title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures IV

city: Aachen

first: Ivan

middle: G.

last: Costa

state: NRW

address: Pauwelsstr. 19

department: RWTH Aachen Medical Faculty

institute: Institute for Computational Genomics

summary:
RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions.
In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including repeat elements.
Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.

overall_design: We have established methods (DNA-IP and SPRI) to enrich RNA regions that are directly associated with DNA (i.e., TriplexRNAs). These RNAs were sequenced as biological replicates (IP_rep1-3 and SPRI_rep1-4). Nuclear and chromatin-associated RNAs were sequenced as control (nuclear_rep1-2 and chromatin_rep1-4). For U2OS cells, biological duplicates were sequenced from TriplexRNA (IP_rep1-2) and nuclear RNA (nuclear_rep1-2).

first: Nevcin

last: Sentürk

first: Chao-Chung

last: Kuo

middle: G

first: Ingrid

last: Grummt


title: Transcriptome analysis of OGT-sufficient and OGT-deficient regulatory T (Treg) cells

status: Public on Dec 21 2018

city: Minneapolis

first: Hai-Bin

last: Ruan

state: MN

department: Integrative Biology and Physiology

country: USA

institute: University of Minnesota

type: Expression profiling by high throughput sequencing

summary: To gain comprehensive insight into the OGT-dependent transcriptional program in Treg cells, we performed RNA-sequencing of isolated YFP+ Treg cells from Foxp3YFP-Cre/wtOgtwt/fl and healthy Foxp3YFP-Cre/wtOgtfl/fl females to avoid secondary changes in gene expression caused by inflammation. We were able to identify 269 differentially expressed genes including 154 downregulated and 115 upregulated with p values less than 0.01, OGT-deficient Treg cells had impaired suppressive function and attenuated IL2/STAT5 signaling pathway.

overall_design: Examination of the function of OGT in Treg cells

first: Bing

last: Liu


title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

status: Public on Dec 20 2018

country: Germany

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

overall_design: Refer to individual Series


title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures I

overall_design: We captured NEAT1-associated DNA from deproteinized chromatin using a biotinylated antisense DNA oligonucleotide (ASO) that binds to NEAT1. A sense oligo that does not hybridize to NEAT1 served as control. Biological triplicates from HeLa S3 cells (rep1-3_antisense and rep1-3_sense) as well as one set from MCF7 cells were sequenced.


title: Identification and validation of a tumor-infiltrating Treg transcriptional signature conserved across species and tumor types.

city: Boston

first: CBDM

last: Lab

state: MA

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: We reported transcriptional characterization of tumor-infiltrating T regulatory cells (TITRs) that is shared between species and among different tumor types and stages. We genomically profiled immunocytes from 2 species: 1. Mouse: CD4+ Treg and Tconv, and CD8+ T cells from tumor and spleen of B16, MC38 or CT26 tumor bearing mice, as well as from spleens of non-tumor bearing mice. 2. Human: CD4+ Treg cells from surgically resected and cryopreserved human colorectal carcinomas or normal colon tissue.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Gene expression profiles of CD4+ Treg and Tconv, and CD8+ T cells from tumor and spleen of B16, MC38 or CT26 tumor bearing mice (biological triplicates), as well as from spleens of non-tumor bearing mice, and CD4+ Treg cells from 12 surgically resected and cryopreserved human colorectal carcinomas or normal colon tissue.

first: Angela

last: Magnuson

first: Christophe

last: Benoist

first: Diane

last: Mathis


title: Malt1 protease is critical in maintaining function of Treg cells and may be a therapeutic target for anti-tumor immunity

city: Beijing

first: Liqing

last: Cheng

state: Beijing

address: Tsinghua University,Hai Dian, Beijing

department: school of medicine

country: China

institute: Tsinghua university

summary: We report that the paracaspase Malt1 is required for the development and function of Treg cells. By generating Malt1 conditional knockout and protease dead mutant mice, we found Malt1-loss in Treg cells would lead to early-onset lethal autoimmune disease and the mice would die within 40 days after birth. Interestingly, mice with Treg specific inhibition of Malt1 protease activity develop spontaneous inflammatory disorders.

overall_design: YFP+ Treg cells sorted from WT, cKO and cKI mice were used for RNA extraction and RNA-sequencing

first: Naixue

last: Yang


title: RNA sequencing of CFU-GM derived from CD34+ cells expressing PRR14L shRNA or a scramble control

city: Oxford

first: Andrea

last: Pellagatti

state: Oxfordshire

address: John Radcliffe Hospital, Headley Way

department: RDM Clinical Laboratory Sciences

country: United Kingdom

institute: University of Oxford

summary: Here we are using RNA-Seq to study the effect of PRR14L knockdown on transcriptome of hematopoietic cells differentiated towards the granulomonocytic lineage.

overall_design: RNAseq was performed on individual CFU-GM with shRNA-mediated PRR14L knockdown and scramble control to study the effects of PRR14L knockdown on the transcriptome of hematopoietic cells differentiated towards the granulomonocytic lineage.

first: Jacqueline

last: Boultwood

first: Shalini

last: Singh


title: Next generation sequencing identifies a highly accurate microRNA panel that distinguishes well-differentiated thyroid cancer from benign thyroid nodules

city: Jerusalem

first: Iddo

middle: Z.

last: Ben-Dov

address: Ein Kerem

department: Nephrology and Hypertension

country: Israel

institute: Hadassah Medical Center

type: Non-coding RNA profiling by high throughput sequencing

summary: Background: Fine needle aspiration biopsy (FNAB) is the gold-standard procedure for diagnosing malignant thyroid nodules. Indeterminate cytology is identified in 10-40% of cases and molecular testing may guide management in this setting. Current commercial options are expensive, and are either sensitive or specific. The aim of this study was to utilize next generation sequencing (NGS) technology to identify informative diversities in the microRNA (miRNA) expression profile of benign versus malignant thyroid nodules. Methods: Ex-vivo FNAB samples were obtained from thyroid specimens of patients that underwent thyroidectomy at a referral center. miRNA levels were determined using NGS and multiplexing technologies. Statistical analyses identified differences between normal and malignant samples and miRNA expression profiles that associate with malignancy were established. The accuracy of the miRNA signature in predicting histological malignancy was validated using a group of patient specimens with indeterminate cytology results. Results: 274 samples were obtained from 102 patients undergoing thyroidectomy. Of these samples, 71% were benign and 29% were malignant. Nineteen miRNAs were identified as statistically different between benign and malignant samples and were used to classify 35 additional nodules with indeterminate cytology (validation). The miRNA panel’s sensitivity, specificity, negative and positive predictive values and overall accuracy were 91%, 100%, 87%, 100% and 94%, respectively. Conclusion: Using NGS technology we identified a panel of 19 miRNAs that may be utilized to distinguish benign from malignant thyroid nodules with indeterminate cytology. Impact: Our panel may classify indeterminate thyroid nodules at higher accuracy than commercially available molecular tests.

overall_design:
Ex-vivo FNAB samples were obtained from thyroid specimens of patients that underwent thyroidectomy at a referral center. miRNA levels were determined using NGS and multiplexing technologies. Statistical analyses identified differences between normal and malignant samples and miRNA expression profiles that associate with malignancy were established. The accuracy of the miRNA signature in predicting histological malignancy was validated using a group of patient specimens with indeterminate cytology results.

Grant ID/title/funding source: I-CORE Program of the Planning and Budgeting Committee and the Israel Science Foundation (grant no. 41/11).

first: Haggi

last: Mazeh

first: Devorah

last: Gur-Wahnon

middle: Z


title: Hepatocytes direct the formation of a pro-metastatic niche in the liver.

city: Philadelphia

first: Gregory

middle: L.

last: Beatty

state: PA

address: 3400 Civic Center Blvd, South Pavilion, Rm 8-107

department: Medicine

institute: University of Pennsylvania

summary: The liver is the most common site of metastatic disease in gastrointestinal malignancies, including pancreatic ductal adenocarcinoma (PDAC). While this metastatic tropism may reflect mechanical trapping of tumor cells that enter the circulation, liver metastasis is also dependent, at least in part, on the formation of a “pro-metastatic” niche that supports tumor cell seeding and colonization in the liver. However, mechanisms that orchestrate the establishment of this niche are poorly understood. Here, we show that hepatocytes coordinate accumulation of myeloid cells and fibrosis within the liver, the two defining features of a pro-metastatic niche. Early during pancreatic tumorigenesis in mice, hepatocytes demonstrate activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling and increased production of serum amyloid A1 and A2 (SAA). Overexpression of SAA by hepatocytes also occurs in PDAC patients with liver metastases, and many patients with locally advanced and metastatic disease display elevated levels of circulating SAA. STAT3 activation in hepatocytes and the subsequent production of SAA are dependent on interleukin 6 (IL-6) that is released into the circulation by non-malignant cells that reside adjacent to malignant cells in the pancreas. Genetic ablation or blockade of components of IL-6/STAT3/SAA signaling in hepatocytes effectively prevents the establishment of a pro-metastatic niche and inhibits metastatic seeding in the liver. Collectively, our data reveal an intercellular network underpinned by hepatocytes that forms the basis for a pro-metastatic niche in the liver and identify new therapeutic targets for pancreatic cancer.

overall_design: RNA profiles of the liver of LSL-KrasG12D/+;LSL-Trp53R127H/+;Pdx-1-Cre (KPC) mice and control mice (LSL-Trp53R127H/+;Pdx-1-Cre (PC)) were generated using QuantSeq 3’ mRNA sequencing.

middle: L

first: Jae

middle: W

last: Lee


title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures II

first: Ivan

last: Costa

overall_design: We used biotinylated RNA oligonucleotides corresponding to two triplex-forming regions in NEAT1 (TFR1 and TFR2) to capture their putative target loci. TFR-associated DNA from biological triplicates was sequenced (TFR1_rep1-3 and TFR2_rep1-3). Experiments with an oligonucleotide that does not contain a potential TFR served as control (Control_rep1-3).

