H3K27me3 profiling for Eed CTR and matKO morula embryos were performed using CUT&RUN.

first: Azusa

last: Inoue

first: Qiangzong

last: Yin

last: Zhang


title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions (mouse NPC RNAseq)

overall_design: This series contains RNAseq from mouse NPC cells.


title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions (human fibroblast RNAseq)

overall_design:
This series contains RNAseq from human fibroblast cells.
Raw sequencing reads protected as patient material, submitted to EGA. Accession: EGAS00001003232


title: C57BL/6 substrain differences in inflammatory and neuropathic nociception and genetic mapping of a major quantitative trait locus underlying acute thermal nociception

city: Medford

first: Eric

last: Reed

address: 419 Boston Ave.

department: Data Intensive Studies Center

institute: Tufts University

summary:
Sensitivity to different pain modalities has a genetic basis that remains  largely unknown. The use of closely related inbred mouse strains can  facilitate gene mapping of nociceptive behaviors in preclinical pain  models. We previously reported enhanced sensitivity to acute thermal  nociception in C57BL/6J (B6J) versus C57BL/6N (B6N) substrains. Here, we  expanded on pain phenotypes and observed an increase in inflammatory  nociceptive behaviors induced by hindpaw formalin injections in B6J versus  B6N mice (Charles River Laboratories). No strain differences were observed  in mechanical or thermal hypersensitivity or in paw diameter following the  Complete Freund s Adjuvant (CFA) model of inflammatory pain, indicating  specificity in the inflammatory nociceptive stimulus. In the chronic nerve  constriction injury (CCI), a model of neuropathic pain, no strain  differences were observed in baseline mechanical threshold or in  mechanical hypersensitivity up to one month post-CCI. We replicated the  enhanced thermal nociception in B6J mice in the 52.5 C hot plate test  relative to B6N mice from The Jackson Laboratory. Using a B6J x B6N-F2  cross (N=164), we mapped a major QTL underlying hot plate sensitivity to  chromosome 7 that peaked at 26 Mb (LOD = 3.81,  8.74 Mb-36.50 Mb) that was  more pronounced in males. Genes containing expression QTLs (eQTLs)  associated with the peak nociceptive marker that have been implicated in  pain and inflammation include Ryr1, Cyp2a5, Pou2f2, Clip3, Sirt2, Actn4,  and Ltbp4 (FDR < 0.05). Future studies involving positional cloning and  gene editing will determine the quantitative trait gene(s) and potential  pleiotropy of this locus across other pain modalities.
RNA-seq data and genotype information from striatum punches of F2 C57BL/6J (B6J) cross C57BL/NJ (B6NJ) oxycodone-treated mice. Genotypes are given relative to B6J allele, eg 0 = homozygous B6J.

overall_design: C57BL/6J (B6J) and C57BL/NJ (B6NJ) mice were purchased from JAX at 7 weeks of age and were habituated in the vivarium one week prior to experimental testing that occurred next door. B6J females were crossed to B6NJ males to generate B6J x B6NJ-F1 mice and B6J x B6NJ F1 offspring were intercrossed to generate B6J x B6NJ F2 mice. Mice were 50-100 days old at the time of testing. F2 mice recieved four daily oxycodone injections (20 mg/kg, i.p.). Ninety SNP markers spaced approximately 30 Mb (approximately 15 cM) apart were genotyped using a custom-designed Fluidigm array.

first: Camron

last: Bryant

first: Deniz

last: Bagdas

first: Lisa

last: Goldberg

first: Tala

last: Khalefa

first: Stacey

middle: L

last: Kirkpatrick

first: Julia

last: Kelliher

first: Melanie

middle: M

first: William

last: Johnson

first: Megan

middle: K

last: Mulligan

first: M

middle: I

last: Damaj


title: Identification of differentially expressed genes between male and female in mouse embryonic fibroblasts (MEFs).

status: Public on Nov 25 2018

city: Toyama

first: Jun-Dal

last: Kim

state: Toyama

address: 2630 Sugitani

department: Department of Complex Biosystem Research (CBR), Institute of National Medicine (INM)

institute: University of Toyama

summary: The transcriptomic profiles of mouse embryonic fibroblasts (MEFs) were investigated using the next-generation RNA sequencing (RNA-Seq). The CLC Genomic Workbench software was used to screen the differentially expressed transcripts. A total of 49 genes with a significantly differential expression (false discovery rate (FDR) p<0.05, fold change >2) in the female group as compared with the male group.

overall_design: mRNA profiles of mouse embryonic fibroblast (MEF) were generated by RNA sequencing using the NextSeq 500 (Illumina).

first: Hayase

last: Mizukami

first: Akiyoshi

last: Fukamizu


title: Genes regulated in HT-1080 cells after 24 hours of treatment with IRAK4 inhibitor

status: Public on Nov 24 2018

first: Andrzej

last: Mazan

summary:
Analysis of HT-1080 fibrosarcoma cells transcriptome following  24 hours treatment with IRAK4 inhibitor was performed.  Results provide insight into mode of action of IRAK4 inhibitor under study.
Project co-financed from European Regional Development Fund under The Operational Program Innovative Economy, 2007-2013, measure 1.4 (UDA-POIG.01.04.00-12-049/11-00)

overall_design: Samples from HT-1080 cells treated for 24 hours with IRAK4 inhibitor and control samples were subjected to RNA sequencing using Ion Torrent Proton. Three biological replicates for a given condition were studied.