first: Nevcin

last: Sentürk

first: Chao-Chung

last: Kuo

first: Ingrid

last: Grummt


title: TREM2-activating antibodies abrogate the negative pleiotropic effects of the Alzheimer’s disease variant TREM2R47H on murine myeloid cell function

city: San Diego

first: Jun

last: Yin

state: CA

address: 10901 N Torrey Pines Rd

department: Bioinformatics Core

institute: Sanford Burnham Prebys Medical Discovery Institute

summary: Triggering receptor expressed on myeloid cells 2 (TREM2) is an orphan immune receptor expressed on cells of myeloid lineage such as macrophages and microglia. The rare–variant R47H TREM2 is associated with an increased risk for Alzheimer’s disease (AD), supporting the hypothesis that TREM2 loss of function may exacerbate disease progression. However, a complete knockout of the TREM2 gene in different genetic models of neurodegenerative diseases has been reported to result in both protective and deleterious effects on disease-related endpoints and myeloid cell function. Here, we describe a TREM2R47H transgenic mouse model and report that even in the absence of additional genetic perturbations, this variant clearly confers a loss of function on myeloid cells. The TREM2R47H variant–containing myeloid cells exhibited subtle defects in survival and migration and displayed an unexpected dysregulation of cytokine responses in a lipopolysaccharide challenge environment. These subtle phenotypic defects with a gradation in severity across genotypes were confirmed in whole-genome RNA-Seq analyses of WT, TREM2−/− and TREM2R47H myeloid cells under challenge conditions. Of note, TREM2-activating antibodies that boost proximal signaling abrogated survival defects conferred by the variant and also modulated migration and cytokine responses in an antibody-, ligand-, and challenge-dependent manner. In some instances, these antibodies also boosted wildtype myeloid cell function. Our studies provide a first glimpse into the boost in myeloid cell function that can be achieved by pharmacological modulation of TREM2 activity that can potentially be ameliorative in neurodegenerative diseases such as AD.

overall_design: Day 6 BMDMs were harvested from TREM2-/-, TREM2R47H and respective wild-type littermates (n=5 animals per genotype) and total RNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA-Seq was performed to study TREM2 disturbed gene expression in BMDMs.

first: Qingwen

first: Jean

last: Danao

first: Santosh

last: Talreja

first: Paul

last: Wen

first: Ning

last: Sun

first: Chi-Ming

last: Li

first: Danny

last: Chui

first: David

last: Tran

first: Samir

last: Koirala

first: Hang

last: Chen

first: Ian

middle: N

last: Foltz

first: Songli

last: Wang

first: Shilpa

last: Sambashivan

first: Jere

last: Meredith


title: Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [scATAC-seq]

city: Stanford

last: Khavari

address: 269 Campus Drive, CCSR 2150

institute: Stanford University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Here we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by Assay of Transposase-accessible Chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ~3,700 single cells, encompassing more than 75 unique genotype-phenotype relationships.

overall_design: Single-cell epigenome and perturbation identification in lymphoblastoid cells and epidermal keratinocytes.

first: Adam

middle: J

last: Rubin

first: Kevin

middle: R

last: Parker

first: Ansuman

middle: T

last: Satpathy

first: Howard

middle: Y

last: Chang

middle: A


title: Transcriptional profiling of MST1 or FOXO1 depleted human umbilical vein endothelial cells

city: Irvine

first: Hosung

last: Bae

address: 839 Health Sciences Road Sprague Hall, Room 150

institute: UC Irvine

summary: Analysis of human umbilical vein endothelial cells depleted for MST1 or FOXO1 by siRNA knockdown to identify the commonly regulated genes by MST1-FOXO1 cascade. Results provide insight into the role of MST1-FOXO1 in endothelial cell.

overall_design: mRNA expression profiles of the human umbilical vein endothelial cells after MST1 or FOXO1 siRNA knockdown by Illumina HiSeq 2500.

first: Gou-Young

last: Koh

first: Yoo-Hyung

last: Kim


title: Expression data from Drosophila tumor models

city: Ribeirao Preto

first: Flavia Cristina de

middle: Paula

last: Freitas

state: Sao Paulo

address: Avenida dos Bandeirantes, 3900

department: Genetics

country: Brazil

institute: University of Sao Paulo

summary:
Epithelial tumors can progress from a benign tissue overgrowth (hyperplasia) to a malignant neoplastic tumor, which is characterized by an increase in motility and invasiveness. The Cohen laboratory has developed an epithelial tumor model in which overexpression of the epidermal growth factor receptor gene (EGFR) leads to benign tissue hyperplasia. When combined with other cooperating factors, EGFR overexpression can lead to neoplasia and malignant metastasis.
Microarray analysis were performed in normal epithelia, hyperplastic, and neoplastic tumors collected from Drosophila wing imaginal discs to identify genes whose misexpression correlates with tumor progression

overall_design: For each sample, approximately 20 pairs of wing discs were dissected and processed for total RNA extraction and hybridization on Affymetrix microarrays. Four biological replicates were analyzed for each of the five tested conditions: EGFR and EGFR+psqRNAi at 12 hr (E-Ctrl and EP-Ctrl); EGFR and EGFR+psqRNAi samples at day 4 (E-EHT and EP-EHT), and from EGFR+psqRNAi at day 11 of tumor growth (EP-LNT). E-EHT rep1  was discarded from further analysis as preliminary analysis pointed to technical problems.

first: Teresa

last: Eichenlaub

first: Flávia

middle: C

first: Jan

last: Gorodkin

first: Héctor

last: Herranz

first: Stephen

middle: M

last: Cohen


title: Distinct stress response and altered striatal transcriptome in alpha-synuclein overexpressing mice

city: Saarbrücken

first: Julia

middle: Maria

last: Schulze-Hentrich

address: Campus, Building A2.4

department: Genetics and Epigenetics

institute: Saarland University

summary: Using a mouse model overexpressing human SNCA and profiling the striatal transcriptome, we assessed gene-environment interactions to reveal perturbations in gene expression and their modulation through chronic unpredictable mild stress (CUMS) exposure.

overall_design: Using a 2x2 factorial design, we cross-compared a line of transgenic mice overexpressing human SNCA with wildtype animals, and the effects of chronic unpredictable mild stress (CUMS) with standard housing conditions. Employing RNA-seq, we profiled gene expression in the striatum of 6-month-old female animals.

first: Thomas

last: Hentrich


title: CRAC of yeast RNA polymerase II in anchor-away and auxin-degron strains.

city: Montpellier

first: Domenico

last: Libri

address: 1919 route de Mende

country: France

institute: IGMM - CNRS

summary: We obtained  RNA polymerase II occupancy profiles across the genome of S.cerevisiae  strains:   Abf1 anchor-away cells after addition of  rapamycin for different time points (including no addition of rapamycin) and  Rap1-AID auxin-degron cells after addition of auxin for different time points (including no addition of auxin) .  This allowed us to compare polymerase occupancy profiles when these proteins are depleted from the nucleus or degraded.

overall_design: The nascent RNA was crosslinked to RNAPII and sequenced after purification of Rpb1 under stringent and denaturing conditions. This reveals the position of the polymerase at the nucleotide resolution level.

first: Drice

last: Challal

first: Mara

last: Barucco

first: Slawomir

last: Kubik

first: Frank

last: Feuerbach

first: Tito

last: Candelli

first: Hélène

last: Géoffroy

first: Chaima

last: Benaksas

first: David

last: Shore


title: RNAseq of germinal center B cells from Blimp-1-GFP reporter mice with an ablatable TfH compartment.

first: Daniel

last: Radtke

address: John Radcliffe Hospital, University of Oxford

department: HIU Human Immunology Unit

institute: Weatheral Institute of Molecular Medicine

summary: RNAseq of germinal center B cells (B220+IgDlowCD95+GL7+) with different Blimp-1-GFP reporter level in the presence of TfH cells or 72 hr after their depletion. Sorted germinal center populations were defined by their expression of Blimp-1-GFP reporter and the cell surface marker CD138. Analysed mice were bone marrow chimeras generated with a bone marrow mixture from CD4-cre+ Rosa26-loxP-stp-loxP-DTR Blimp-1GFP/+ (10%) and Rag1-/- OTII (90%) mice. In these mice TfH cells can be depleted by the injection of human diphteria toxin (DT).

overall_design: We used RNAseq to find genes differentially regulated in various splenic Blimp-1-GFP expressing germinal center populations in the presence of TfH cells or after their depletion. Cells were sorted from the spleen at day 10 after intraperitoneal immunisation with sheep red blood cells.

first: Oliver

last: Bannard


title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures III

first: Ivan

middle: G.

last: Costa

overall_design: We have established a ligation-mediated RNA pulldown method (TriplexDNA-seq) to enrich DNA regions that are directly associated with RNA. DNA from biological quadruplicates was sequenced (rep1-4_plus_RNA_ligation), samples omitting ligation serving as control (rep1-4_minus_RNA_ligation).

first: Nevcin

last: Sentürk

first: Chao-Chung

last: Kuo

first: Ingrid

last: Grummt


title: m6A-seq  data analysis of control and PCIF1 knockdown transcriptome

city: BeiJing

first: Kai

last: Li

address: Yi He Yuan Street No.5

department: School of Life Science

institute: Peking University

summary: Purpose: To identify the in vivo RNA targets of PCIF1, we performed meRIP experiments for PCIF1 KD and control cells using an anti-m6A antibody. We envisioned that the cap-specific PCIF1 should selectively alter the m6Am modification at the 5-terminal region of transcripts. Indeed, we observed a reduction of modification peaks at the 5’-end but not the 3’-UTR regions of mRNA upon PCIF1 KD.

overall_design: Methods: Immunoprecipitated RNA fragments were subjected to library construction using NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Library sequencing was performed on llumina Hiseq X10 with paired-end 2x150 bp read length. Processed reads were mapped to human genome (hg19, UCSC Genome Browser) using tophat2 aligner (v2.0.13) with default parameters.

first: Meiling

last: Zhang

first: Hanxiao

last: Sun


title: Genome wide identification of acetyl-CoA regulated loci

first: Kathryn

last: Wellen

address: 421 Curie Blvd BRB II/III RM 632

department: Cancer Biology

summary: In order to identify regions of the genome that display quantitative changes in acetyl-CoA, we utilized an established “spike-in” normalization method that allows the quantitative comparison of histone modifications across cell populations using defined quantities of a reference epigenome. We found that some regions of the genome are more sensitive to changes in acetyl-CoA compared to others.

overall_design: ChIP-Seq of histone H3K27ac in glioblastoma samples treated with different amounts of glucose or acetate with a drosophila cell line as the reference epigenome spike-in.

first: Joyce

middle: V

last: Lee

first: Payel

last: Sen

first: Taehyong

last: Kim

middle: E


title: Heavy chain immunoglobulin sequencing of Lyme disease PBMCs

first: Lisa

middle: K

last: Blum

address: 269 Campus Drive

summary: Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity world-wide. Here, we investigated B cell responses to Lyme disease through molecular identifier-enabled antibody heavy chain sequencing of bulk B cells from PBMCs. Single-cell immunoglobulin sequencing of paired heavy- and light-chain genes from this project will also be separately deposited. Additional information regarding patient characteristics and overlap with other data from the SLICE study is available upon request.

overall_design:
Total mRNA was isolated from peripheral blood mononuclear cells (PBMCs) of Lyme disease patients at four timepoints:
Visit 1, Untreated acute infection; Visit 3, one month after completion of treatment; Visit 5, six months after treatment; Visit 7, two years after treatment.
Total mRNA was isolated from peripheral blood mononuclear cells (PBMCs) of Healthy controls at three timepoints:
Visit H2, initial visit; Visit H3, six months after initial; Visit H4, one year after initial visit.
cDNA was tagged with unique molecular identifiers, amplified with primers specific for immunoglobulin constant regions, and sequenced by HiSeq 2500.

first: William

middle: H

last: Robinson

first: Alison

last: Rebman


title: Derivation of kidney organoids from human pluripotent stem cells [RNA-Seq: Data Set 2]

city: Barcelona

first: Nuria

last: Montserrat

address: Baldiri Reixac, 10-12

country: Spain

institute: Institute for Bioengineering of Catalonia (IBEC)

summary: We have developed a robust methodology for the derivation of kidney organoids from human pluripotent stem cells further vascularized taking advantage of several in vivo approaches. Our methodology suffices for the generation of kidney derivatives transcriptomically comparable with 22 weeks of gestation kidney tissues and highlight the use of longer periods of exposure to 3D in order to promote renal differentiation in a time frame of 15 days. In order to ascertain the impact of extracellular matrix microenvironment in renal differentiation undifferentiated human pluripotent stem cells were further cultured in compliant substrates mirroring embryo-ECM microenvironment (1kPa) in order to promote renal differentiation and compared these conditions in front of rigid substrates (60 kPa) mimicking hard substrates normally used when differentiating human pluripotent stem cells

overall_design: mRNA profiles of undifferentiated and differentiatig cells were generated by deep sequencing, in duplicate, using Illumina system.

first: Carolina

last: Tarantino

first: Luca

last: Cozzuto


title: Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Bulk ATAC-seq]

first: Paul

last: Khavari

first: Adam

last: Rubin

first: Kevin

last: Parker

first: Ansuman

last: Satpathy

first: Howard

last: Chang


title: Primary term human cytotrophoblast, untreated [ChIP-Seq]

status: Public on Dec 19 2018

city: New Brunswick

first: Bingbing

last: Wang

state: New Jersey

address: 125 Paterson St Room 2143

institute: Rutgers RWJMS

summary: Chromatin alterations are important mediators of gene expression changes. We have recently shown that activated non-canonical NF-κB signaling (RelB/p52) recruits histone acetyltransferase CBP and deacetylase HDAC1 to selectively acetylate H3K9 (H3K9ac) to induce expression of corticotropin-releasing hormone (CRH) and prostaglandin-endoperoxide synthase-2 (PTGS2) in the human placenta. Both of these genes play a role in initiating. We performed chromatin immunoprecipitation followed by gene sequencing (ChIP-seq) in primary term human cytotrophoblast (CTB) with use of antibodies to RelB, CBP, HDAC1 and H3K9ac. We further associated these chromatin alterations with gene expression changes from mid-trimester to term in CTB by RNA sequencing (RNA-seq).   We detected a genome-wide differential gene enrichment between mid-trimester and term human placenta. Pathway analysis identified that cytokine-cytokine receptor interaction, NF-κB, and TNF are the leading pathways enriched in term placenta and associated with these chromatin alterations. Our analysis has provided the first-time characterization of the key players of human placental origin with molecular changes resulting from chromatin modifications, which could drive human labor.

overall_design: 3 individual placental specimens from full-term pregnancies were collected from healthy women with estimated gestational age (EGA) of 38 and 40 weeks who were delivered by Cesarean section (C-section). 3 individual mid-trimester placentas were collected from healthy individuals with EGA between18 to 22 weeks following elective termination of pregnancy.


title: Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation [histone marks]

first: Daniel

last: Fuentes

address: 265 Campus Drive, G3065

department: Chemical and Systems Biology

summary: Recent work suggests extensive adaptation of transposable elements (TEs) for host gene regulation. However, high numbers of integrations typical of TEs, coupled with sequence divergence within families, have made systematic interrogation of the regulatory contributions of TEs challenging. Here, we employ CARGO, our recent method for CRISPR gRNA multiplexing, to facilitate targeting of LTR5HS, a higher primate-specific class of HERVK (HML-2) LTRs that is active during early development and present in ~700 copies throughout the human genome. We combine CARGO with CRISPR activation or interference to, respectively, induce or silence LTR5HS en masse, and demonstrate that this system robustly targets the vast majority of LTR5HS insertions. Remarkably, activation/silencing of LTR5HS is associated with reciprocal up- and down-regulation of hundreds of human genes. These effects require presence of retroviral sequences, but occur over long genomic distances, consistent with a pervasive function of LTR5HS elements as early embryonic enhancers in higher primates.

overall_design: Examine histone marks and dCas9 occupancy in WT NCCIT or NCCIT expressing dCas9-VPR or dCas9-KRAB, with CARGO gRNA array targeting LTR5HS

first: Joanna

last: Wysocka


title: Genome expression profiling of Chick pea under Abiotic Stress

city: Allahabad

first: Ashutosh

last: Mani

address: Teliarganj

country: India

institute: MNNIT, Allahabad

overall_design: Organism : Chickpea, Agilent custom Chickpea Gene Expression  8x60k Array (AMADID: 037094) designed by Genotypic Technology Private Limited

first: Birendra

middle: S

last: Yadav


title: Chronic Circadian Misalignment Results in Reduced Longevity and Large-Scale Changes in Gene Expression in Drosophila

city: Chicago

last: Cavanaugh

state: IL

address: 1050 W Sheridan Rd., Life Sciences Building Room 215

department: Biology

institute: Loyola University Chicago

summary: We investigated the consequences of chronic circadian misalignment (CCM) in the powerful model system of the fruit fly, Drosophila melanogaster. We subjected flies to daily 4-hr phase delays in the light-dark schedule and used the Drosophila Activity Monitoring (DAM) system to continuously track locomotor activity and sleep while simultaneously monitoring fly lifespan. Consistent with previous results, we find that exposing flies to CCM leads to a ~15% reduction in median lifespan in both male and female flies. Importantly, we demonstrate that the reduced longevity occurs independent of changes in overall sleep or activity. To uncover potential molecular mechanisms of CCM-induced reduction in lifespan, we conducted whole body RNA-sequencing to assess differences in gene transcription between control and misaligned flies. CCM caused progressive, large-scale changes in gene expression characterized by upregulation of genes involved in response to toxic substances, aging and oxidative stress, and downregulation of genes involved in regulation of development and differentiation, gene expression and biosynthesis. Many of these gene expression changes mimic those that occur during natural aging, consistent with the idea that CCM results in premature organismal decline, however, we found that genes involved in lipid metabolism are overrepresented among those that are differentially regulated by CCM and aging. This category of genes is also among the earliest to exhibit CCM-induced changes in expression, thus highlighting altered lipid metabolism as a potentially important mediator of the negative health consequences of CCM.

overall_design: Humidity and temperature-controlled incubators were used to expose male and female iso31 flies to either a 24-hour schedule (LD 12:12; control group), or a 28-hour schedule (LD 14:14; chronic circadian misalignment (CCM) group) for 2 or 3 weeks. Flies were fed 5% sucrose/ 2% agar food. 10 male and 10 female flies were collected for each sample (3 CCM and 3 control samples at each time point; 12 total) and total RNA was extracted at ZT0-3


title: RNA Sequencing Facilitates Quantitative Analysis of Transcriptomes of human ESCs early hematopoietic differentiation

city: Tianjin

first: Jiaxi

last: Zhou

state: Tianjin

address: 288# Nanjing Road, He ping district

institute: PUMC

summary:
Purpose:hESCs hematopoietic differentiation recapitulates embryonic hematopoiesis in vivo and occurs through three main stages: mesoderm commitment, hemogenic endothelium (HE) formation and hematopoietic specification. The goal of this study are to increase the understanding of hESCs hematopoietic differentiation process and its regulatory mechanism.
Methods: hESC samples were collected through flow cytometry sorting.mRNA profiles of  this samples were generated by deep sequencing using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays
Conclusions: Compared with other three populations, 57 cell surface markers were highly enriched in CD31+CD34+ endothelial cells.

overall_design: hESCs hematopoietic differentiation

first: Hongtao


title: mRNASeq and miRNASeq of z_Q175 knock-in mouse lines with and without neo cassette

city: Princeton

first: Jeff

last: Aaronson

address: 155 Village Boulevard, Suite 200

department: Discovery & Systems Biology

institute: CHDI

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

overall_design: Refer to individual Series


title: miRNASeq of z_Q175 knock-in mouse lines with and without neo cassette

summary: In order to determine the effect of neoR gene in Q175 locus, miRNASeq was performed to compare the transcriptional signatures of zQ175 knock-in neo-in (z_Q175 KI) (CHDI-81003003) and zQ175 KI neo-out (Z_Q175 (neo -) KI) (CHDI-81003019) mouse lines. The z_Q175 KI is a knock-in mouse line with humanized exon 1 (190-200 pure CAG repeats) derived from Q140 KI colony. It contains floxed neo cassette upstream of exon 1. The Z_Q175 KI (neo -) KI was generated by crossing the Z-Q175 KI line with a zp3-cre transgenic line to delete out the neo cassette.

overall_design: miRNASeq was performed on samples from z_Q175 KI (CHDI-81003003) and Z_Q175 (neo -) KI (CHDI-81003019) mice and corresponding wild type littermate controls. Striatum, cortex, and liver tissues were collected N = 8 per group (4M/4F).

first: Jim

last: Rosinski


title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

city: Austin

first: Jonghwan

last: Kim

state: TX

address: 2506 Speedway

department: Molecular Biosciences

institute: University of Texas at Austin

summary: While Yap1, a Hippo pathway transcriptional effector, plays numerous roles in development and cancer, its functions in embryonic stem (ES) cell differentiation remain poorly characterized. It is known that approximately 30% of ES cells die after exiting self-renewal to differentiate, but upstream regulators of this process are not well known. We first observe that ES cells lacking Yap1 experience massive cell death upon the exit from self-renewal. We subsequently reveal that Yap1 contextually protects differentiating, but not self-renewing, mouse ES cells from hyperactivation of the apoptotic cascade. Mechanistically, Yap1 strongly activates anti-apoptotic genes via directly occupying their regulatory elements while mildly suppressing pro-apoptotic genes. Our demonstration of the pro-survival functions of Yap1 during ES cell differentiation expands our understanding of upstream regulators of the balance between survival and death during cell fate changes.