first: Anna

last: Wróbel

first: Karolina

last: Pyziak

first: Agnieszka

last: Sroka-Porada

first: Mateusz

last: Ogórek

first: Monika

last: Dobrzańska

first: Piotr

last: Kowalczyk

first: Claude

last: Commander

first: Katarzyna

last: Wiklik

first: Federico

last: Malusa

first: Michał

last: Combik

first: Maria

last: Kulecka

last: Mikula

last: Paziewska


title: Spo11-oligo mapping of double-strand breaks in pch2 and sir2 mutants

city: New York

first: Scott

last: Keeney

address: 1275 York Ave

department: Molecular Biology Program

institute: Memorial Sloan Kettering Cancer Center

summary: The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to map the distribution of meiotic DSBs in pch2 and sir2 mutant strains of Saccharomyces cerevisiae.

overall_design: Two samples from each strain (four total) collected at 4 hr after transfer to sporulation medium

first: Xuan

last: Zhu


title: Single-cell transcriptomics characterizes cell types in the subventricular zone and uncovers molecular defects impairing adult neurogenesis.

city: Berlin

first: Nikolaus

last: Rajewsky

address: Robert-Rössle-Str. 10

department: BIMSB

country: Germany

institute: MDC Berlin

summary: Neural stem cells (NSCs) contribute to plasticity and repair of the adult brain. Niches harboring NSCs regulate stem cell self-renewal and differentiation. We used comprehensive and untargeted single-cell RNA profiling to generate a molecular cell atlas of the largest germinal region of the adult mouse brain, the subventricular zone (SVZ). We characterized > 20 neural and non-neural cell types and gained insights into the dynamics of neurogenesis by predicting future cell states based on computational analysis of RNA kinetics. Furthermore, we applied our single-cell approach to document decreased numbers of NSCs, reduced proliferation activity of progenitors, and perturbations in Wnt and BMP signaling pathways in mice lacking LRP2, an endocytic receptor required for SVZ maintenance. Our data provide a valuable resource to study adult neurogenesis and a proof-of-principle for the power of single-cell RNA-sequencing to elucidate neural cell type-specific alterations in loss-of-function models.

overall_design: We subjected cells derived from the adult subventricular zone neurogenic niche to single-cell RNA sequencing without pre-selection and sorting.

first: Vera

last: Zywitza

first: Aristotelis

last: Misios

first: Lena

last: Bunatyan

first: Thomas

last: Willnow


title: β2 adrenergic receptor-mediated negative regulation of group 2 innate lymphoid cell responses

first: Gregory

last: Putzel

address: 430 East 29th Street

department: Microbiology

institute: NYU Langone Health

summary: RNA sequencing of ILC2s sorted from β2 adrenergic receptor agonist-treated and non-treated mice

overall_design: RNAs of ILC2s sorted as KLRG1+CD127+CD90+Lin-CD45+ from β2 adrenergic receptor agonist-treated and non-treated mice mLNs 4 days post N. brasiliensis infection were analyzed

first: David

last: Artis


title: Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions

first: Duncan

last: Sproul


title: Genes regulated in HT-1080 cells after 4 hours of treatment with IRAK4 inhibitor

first: Andrzej

last: Mazan

institute: Selvita S.A.

summary:
Analysis of HT-1080 fibrosarcoma cells transcriptome following 4 hours treatment with IRAK4 inhibitor was performed.  Results provide insight into mode of action of IRAK4 inhibitor under study.
Project co-financed from European Regional Development Fund under The Operational Program Innovative Economy, 2007-2013, measure 1.4 (UDA-POIG.01.04.00-12-049/11-00)

overall_design: Samples from HT-1080 cells treated for 4 hours with IRAK4 inhibitor and control samples were subjected to RNA sequencing using Ion Torrent Proton. Three biological replicates for a given condition were studied.

first: Anna

last: Wróbel

first: Karolina

last: Pyziak

first: Agnieszka

last: Sroka-Porada

first: Mateusz

last: Ogórek

first: Monika

last: Dobrzańska

last: Kowalczyk

last: Commander


title: Gene expression of cultured human choroidal stromal fibroblasts and retinal pigment epithelial (RPE) cells

city: Québec

first: Stéphanie

last: Proulx

state: Qc

address: 1050 Chemin Ste-Foy

institute: Université Laval

summary: Retinal pigment epithelial (RPE) cells and choroidal stromal fibroblast (CSF) were isolated from healthy human donor eyes. Cells were cultured and RNA extracted.

overall_design: Three human choroidal stromal fibroblast cell populations and one human retinal pigment epithelial cell population (primary cells) were cultured and analyzed by gene profiling.

first: Aïcha

last: Dede Djigo

first: Solange

last: Landreville

first: Julie

last: Bérubé