overall_design: ChIP sequencing was performed for Yap1 in male mouse ES cells (strain J1). To perform ChIP-seq for Yap1, we generated Yap1-overexpressing FLAG/biotin-tagged clones, differentiated them for 3 days in ES- medium, and proceeded to formaldehyde-assisted crosslinking. Yap1 was pulled down along with associated DNA fragments using magnetic streptavidin-conjugated beads. ChIP-seq was also performed on BirA cells as a control, which express the BirA enzyme responsible for biotinylation, but do not overexpress any gene of interest.


title: Gene expression profiling of PDX-derived lung squamous carcinoma cells (TUM622) grown in 2D vs. 3D culture

city: Pearl River

first: Kenneth

last: Geles

state: New York

address: 401 N Middletown Rd

department: Oncology Research Unit

institute: Pfizer

summary:
Tumorigenesis depends on intricate interactions between genetically altered tumor cells and their surrounding tumor microenvironment (TME). Investigation of tumor cell-TME interactions could be greatly facilitated by models that mimic human disease on both the genotypic and phenotypic levels. Three dimensional (3D) cultures represent an important means to study the impact of tumor cell-TME interactions on specific aspects of neoplastic phenotypes. Here, we developed a novel 3D culture system (termed TUM622) derived from a patient-derived xenograft (PDX) of a lung squamous carcinoma (LUSC) that was grown in extracellular matrix (Damelin et al, Cancer Research; 71(12) June 15, 2011). Single TUM622 cells established acinar-like structures with proper apical-basal polarity, remained non-motile and demonstrated heterogeneous expression of stem-like and differentiation markers similar to the original PDX model and patient tumor, but nonetheless exhibited hyperplasia. Transcriptional profiling and gene set enrichment analysis (GSEA) revealed many similarities to the established acinar models, as well as modulation of the Wnt signaling pathway and stem cell signatures.  These results demonstrate that the tumor microenvironment significantly influences the plasticity of NSCLC tumor cells.
In order to gain a better understanding of the cell intrinsic mechanisms regulating TUM622 acinar morphogenesis in 3D, we performed transcriptional profiling of TUM622 cells collected from 2D and 3D cultures.We identified genes that vary more than 1.5-fold with reproducible changes among the biological replicates (p < 0.05) and found a total of 9227 genes differentially expressed, indicating that changing culture conditions from 2D to 3D exerts a major influence on gene expression in TUM622 cells.

overall_design: A total of 6 samples, including a triplicate of TUM622 cells in 2D culture and a triplicate of TUM622 cells in 3D culture were harvested by FACS (EpCAM+ cells) and then used for gene expression profiling with the human U133+2 GeneChip

first: Sarah

last: Wyman

first: Wenyan

last: Zhong

first: Christine

last: Loreth


title: MicroRNA-31 reduces the motility of proinflammatory T helper 1 lymphocytes

city: Berlin

first: Pawel

last: Durek

address: Charitéplatz 1

institute: Deutsches Rheuma-Forschungszentrum

summary: We performed  total RNA-Seq of murine Th1 cells which were four times reactivated in vitro in the presence of irradiated APC'srepeatedly activated in vitro.

overall_design: CD4+CD62Lhi (naive) cells were isolated from C57BL/6 mice, activated with aCD3 and aCD28 an cultured under Th1 polarizing conditions in the presence of irradiated APCs. Every sixth day cells were harvested, restimulated with aCD3 and aCD28 and cultured under Th1 polarizing conditions in the presence of irradiated APCs APCs. After four rounds of restimulation, total RNA was extracted and cDNA libraries for total RNA sequencing were generated using “TruSeq® Stranded Total RNA Library” kit (Illumina, San Diego, CA, USA).

first: Markus

last: Bardua

first: Claudia

last: Haftmann

first: Kerstin

last: Westendorf

first: Antje

last: Buttgereit

first: Cam

middle: L

last: Tran

first: Mairi

last: McGrath

first: Melanie

last: Weber

first: Katrin

last: Lehmann

first: Richard

last: Addo

first: Gitta

last: Heinz

first: Anna-Barbara

last: Stittrich

first: Patrick

last: Maschmeyer

first: Helena

last: Radbruch

first: Michael

last: Lohoff

first: Hyun-Dong

first: Andreas

first: Mir-Farzin

last: Mashreghi


title: RNA-Seq of germinal center B cells from Blimp-1-GFP reporter mice

city: Oxford

first: Daniel

last: Radtke


title: Deconstructing Retinal Organoids: single cell RNA-Seq reveals the cellular components of human pluripotent stem cell-derived retina

city: Newcastle Upon Tyne

first: Majlinda

last: Lako

address: International Centre for Life, Central Parkway

department: Institute of Genetic Medicine and Institute for Ageing

institute: Newcastle University

summary: The rapid improvements in single cell sequencing technologies and analyses methods afford greater scope for dissecting organoid cultures composed of multiple cell types and create an opportunity to interrogate these models to understand tissue biology, cellular behaviour and interactions. To this end, retinal organoids generated from human embryonic stem cells (hESCs) were analysed by single cell RNA-Sequencing at three time points of differentiation. Combinatorial data from all time points revealed the presence of nine clusters, five of which corresponded to key retinal cell types, namely retinal pigment epithelium (RPE), retinal ganglion cells (RGCs), cone and rod photoreceptors and Müller glia cells. The remaining four clusters expressed genes typical of mitotic cells, extracellular matrix (ECM) components and those involved in retinal homeostasis. The cell clustering analysis revealed the decreasing presence of mitotic cells and RGCs, formation of a distinct RPE cluster, the emergence of cone and rod photoreceptors from photoreceptor precursors and an increasing number of Müller Glia cells over time. The pseudotime analysis resembled the order of cell birth during retinal development, with the mitotic cluster commencing the trajectory and the large majority of Müller glia being the latest. Together, these data demonstrate the feasibility and potential of single cell RNA-Seq to dissect the inherent complexity of the organoids and the orderly birth of key retinal cell types.

overall_design: A hESC (H9) cell line harbouring a CRX-GFP reporter was differentiated to retinal organoids 25. Samples were collected at 60, 90 and 200 days, dissociated, partitioned into single cells using the Fluidigm C1 Single-Cell mRNA-Seq HT IFC and processed for scRNA-Seq.

first: Joseph

last: Collin


title: Single cell transcriptome profiling of mouse and hESC-derived pancreatic progenitors

city: Vancouver

first: Francis

last: Lynn

state: BC

address: 950 28th Ave W

department: Surgery

country: Canada

institute: University of British Columbia

summary: Human embryonic stem cells (hESCs) are a potential unlimited source of insulin-producing β-cells for diabetes treatment. A greater understanding of how β-cells form during embryonic development will improve current hESC differentiation protocols. As β-cells are formed from NEUROG3-expressing endocrine progenitors, this study focused on characterizing the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors. To do this, 7,223 E15.5 and 6,852 E18.5 single cells were isolated from Neurog3-Cre; Rosa26mT/mG embryos, allowing for enrichment of endocrine progenitors (yellow; tdTomato + EGFP) and endocrine cells (green; EGFP). From a NEUROG3-2A-eGFP CyT49 hESC reporter line (N5-5), 4,497 hESC-derived endocrine progenitor cells were sequenced. Differential expression analysis reveals enrichment of markers that are consistent with progenitor, endocrine, or novel cell-state populations. This study characterizes the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors and serves as a resource (https://lynnlab.shinyapps.io/embryonic_pancreas/) for improving the formation of functional β-like cells from hESCs.

first: Nicole

last: Krentz

first: Michelle

last: Lee

first: Eric

last: Xu

first: Shugo

last: Sasaki

first: Sasha

last: Maslova

first: Shannon

last: Sproul


title: Coupled single-cell epigenome editing and profiling reveals causal gene regulatory networks [Perturb-ATAC (ATAC)]

city: Stanford

first: Paul

last: Khavari

address: 269 Campus Drive, CCSR 2150

institute: Stanford University

summary: Here we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by Assay of Transposase-accessible Chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ~3,700 single cells, encompassing more than 75 unique genotype-phenotype relationships.

overall_design: Single-cell epigenome and perturbation identification in lymphoblastoid cells and epidermal keratinocytes.

first: Adam

last: Rubin

first: Kevin

last: Parker

first: Ansuman

last: Satpathy

first: Howard

last: Chang


title: RNAseq of germinal center B cells from Blimp-1-GFP reporter mice

address: John Radcliffe Hospital, University of Oxford

summary:
RNAseq of germinal center B cells with different Blimp-1-GFP reporter level. Sorted germinal center populations were defined by their expression of Blimp-1-GFP reporter and the cell surface marker CD138 before they were further split into dark zone or light zone / dark zone like or light zone like cells based on their expression of the surface marker CXCR4 and CD83.
We used RNAseq to find genes differentially regulated in various Blimp-1-GFP expressing germinal center populations from the spleen at day 10 after intraperitoneal immunisation with sheep red blood cells.

overall_design: Germinal center B cells (B220+IgDlowCD95+GL7+) from spleens of sheep red blood cell immunised mice with different Blimp-1-GFP expression levels were sorted. In addition naïve B cells (B220+IgD+) and plasma cells (B220intCD138+Blimp-1-GFPhigh) were sorted as a reference. 100 cells per sample and population were sorted in five biological replicates except for naive cells of which three biological replicates were sorted.

first: Oliver

last: Bannard


title: RNA expression in BRCA2-depleted breast cancer cell lines

city: Groningen

first: Francien

last: Talens

address: Hanzeplein 1

department: Medical Oncology

country: Netherlands

institute: University Medical Center Groningen

summary: Next generation RNA sequencing was performed to analyze changes in gene expression upon BRCA2 depletion. BRCA2 depletion was introduced in two human breast cancer cell lines with doxycyclin inducible short hairpin RNAs.

overall_design: Examination of BRCA2 deficiency in 2 different breast cancer cell lines.

first: Marcel

last: van Vugt

first: Anne

last: Heijink

first: Stephanie

last: van Gijn

first: Rudolf

last: Fehrmann


title: Ezh2 knockout and gene expression profiling in CD8+ T cells

city: Ann Arbor

first: Rork

last: Kuick

state: MI

address: University of Michigan, SPH II, 1415 Washington Heights, Room M2533

institute: University of Michigan

summary:
We used 6 male 4 week old C57BL/6 (B6) mice, three with wild-type (WT) genes, the other 3 with conditional knockout (KO) of both Ezh2 alleles. Knockout was achieved by breading B6 mice with floxed alleles of Ezh2 (Ezh2-fl/fl) to B6 mice expressing Cre recombinase under the control of a CD4 promotor to generate T-cell specific homozygous Ezh2 conditional knockout (CKO) mice.  CD8 T-cells were purified using anti-mouse CD8 antibody (Ab)-conjugated magnetic beads from the spleen and lymph nodes of WT and CKO mice. The purity was consistently more that 93% as assessed by flow cytometry analysis. Freshly isolated mature T cells were named naïve CD8 T cells. These naïve CD8 T cells (two million cells / ml) were cultured in media containing 10% fetal bovine serum-IMDM in the presence of plate-coated anti-CD3 Ab (3 mg /ml) + anti-CD28 Ab (3 mg /ml). Three days later, these in vitro stimulated CD8 T cells were collected and named activated CD8 T cells.  About 200 ng total RNA was extracted, and amplified using small-sample methods to make biotin-labeled cDNA targets that were hybridized to Affymetrix HT Mouse 430 PM arrays. This resulted in 4 groups of samples: naive or activated cells that were either KO or WT for Ezh2, each group having three biological replicates. The samples are in theory paired, since pairs of samples came from the same mouse, however we did not find many significant mouse to mouse effects, and do not think they need to be modeled.  We provide a supplementary Excel workbook that holds the expression data, but it also holds probe-set annotation, and some statistical calculations (and the functions used to compute them), and may be convenient for some users.
As of Dec 20, 2018, we were not expecting to use this data in a publication in the near future, and so released it to the public.

overall_design: We used 6 male 4 week old C57BL/6 (B6) mice, three with wild-type (WT) genes, the other 3 with conditional knockout (KO) of both Ezh2 alleles.  Knockout was achieved by breading B6 mice with floxed alleles of Ezh2 (Ezh2-fl/fl) to B6 mice expressing Cre recombinase under the control of a CD4 promotor to generate T-cell specific homozygous Ezh2 conditional knockout (CKO) mice. CD8 T cells were purified using anti-mouse CD8 antibody (Ab)-conjugated magnetic beads from the spleen and lymph nodes of WT and CKO mice. The purity was consistently more that 93% as assessed by flow cytometry analysis. Freshly isolated mature T cells were named naïve CD8 T cells. These naïve CD8 T cells were cultured in media containing 10% fetal bovine serum-IMDM in the presence of plate-coated anti-CD3 Ab (3 mg /ml) + anti-CD28 Ab (3 mg /ml). Three days later, these in vitro stimulated CD8 T cells were collected and named activated CD8 T cells.  About 200 ng total RNA was extracted, and amplified using small-sample methods to make biotin-labeled cDNA targets that were hybridized to Affymetrix HT Mouse 430 PM arrays. This resulted in 4 groups of samples: naive or activated cells that were either KO or WT for Ezh2, each group having three biological replicates.

first: Yi

last: Zhang


title: Derivation of kidney organoids from human pluripotent stem cells

first: Nuria

last: Montserrat


title: Derivation of kidney organoids from human pluripotent stem cells [RNA-Seq: Data Set 1]

summary: We have developed a robust methodology for the derivation of kidney organoids from human pluripotent stem cells further vascularized taking advantage of several in vivo approaches. Our methodology suffices for the generation of kidney derivatives transcriptomically comparable with 22 weeks of gestation kidney tissues and highlight the use of longer periods of exposure to 3D in order to promote renal differentiation in a time frame of 15 days.

first: Carolina

last: Tarantino

first: Luca

last: Cozzuto


title: Effect of Smurf1 or Smurf2 deficiency on Gene Expression Profiling of aged mouse liver tissues

city: Bethesda

first: Ying

state: Maryland

address: 37 Convent Dr., Bldg.37, Rm.2056

department: LCMB/CCR

institute: National Cancer Institute, NIH

summary:
Transcriptome analysis of RNAs extracted from livers of wild type or Smurf1 knock out (KO) or Smurf2 KO mice at age of 11 month old.
We prepared RNA from the following groups: livers of Wild type (WT) or Smurf1 KO or Smurf2 KO mouse from mixed Black Swiss/129SvEv background at age of 11 month old. The gene expression profiles were compared, and selected genes that showed either increased or decreased expression by a cut-off of 1.5 folds, p< 0.05.  The results showed that   many genes that are involved in lipid metabolism were upregulated in Smurf1-/- livers.  These results also indicate that Smurf1 plays a more prominent role in the liver than Smurf2.

overall_design: We analyzed total RNA from mouse livers of the following group: Wild type (WT), Smurf1 knock out (KO) or Smurf2 KO from mixed Black Swiss/129SvEv background at age of 11 month old using the Affymetrix GeneChip® Mouse Gene 1.0 ST arrayArray platform. Biological Duplicates were performed, no techinical replicates were performed.

first: Kun

last: Zhu


title: Coupled single-cell CRISPR screening and epigenomic profiling reveals causal gene regulatory networks

first: Paul

last: Khavari


title: Xenopus hybrids provide insight into cell and organism size control

city: Ulsan

first: Taejoon

last: Kwon

address: 50 Unist-gil

department: Department of Biomedical Engineering

country: South Korea

institute: Ulsan National Institute of Science and Technology

summary: Determining how size is controlled is a fundamental question in biology that is poorly understood at the organismal, cellular and subcellular levels. The Xenopus species, X. laevis and X. tropicalis differ in size at all three of these levels. Despite these differences, fertilization of X. laevis eggs with X. tropicalis sperm gives rise to viable hybrid animals that are intermediate in size. Hybrid and X. laevis embryos initially start from the same size egg and develop at the same rate, but hybrid animals were smaller by the tailbud stage, and reduced nuclear to cell size ratios were observed shortly after zygotic genome activation (ZGA), indicating that differential gene expression may contribute to size differences. Transcriptome analysis at the onset of ZGA identified twelve transcription factors paternally expressed in hybrids. A screen of these X. tropicalis factors by expression in X. laevis embryos revealed that Hes7 and Ventx2 were sufficient to significantly reduce X. laevis organismal size by the tailbud stage, although nuclear to cell size ratios were not significantly affected. Together, these results suggest that transcriptional regulation contributes to biological size control in Xenopus.

sample_organism:
Xenopus laevis
Xenopus tropicalis
Xenopus tropicalis x Xenopus laevis

overall_design: Collect mRNA from whole embryos; three biological replicates were analyzed

first: Romain

last: Gibeaux

first: Kelly

last: Miller

first: Rachael

last: Acker

first: Rebecca

last: Heald


title: A High-Throughput Screen Identifies DYRK inhibitor ID-8 that Stimulates Human Kidney Tubular Proliferation

city: Boston

first: Lorena

last: Pantano

address: 655 Huntington Ave

department: Biostatistics

institute: Harvard Chan School of Public Health

summary: Use NGS-transcriptome profiling (RNA-seq) to investigate deregulated genes involved in the proliferative effects of ID-8 and Harmine after hypoxia-induced damage in primary human proximal tubular epithelial cells (HPTECs)

overall_design: Examination of differentially expressed genes in HPTECs treated with 1uM of ID-8; or 1uM of Harmine; or EGF in comparison to cells without treatment after 24 hours of hypoxia, in triplicates

first: Maria

middle: B

last: Monteiro


title: Development of a 3D Bone Marrow Adipose Tissue Model

city: Northfield

first: Heather

middle: Elizabeth

last: Driscoll

state: VT

address: 158 Harmon Drive

institute: Norwich University

summary:
Herein we compare mouse (C57B6/J background, 16 week old) adherent bone marrow stromal cell gene expression after 4 weeks of adipogenic differentiation in 3D versus 2D culture.
Adipogenic differentiation was compared on a 3D silk scaffold versus a 2D (tissue culture plastic) surface using an microarray mRNA analysis.

overall_design: Total RNA was recovered from 10 mouse adipose tissue cultures differentiated from whole bone marrow from 5 mice. Five replicates from 3D silk scaffold cultures and five replicates from 2D tissue culture plastic were run on Affymetrix® ClariomTM S Arrays.

first: Michaela

last: Reagan

middle: F

last: Campbell


title: Molecular variability of patient-derived colon tumor xenografts

city: Warszawa

first: Krzysztof

last: Goryca

address: Banacha 2c

department: CeNT

country: Poland

institute: Uniwersytet Warszawski

summary: Primary tumors and their corresponding P2 mouse xenografts were surveyed for gene expression using the RNA-sequencing approach.

overall_design: 20 primary colon tumours ("p0"), their respective P2 xenografts ("p2") and 17 primary colon tumours for which creating a xenograft failed ("noPDX").

first: Michal

last: Mikula

first: Agnieszka

last: Paziewska

first: Katarzyna

last: Paczkowska

first: Michalina

last: Dabrowska

first: Magdalena

last: Cybulska

first: Jerzy

last: Ostrowski


title: Colorectal Adenomas and Consensus Molecular Subtyping

city: Houston

first: Eduardo

last: Vilar

address: 1515 Holcombe Blvd., Unit 1360

department: Clinical Cancer Prevention

institute: The University of Texas MD Anderson Cancer Center

summary: Consensus molecular subtyping in adenomatous and serrated polyps

overall_design: 5 samples. Colonic tubular adenoma from patient with Lynch Syndrome (LS)


title: mRNASeq of z_Q175 knock-in mouse lines with and without neo cassette

first: Jeff

last: Aaronson

summary: In order to determine the effect of neoR gene in Q175 locus, mRNASeq was performed to compare the transcriptional signatures of zQ175 knock-in neo-in (z_Q175 KI) (CHDI-81003003) and zQ175 KI neo-out (Z_Q175 (neo -) KI) (CHDI-81003019) mouse lines. The z_Q175 KI is a knock-in mouse line with humanized exon 1 (190-200 pure CAG repeats) derived from Q140 KI colony. It contains floxed neo cassette upstream of exon 1. The Z_Q175 KI (neo -) KI was generated by crossing the Z-Q175 KI line with a zp3-cre transgenic line to delete out the neo cassette.

overall_design: mRNASeq was performed on samples from z_Q175 KI (CHDI-81003003) and Z_Q175 (neo -) KI (CHDI-81003019) mice and corresponding wild type littermate controls. Striatum, cortex, and liver tissues were collected N = 8 per group (4M/4F).

first: Jim

last: Rosinski


title: REV-ERBa regulates TH17 cell development and autoimmunity

city: La Jolla

first: Alain

last: Domissy

address: 10550 North Torrey Pines Road

department: Immunology

institute: The Scripps Research Institute

summary: RORγt is well recognized as the lineage defining transcription factor for TH17 cell development.  However, the cell-intrinsic mechanisms that negatively regulate TH17 cell development and autoimmunity remain poorly understood.  Here we demonstrate that the transcriptional repressor REV-ERBa is exclusively expressed in TH17 cells, competes with RORγt for their shared DNA consensus sequence, and negatively regulates TH17 cell development via repression of genes traditionally characterized as RORγt-dependent, including Il17a. Deletion of REV-ERBa enhanced TH17-mediated pro-inflammatory cytokine expression, exacerbating experimental autoimmune encephalomyelitis (EAE) and colitis. Treatment with REV-ERB-specific synthetic ligands, which have similar phenotypic properties as RORγ modulators, suppressed TH17 cell development, was effective in colitis intervention studies, and significantly decreased the onset, severity, and relapse rate in several models of EAE without affecting thymic cellularity.  Our results establish that REV-ERBa negatively regulates pro-inflammatory TH17 responses in vivo and identifies the REV-ERBs as potential targets for the treatment of TH17-mediated autoimmune diseases.

overall_design: 10 samples; 5 conditions with 2 replicates per condition

first: Laura

last: Solt


title: Transgenerational inheritance of chronic adolescent stress: Effects of stress response and the amygdala transcriptome

city: Philadelphia

first: Melissa

middle: T

last: Manners

address: 125 S 31st Street

institute: University of Pennsylvania

summary: Adolescent stress can impact health and well-being not only during adulthood of the exposed individual but even in future generations. To begin to unravel the molecular mechanisms underlying these long-term effects, we determined if stress administered to males during adolescence (F0) altered anxiety behaviors and gene expression profiles in the amygdala – a critical region in the control of emotional states – of their progeny in two generations (F1, F2). Male C57BL/6 mice underwent chronic unpredictable stress (CUS) for two-weeks during adolescence and were used to produce two generations of offspring. Male and female F1 and F2 animals were tested in behavioral assays to measure affective behavior and stress reactivity. Remarkably, transgenerational inheritance of paternal stress exposure produced a protective phenotype in the male, but not the female lineage. In behaviorally naïve mice (n = 5), mRNA from the amygdala was sequenced to determine the total transcriptomes and pathway analysis was employed to identify differentially expressed genes of functional interest.  RNA-Seq analysis of the amygdala from F1 and F2 male offspring identified genes with altered expression in mice derived from fathers exposed to CUS. Among the differentially expressed pathway was ‘notch signaling’, which was significantly altered in F2 males. Therefore, we show that paternal stress exposure impacts future generations which manifest in behavioral changes and molecular adaptations.

overall_design: Amygdala of F1 or F2 mice from stress or no stress lineage. All conditions have 5 biological replicates

first: Nicole

last: Yohn

first: Nicholas

last: Lahens

first: Gregory

middle: R

last: Grant

first: Marisa

last: Bartolomei

first: Julie

last: Blendy


title: Mechanistic and pharmacological assessment of murine IL-23 mediated psoriasiform dermatitis; implications for drug discovery

status: Public on Dec 18 2018

city: Cambridge

first: Radhika

last: Goenka

address: 200 Sidney St.

institute: Abbvie

summary: The goal of our study is to  is to provide a comprehensive disease and pharmacological assessment of IL-23 driven skin inflammation and its similarity to human psoriatic skin.

overall_design: RNA-seq of vehicle and IL-23 treated mice

first: Stephen

last: Gauld


title: Genome-wide RNA-seq and ATAC-seq analysis of an in vivo human immune response to acute Zika virus infection

first: Aaron

last: Carlin

state: California

address: 9500 Gilman Drive GPL Rm 118

department: Pathology and Medicine

institute: University of California, San Diego

summary: A longitudinal systems immunologic investigation of acute Zika virus infection in an individual infected with ZIKV while traveling to Caracas, Venezuela during the zika virus epidemic. Includes longitudinal RNA-seq and ATAC-seq characterization of innate and adaptive immune responses during an acute ZIKV infection.

overall_design: Blood was collected from an individual with NAAT confirmed acute Zika virus infection on four occasions d3, d6, d17, and d48 post-onset of symptoms (POS). Peripheral blood mononuclear cells were isolated from heparinized blood using Histopaque-1077 per manufacturers instructions and subjected to flow activated cell sorting (FACS) to characterize and isolate innate and adaptive immune cell populations. 5000-15,000 live PBMCs or individual cell populations were used to perform low input RNA-seq using the Smart-seq2 method with some modifications. 50,000 FACS isolated live classical monocytes or NK cells were used for ATAC-seq analysis.

first: Sujan

last: Shresta

first: Chris

last: Glass


title: NextPBM - A platform to study cell-specific transcription factor binding and cooperativity

first: Trevor

last: Siggers

address: 24 Cummington Mall

department: Department of Biology

institute: Boston University

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

overall_design: Refer to individual Series


title: NextPBM - A platform to study cell-specific transcription factor binding and cooperativity [array]

summary: High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM (nuclear extract protein-binding microarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase-treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity at single-nucleotide resolution.

overall_design: Protein-binding microarray experiments performed using nuclear extract and designed to profile cell-specific PU.1 and IRF8 binding to genomic canonical ETS elements and ETS-IRF composite elements. Nuclear extract conditions include: untreated THP-1 nuclear extract (2 replicates), nuclear extract from a line of IRF8 (-/-) THP-1 cells, pan-phosphatase-treated THP-1 nuclear extract, IRF8 immune-depleted THP-1 nuclear extract, and a THP-1 nuclear extract concentration gradient (1/2, 1/4, and 1/8). Results were compared to those obtained using in vitro transcribed/translated PU.1. Binding of PU.1 in the untreated THP-1 nuclear extract is compared to related ETS factor FLI1 as a control.

first: David

last: Bray


title: Metabolism as an early predictor of DPSCs aging

city: Seattle

first: Hannele

last: Ruohola-Baker

state: WA

address: 850 Republican Street

institute: Institute for Stem Cell and Regenerative Medicine

summary: Pluripotent stem cells can switch their unique metabolic requirements to facilitate cellular changes but it is not clear if adult stem cells utilize metabolism in a similar manner. Here we studied the metabolism of a human adult stem cell: dental pulp stem cells (DPSCs).  The dental pulp from third molars of a diverse patient group was surgically extracted, generating cells that had a high percentage of mesenchymal stem cell markers CD29, CD44, CD146 and Stro1 and had the ability to differentiate into osteogenic and adipogenic lineages. Through RNA seq analysis we identified homeobox protein, Barx1, as a marker for DPSCs. Furthermore, using high throughput proteomic analysis we identified markers for DPSC populations with accelerated replicative senescence. In particular, we show that the transforming growth factor-beta (TGF-β) pathway and the proteins associated with muscle contraction are upregulated in rapid aging DPSCs, indicating a loss of stem cell characteristics and spontaneous initiation of terminal differentiation. Importantly, using metabolic flux analysis, we identified a metabolic signature for the rapid aging DPSCs.  This metabolic signature can be used to predict the onset of replicative senescence phenotypes.  Hence, the present study identifies Barx1 as a DPSCs marker and dissects the first predictive metabolic signature for DPSCs aging.

overall_design: We did RNA-seq of dental pulp stem cells (DPSC) using our own approach (ID# 29, 43, 44, 45), as well as commercial DPSC and mesenchymal stem cells (MCS) from Lonza.

first: Yuliang

last: Wang


title: DNA methylation analysis of melanoma cell lines SK28 and MEL501

city: Camden

first: Jaroslav

last: Jelinek

state: NJ

address: 403 Haddon Avenue

department: Research Labs

institute: Coriell Institute for Medical Research

type: Methylation profiling by high throughput sequencing

summary: Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment.  In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints.  As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed.  We reasoned that the base excision repair enzyme Thymine DNA Glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation.  Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence and death by mitotic alterations, alters the transcriptome and methylome, and impairs xenograft tumor formation.  Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity.  These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target.  By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.

overall_design: Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SK28 and MEL501 melanoma cells with TDG knockdown by lentiviral shRNA and empty vector control, both in triplicate.  Briefly, genomic DNA was sequentially digested by SmaI and XmaI, which both recognize the sequence CCCGGG. SmaI is methylation sensitive, where XmaI is methylation insensitive. Distinct signatures, 5’-GGG at unmethylated sites or 5’-CCGGG at methylated sites were created by enzyme digestion and ultimately high througput sequencing was used to map these sites to the genome. Methylation ratios for each individual CCCGGG site were calculated as a proportion of methylated counts to the sum of unmethylated and methylated counts, and subsequently adjusted for differences in restriction enzyme efficiency using values obtained from spiked in standards.

first: Pietro

last: Mancuso

first: Rossella

last: Tricarico

first: Vikram

last: Bhattacharjee

last: Cosentino

first: Yuwaraj

last: Kadariya

first: Emmanuelle

last: Nicolas

first: Margret

last: Einarson

first: Neil

last: Beeharry

first: Karthik

last: Devarajan

first: Richard

middle: A

last: Katz

first: Dorjbal

middle: G

last: Dorjsuren

first: Hongmao

last: Sun

first: Anton

last: Simeonov

first: Antonio

last: Giordano

first: Joseph

last: Testa

first: Guillaume

last: Davidson

first: Irwin

first: Lionel

last: Larue

first: Robert

middle: W

last: Sobol

first: Timothy

last: Yen

first: Alfonso

last: Bellacosa


title: Translational profiling of dorsal root ganglia and spinal cord in a mouse model of neuropathic pain

city: Montreal

first: Arkady

last: Khoutrosky

state: Quebec

address: 3655 Prom. Sir-William-Osler

department: Department of Anesthesia

institute: McGill University

summary: Acute pain serves as a protective mechanism, guiding the organism away from actual or potential tissue injury. In contrast, chronic pain is a debilitating condition without any obvious physiological function. The transition to, and the maintenance of chronic pain require new gene expression to support biochemical and structural changes within the pain pathway. The regulation of gene expression at the level of mRNA translation has emerged as an important step in the control of protein expression in the cell. Recent studies show that signaling pathways upstream of mRNA translation, such as mTORC1 and ERK, are upregulated in chronic pain conditions, and their inhibition effectively alleviates pain in several animal models. Despite this progress, mRNAs whose translation is altered in chronic pain conditions remain largely unknown. Here, we performed genome-wide translational profiling of dorsal root ganglion (DRG) and spinal cord dorsal horn tissues in a mouse model of neuropathic pain, spared nerve injury (SNI), using the ribosome profiling technique. We identified distinct subsets of mRNAs that are differentially translated in response to nerve injury in both tissues. We discovered key converging upstream regulators and pathways linked to mRNA translational control and neuropathic pain. Our data are crucial for the understanding of mechanisms by which mRNA translation promotes persistent hypersensitivity after nerve injury.

overall_design: Four conditions were analysed, with each condition having duplicate samples. That is, eight tissue samples were collected. The eight samples were lysed and the lysate was divided into two parts, one was used to prepare a ribosome footprint library and the other fraction was used to prepare whole mRNA library. Thus, we had 16 libraries in total for sequencing. The four conditions studied were: spinal_cord_SNI with spinal_cord_SHam as control and DRG_SNI with SRG_sham as control.

first: Sonali

last: Uttam

first: Calvin

last: Wong

first: Inês

last: Amorim

first: Seyed

middle: M

last: Jafarnejad

first: Shannon

middle: N

last: Tansley

first: Jieyi

last: Yang

first: Masha

last: Prager-Khoutorsky

first: Jeffrey

last: Mogil

first: Christos

last: Gkogkas

last: Khoutorsky


title: HSCs contribute actively to native multilineage hematopoiesis but with reduced differentiation capacity upon aging

city: Lund

first: Shamit

last: Soneji

address: Solvegatan 19

country: Sweden

institute: BMC

summary: A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age

overall_design: Lineage tracing from adult/aged HSCs in steady state

first: Petter

last: Säwén

first: Eva

last: Erlandsson

first: David

last: Bryder


title: Argonaute 2 drives miR-145-dependent gene expression program in breast cancer cells

city: rome

first: andrea

last: sacconi

address: via elio chianesi

country: Italy

institute: regina elena cancer institute

summary: miR-145 acts as a tumor suppressor miRNA in breast cancer cells expressing Argonaute-2 protein (AGO2), while in the absence of AGO2 miR-145 loses its inhibitory effect on migration. To identify mRNAs that are putatively targeted by miR-145 in breast cancer cells, we exogenously expressed miR-145 in MDA-MB-231 cells, in presence (mimic145) or absence (sirnaAGO2-mimic145) of AGO2 expression. mRNAs modulated by interference of AGO2 alone were also evaluated (sirnaAGO2).

overall_design: Experiment was performed in biological triplicate and includes: 1) MDA-MB-231 cells of control (cntrl), transfected with mimic and siRNA control oligonucleotides; 2) MDA-MB-231 cells overexpressing miR-145 (mimic145); 3) MDA-MB-231 cells interfered for AGO2 expression (sirnaAGO2); 4) MDA-MB-231 cells overexpressing miR-145 and interfered for AGO2 expression (sirnaAGO2+mimic145).

first: Francesco

last: Fazi

first: Giovanni

last: Blandino


title: NextPBM - A platform to study cell-specific transcription factor binding and cooperativity [ChIP-Seq]

first: Trevor

last: Siggers

state: MA

summary: We analyzed binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity using nextPBM, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative factors (C/EBPa)

overall_design: Genome-wide binding of 3 monocyte/macrophage lineage-determining TFs (PU.1, C/EBPa, and IRF8) and locations of enhancer-related histone marks (H3K4me1 and H3K27ac) in THP-1 cells. Each experiment was performed across 2 biological replicates. Biological replicates were analyzed separately up until the peak filtering step where the Irreproducible Discovery Rate (IDR) was used as a cross-replicate reproducibility filter. Results were then used in the design of a custom array for the nextPBM protein-binding experiments

last: Bray


title: Ezh2 regulates Langerhans cell migration and host protection against allergic contact dermatitis

city: Singapore

first: Kaibo

last: Duan

address: 8A Biomedical Grove, Immunos Building, Level 4

country: Singapore

institute: Singapore Immunology Network

summary: We analysed  by bulk RNA seq the impact of the depletion of EZH2 on Langerhans cells

overall_design: Langerhans cell from the skin of WT and EZH2 KO mice have been sorted and their transcriptomic profile has been analysed by bulk RNA seq

first: Jia

last: Loh

first: Baptiste

last: Janela

first: Florent

last: Ginhoux

first: I-hsin

last: Su


title: Mitochondrial complex III is essential for regulatory T cell suppressive function

city: Chicago

first: Benjamin

middle: D.

last: Singer

state: Illinois

address: 303 E. Superior St., Simpson Querrey 5th floor

institute: Northwestern University Feinberg School of Medicine


title: Mitochondrial complex III is essential for regulatory T cell suppressive function. [DNA methylation/RRBS]

summary: Regulatory T cells (Treg cells), a distinct subset of CD4+ T cells, are necessary for the maintenance of immune self-tolerance and homeostasis. Recent studies have demonstrated that Treg cells display a unique metabolic profile characterized by an increase in mitochondrial metabolism relative to other CD4+ effector subsets. Furthermore, the Treg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration; however, it remains unknown whether the mitochondrial respiratory chain is required for Treg cell suppressive capacity, stability, and survival. Here we report that Treg cell-specific ablation of mitochondrial respiratory chain complex III results in the development of a fatal inflammatory disease early in life, without impacting Treg cell number. Mice lacking complex III specifically in Treg cells displayed a loss of Treg cell suppressive capacity without altering Treg cell proliferation and survival. Treg cells deficient in complex III display decreased expression of genes associated with Treg function while maintaining stable FOXP3 expression. Complex III-null Treg cells displayed increased DNA methylation at the loci of differentially down-regulated genes without a global increase in DNA methylation. Loss of complex III in Treg cells resulted in buildup of the metabolites 2-hydroxyglutarate (2-HG) and succinate that can function as inhibitors of α-ketoglutarate (α−KG)-dependent dioxygenase reactions such as the ten-eleven translocation (TET) family of DNA demethylases. Thus, Treg cells require mitochondrial respiration to maintain immune regulatory gene expression and suppressive function.

overall_design: Each experiment contains either WT controls animals compared to KO animals, or untreated controls compared to treated conditions.

first: Samuel

last: Weinberg

middle: D

first: Elizabeth

last: Steinert

first: Carlos

last: Martinez

first: Manan

last: Mehta

first: Inmaculada

last: Martinez-Reyes

first: Peng

last: Gao

first: Kathryn

last: Helmin

first: Hiam

last: Abdala-Valencia

first: Laura

last: Sena

first: Paul

last: Schumacker

first: Laurence

last: Turka

first: Navdeep

last: Chandel


title: Mitochondrial complex III is essential for regulatory T cell suppressive function. [gene expression/RNA-seq]

middle: E


title: Genome wide analysis of chick limb bud tissue treated with FGF2 for 24hrs (microarray)

city: Loma Linda

first: Kerby

middle: Clyde

last: Oberg

state: CA

address: 24785 Stewart Street

department: Pathology and Human Anatomy

institute: Loma Linda University

summary:
During limb development, fibroblast growth factors (FGFs) govern proximal-distal outgrowth and patterning.  FGFs also synchronize developmental patterning between the proximal-distal and anterior-posterior axes by maintaining sonic hedgehog (SHH) expression in cells of the zone of polarizing activity (ZPA) in the distal posterior mesoderm. SHH, in turn, maintains FGFs in the apical ectodermal ridge (AER) which caps the distal tip of the limb bud. Crosstalk between FGF and SHH signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well characterized.
Implantation of FGF beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3hrs after application), while prolonged exposure (24hrs) can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations enriched molecules required for FGF-mediated SHH regulation.

overall_design:
Total RNA was extracted from SHH-expressing chick limb bud tissue following 24hrs of FGF2 treatment and compared to PBS-treated control
-------------------------------
Authors state that they are "unable to locate the CEL files".

first: Billy

last: Watson

first: Jennifer

last: Feenstra

first: John

last: Van Arsdale

first: Karndeep

last: Rai-Bhatti

first: Diana

last: Kim

first: Ashley

last: Coggins

first: Gennaya

last: Mattison

first: Eric

last: Steinman

first: Charmaine

middle: U

last: Pira

first: Brendan

last: Gongol

middle: C


title: Systematic Analysis of Transcriptomic Profile of Chondrocytes in Osteoarthritis Using Next-Generation Sequencing and Bioinformatics

city: Kaohsiung

first: Yi-Jen

last: Chen

address: 100, Shih-Chuan 1st Road

department: Graduate Institute of Clinical Medicine, College of Medicine

country: Taiwan

institute: Kaohsiung Medical University

summary: The phenotypic change of chondrocytes and the interplay between cartilage and subchondral bone in osteoarthritis (OA) has received much attention. Structural changes with nerve ingrowth and vascular penetration within OA cartilage may contribute to arthritic joint pain. The aim of this study was to identify differentially expressed genes and potential miRNA regulations in OA knee chondrocytes through next-generation sequencing and bioinformatics analysis. Results suggested the involvement of SMAD family member 3 (SMAD3) and Wnt family member 5A (WNT5A) in the growth of blood vessels and cell aggregation, representing features of cartilage damage in OA. Additionally, 26 dysregulated genes with potential miRNA⁻mRNA interactions were identified in OA knee chondrocytes. Myristoylated alanine rich protein kinase C substrate (MARCKS), epiregulin (EREG), leucine rich repeat containing 15 (LRRC15), and phosphodiesterase 3A (PDE3A) expression patterns were similar among related OA cartilage, subchondral bone and synovial tissue arrays in Gene Expression Omnibus database. The Ingenuity Pathway Analysis identified MARCKS to be associated with the outgrowth of neurite, and novel miRNA regulations were proposed to play critical roles in the pathogenesis of the altered OA knee joint microenvironment. The current findings suggest new perspectives in studying novel genes potentially contributing to arthritic joint pain in knee OA, which may assist in finding new targets for OA treatment.

overall_design: Next-generation sequencing for mRNA and small RNA of normal and OA chondrocytes

first: Wei-An

last: Chang

first: Ling-Yu

last: Wu

first: Ya-Ling

last: Hsu

first: Chia-Hsin

first: Po-Lin

last: Kuo


title: ChIP-Seq: Unstimulated and BMP9/10 Stimulated Endothelial Cells

city: New Orleans

first: Stryder

last: Meadows

state: LA

address: 6400 Freret

department: Cell and Molecular Biology

country: USA

institute: Tulane University

summary: To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed ChIP-sequencing experiments on BMP9 stimulated Ms1 endothelial cells (ECs). This data provides a comprehensive list of all SMAD4 binding sites in endothelial cells.

overall_design: We sequenced a total of 8 samples for this experiment. For the nonstimulated dataset, we have 2 input samples and 2 Smad4 IP samples. The SMAD4 antibody used for immunoprecipitation was Cell Signaling 46535. The BMP9/10 used for stimulation was obtained through R&D catalog #5566 and #6038 (see publication for further information). Likewise for the stimulated dataset, we have two input samples and two SMAD4 IP samples.

first: A

last: Crist

first: J

last: Garai

last: Zabaleta

first: S


title: RNA-Seq: Smad4-iECKO vs Smad4f/f P7 isolated retinal endothelial cells (iECKO=inducible, endothelial cell specific knockout)

summary: To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed RNA-seq on endothelial cells (ECs) isolated from a Smad4 inducible, EC specific knockout (Smad4-iECKO; Smad4f/f;Cdh5-CreERT2) mouse model that develops retinal AVMs.

overall_design:
We sequenced a total of 6 samples. We used three wild type samples (Smad4f/f- samples names: Lit38s45, Lit38s6, Lit40s56) and three mutant samples (Smad4f/f;Cdh5-CreERT2- sample names: Lit38s12, Lit38s37, Lit40s12).

For more detailed information please see supplemental document: GSE116230_Smad4ff_vs_Smad4iECKO.report.pdf


title: Genome wide analysis of chick limb bud tissue treated with FGF2 for 3hrs (RNA-Seq)

first: Kerby

last: Oberg

summary: During limb development, in addition to  proximal to distal outgrowth, FGF regulates a number of factors necessary for SHH expression. To elucidate the molecules involved in FGF-mediated SHH expression we implanted FGF2 beads in HH23 chicken forelimb buds and excised the SHH-expressing tissue for transcriptome analysis. The hypothesis is that this transcriptome data along with data from SHH-expressing tissue after a 24hr treatment would highlight molecules necessary for SHH FGF-mediated SHH expression. Results from the overlap provide a short-list of SHH-related genes which require further testing to assess their role in mediating SHH expression.

overall_design: Total RNA extracted from SHH-expressing chick limb bud tissue following 3hrs of FGF2 treatment compared to PBS-treated controls. Tissue from 15-20 limbs were pooled based on the treatments groups (PBS vs FGF) for each sample. 3 FGF and 3 PBS samples were submitted for RNA-seq analysis.

first: Billy

last: Watson

first: Jennifer

last: Feenstra

first: John

last: Van Arsdale

first: Karndeep

middle: S

middle: H

middle: L


title: Genome wide analysis of chick limb bud tissue treated with FGF2 for 3 hrs


title: CNV Identification: Prostate Cancer (PCa) vs Benign Prostatic Hyperplasia (BPH) Human DNA Samples

city: Wilayah Persekutuan Kuala Lumpur

first: Prevathe

last: Poniah

address: Lembah Pantai

department: Pharmacology

country: Malaysia

institute: University of Malaya

type: Genome variation profiling by genome tiling array

summary: Copy number variations (CNVs) in the human genome have been linked to various carcinomas including prostate cancer (PCa). This study was conducted to identify CNVs in high grade PCa. We performed a pilot genome-wide CNV analysis in 36 subjects (18 high grade PCa and 18 benign prostatic hyperplasia) using array comparative genomic hybridization (aCGH) technique. Array results were validated using PCR-based copy number counting method. A total of 339 CNV regions were found to be unique to PCa subjects in this cohort (P < 0.05). Data segregation and filtering revealed six putative CNV loci associated with susceptibility to PCa. Of these, four were rare (1q21.3, 15q15, 3q27.2 and 7p12.1) and one was a novel copy number gain (12q23.1), harbouring genes such as the ARNT, THBS1, SLC5A8 and DDC which are crucial in the p53 and cancer pathways. Another CNV was a loss at 8p11.21 which contains the SFRP1 gene from the Wnt signalling pathway, known for its interaction with androgen receptors as reported for urological malignancy. Cross comparison analysis with genes already known to be associated with PCa revealed significant CNVs involved in crucial biological processes that elicit cancer pathogenesis via cytokine production, disease progression through endothelial cell proliferation and xenobiotic metabolism. In conclusion, these findings suggest that the CNV regions identified could provide an insight into the development of high grade PCa.

overall_design: 36 samples were analysed for the purpose of CNV identification (discovery cohort) and 46 other samples ( a different subset of samples) were included in the validation cohort. For the discovery cohort, 18 PCa and 18 BPH samples were used as case and control respectively. Controls in this study was used as the reference itself in order to avoid misinterpretation of data due to reference sample of a different genetic makeup i.e. Yoruba Male- provided in the kit.

first: Shamsul

last: Zain

first: Azad

last: Razack

first: Shanggar

last: Kuppusamy

first: Zahurin

last: Mohamed


title: Next-generation sequencing analysis of Abcg2-expressing vascular endothelial stem cells and mature endothelial cells

city: New York

first: Yang

last: Lin

state: NY

address: 455 Main Street 06W

institute: Weill Cornell Medicine

summary: We have identified that ATP binding Cassette Subfamily G Member 2 (Abcg2) lables vascular endothelial stem cells (VESC) that can for endothelial cell (EC) colonies in vitro and functional vessels in vivo. Here we compared the transcriptome between Abcg2-expressing VESC and Abcg2- mature EC from multiple murine tissues.

overall_design: Neonatal or 6-week-old Abcg2CreERT;ROSATdTomato mice were given tamoxifen injection one day before dissection. CD31+CD45-Ter119-TdTomato+ VESC and CD31+CD45-Ter119- mature EC from lung, heart and muscle were sorted by flow cytometry. RNA isolation and next-generation sequencing were then performed.

first: Mervin

last: Yoder


title: Cell type specific gene expression patterns associated with posttraumatic stress disorder in World Trade Center responders

status: Public on Dec 17 2018

city: BUFFALO

first: Eduardo

last: Cortes

address: ELM AND CARLTON STREETS

department: Biostatistics and Bioinformatics

institute: ROSWELL PARK CANCER INSTITUTE

summary: Posttraumatic stress disorder (PTSD) has been linked to immunologic dysregulation. Gene expression profiling has emerged as a promising tool for understanding the pathophysiology of PTSD. However, to date, all but one gene expression study was based on whole blood or unsorted peripheral blood mononuclear cell (PBMC), a complex tissue consisting of several populations of cells. The objective of this study was to utilize RNA sequencing to simultaneously profile the gene-expression of four immune cell subpopulations in  World Trade Center responders. Pathway analyses identified gene sets related to immune response and inflammation as being among the differentially expressed genes in PTSD, including mast cell activation and regulation in CD4T, interferon-beta production in CD8T, and neutrophil related gene sets in monocytes. These findings are suggestive that immune cell dysregulation involves gene expression in various cell populations.

overall_design: 195 samples are submitted. 39 subjects with 5 different cell sorting sequencing runs. 20 of the subjects are diagnosed with PTSD and19 are not.

first: Benjamin

middle: J

last: Luft

first: Pei-Fen

last: Kuan


title: Analysis of metastasis related gene in melanoma cells

city: Suita city

first: Yumiko

last: Hayashi

address: 3-1 Yamadaoka

country: Japan

institute: Osaka University

summary: We show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. We found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro, and increased the number of metastatic foci in vivo.

overall_design: We search for metastatic related gene in melanoma cells. Cells were removed in culture dish and total RNA was extracted from cells using Rneasy-plus mini kits. We compared the gene expression of B16 cells and B16/BL6 cells.

first: Nobuyuki

last: Takakura


title: RNA-sequencing of 7 weeks old mouse testes

first: Demis

last: Menolfi

address: 1130 St. Nicholas Ave, Herbert Irving Cancer Center, Room 501

department: Institute for Cancer Genetics

institute: Columbia University

summary: We performed RNA-sequencing of testes derived from Atr+/+ or Atr+/KD 7 weeks old mice. Since Atr+/KD male mice are sterile and present severe spermatogenesis defects, we aimed at analyzing the possible changes in gene expression given by the ATR kinase-dead (KD) mutation

overall_design: Evalutation of differential gene expression in Atr+/+ or Atr+/KD mice testes.

first: Wenxia

last: Jang

first: Brian

last: Lee

first: Tatiana

last: Moiseeva

first: Zhengping

last: Shao

first: Verna

last: Estes

first: Mark

last: Frattini

first: Christopher

last: Bakkenist

first: Shan

last: Zha


title: In vivo RNA editing of point mutations via RNA-guided adenosine deaminases

city: La Jolla

first: Dario

last: Meluzzi

address: 9500 Gilman Drive #0648

department: School of Medicine

institute: UC San Diego

summary: We investigated the specificity profiles of a variety of RNA guided adenosine deaminases while exploring roles of NLS/NES and hyperactive mutants via analysis of the transcriptome-wide off-target A->G editing effected by these tools. To this end, HEK 293T cells were transfected with each construct and analyzed by RNA-seq. Untransfected cells were included as controls. From each sample, we collected ~40 million uniquely aligned sequencing reads. We then used Fisher’s exact test to quantify significant changes in A->G editing yields, relative to untransfected cells, at each reference adenosine site having sufficient read coverage. The number of sites with at least one A->G editing event detected in any of the samples was computed.

overall_design: Study of transcriptome wide A->G off-targets arising due to the overexpression of a variety of RNA guided adenosine deaminases.

first: Dhruva

last: Katrekar

first: Prashant

last: Mali


title: Calibrated CAR activation potential directs alternative T cell fates and therapeutic potency

first: Yu-Jui

last: Ho

address: 417 E 68th St

department: Cancer Biology & Genetics Program

institute: Memorial Sloan Kettering Cancer Center

summary: CD19-specific CARs that comprise CD28 and CD3z signaling domains program highly performing effector functions that mediate potent tumor elimination, but they impart a relatively limited T cell lifespan. Increasing functional T cell persistence without reducing effector potency is therefore likely to further enhance the therapeutic success of 1928z CAR T cells. We demonstrate that the number and position of ITAMs in 1928z CAR T cells influence functional, phenotypic and transcriptional programs, resulting in profound effects on antitumor efficacy. Improved therapeutic potency of CAR T cells can thus be achieved by calibrating activation strength, thereby retaining memory functions and preventing exhaustion, without compromising effector functions. Our transcriptional analysis underscores the potential of ITAM dosage and position to direct different T cell fates. We were able to identify a novel CAR design, termed 1XX, which programs a favorable balance of effector and memory signatures, inducing increased persistence of highly functional CARs with the replicative capacity of long-lived memory cells and potent effector functions.

overall_design: In order to assess the different phenotypic and functional patterns of CARs encoding a single immunoreceptor tyrosine-based activation motif (ITAM), we compared the genome-wide transcriptional profiles of 1928z, 1XX and XX3 after CD19 antigen stimulation of TRAC-edited naïve T cells. Sorted naïve (TN), stem cell memory (TSCM) and effector (TEFF) CD8+ T cells served as controls.

first: Judith

last: Feucht

first: Jie

last: Sun

first: Justin

last: Eyquem

first: Michel

last: Sadelain