title: Epithelial endoplasmic reticulum stress orchestrates a protective IgA response II

first: Niklas

summary: Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype at mucosal surfaces where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IEC). IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response which protects from small intestinal inflammation. IEC ER stress causes expansion and activation of peritoneal B1b cells independent of microbiota and T cells that culminates in increased lamina propria and luminal IgA. Xbp1dIEC mice exhibit IEC ER stress by conditional deletion of X-box-binding protein 1 (XBP1). Here we examine single-cell transcriptomes of peritoneal cavity cells of germ-free Xbp1dIEC mice (KO) compared to littermate controls (WT).

overall_design: Single-cell gene expression profiles of peritoneal cavity cells of 10-week-old germ-free Xbp1dIEC and WT mice were generated using a droplet-based system (10X Genomics Chromium).

first: Shengbao

last: Suo

first: Adrienne

last: Luoma

middle: W

last: Wucherpfennig

first: Guo-Cheng

last: Yuan


title: Imprinted gene expression at the Dlk1-Dio3 cluster is controlled by both maternal and paternal IG-DMRs in a tissue-specific fashion.

status: Public on Feb 28 2019

city: Philadelphia

first: Katherine

middle: Ann

last: Alexander

state: PA

address: 3400 Civic Center Blvd.

country: USA

institute: University of Pennsylvania

type: Expression profiling by high throughput sequencing

summary: Imprinting at the Dlk1-Dio3 cluster is controlled by the IG-DMR, an imprinting control region differentially methylated between maternal and paternal chromosomes. The maternal IG-DMR is essential for imprinting control, functioning as a cis enhancer element. Meanwhile, DNA methylation at the paternal IG-DMR is thought to prevent enhancer activity. To explore whether suppression of enhancer activity at the methylated IG-DMR requires the transcriptional repressor TRIM28, we analyzed Trim28chatwo embryos and performed epistatic experiments with IG-DMR deletion mutants. We found that while TRIM28 regulates the enhancer properties of the paternal IG-DMR, it also controls imprinting through other mechanisms. Additionally, we found that the paternal IG-DMR, previously deemed dispensable for imprinting, is required in certain tissues, demonstrating that imprinting is regulated in a tissue-specific manner. Using ChRO-seq to analyze nascent transcription, we show that different tissues have a distinctive regulatory landscape at the Dlk1-Dio3 cluster, providing insight into potential mechanisms of tissue-specific imprinting control. ChRO-seq identified 30 novel transcribed regulatory elements, including a candidate regulatory region that depends on the paternal IG-DMR. Together, our findings challenge the model that Dlk1-Dio3 imprinting is regulated through a single mechanism and demonstrate that different tissues use distinct strategies for imprinting control.

overall_design: Duplicates of PRO-seq in E14.5 liver and yolk sac samples obtained from mating C57BL/6J females with males of a mixed genetic background (C57BL/6J  and CAST/EiJ). Males in this cross were selected to exclusively carry CAST/EiJ polymophisms at the Dlk1/Gtl2 locus on chromosome 12

middle: A

first: Maria

middle: J

last: Garcia-Garcia


title: RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena

city: Ann Arbor

first: Fengbiao

last: Mao

state: Michigan

address: 1301 Catherine St

department: Department of Pathology

institute: University of Michigan

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Integrating ATAC-seq and RNA-seq reveals tissue-specific genes and repressed networks in the placenta

city: Ames

first: Geetu

last: Tuteja

state: IA

address: 2106 Molecular Biology Building

department: Genetics, Development and Cell Biology

institute: Iowa State University

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
We performed ATAC-seq on placenta dissected from e9.5 timed-pregnant mice in order to generate genome-wide chromatin accessibility maps. Integrating this data with previously published RNA-seq data showed that: 1) genes that have high expression and high ATAC-seq read coverage at the transcription start site (TSS) were enriched for house-keeping functions; 2) genes that have high expression and medium-low coverage at the TSS were enriched for placenta-specific genes and functions; 3) genes that have low expression and high TSS coverage are actively repressed. From the actively repressed genes, we extracted a protein-protein interaction network enriched for neuronal functions. These data increase our understanding of active and repressed gene networks in the e9.5 placenta. The analysis also provides a method for identifying repressed networks and tissue-specific genes by integrating ATAC-seq and RNA-seq data.

Please note that the RNA-Seq data are already deposited and available under GSE65808.

overall_design: Examination of chromatin accessibility in the e9.5 mouse placenta

first: Rebekah

middle: R

last: Starks


title: Mitochondrial hexokinase-II controls TCR signaling duration and IFN-g production in NKT cells via mTORC2-Akt/PKCq pathways

city: Hefei

first: Shasha

last: Zhu

address: Jixi

country: China

institute: 1st affiliated hospital of Anhui medical university

summary: We performed RNA-seq of purified NKT cells stimulated with either plate-coated mCD1d-PBS57 tetramers or not from livers of Va14 Tg.cxcr6gfp/+ mice by FACS Aria.

overall_design: Examination of  different gene expression in NKT cell before and after stimulation

first: Zonghong

last: Li


title: Sensitivity of MiaPaCa2 cells to E2F inhibitor Ly101-4B.

city: Quebec

first: Ezequiel

middle: L

last: Calvo

state: Quebec

address: 2705 Boul. Laurier

department: Molecular Endocrinilogy

country: Canada

institute: CRCHUL

summary: MiaPaCa2 cells where screened with a set of original compounds with anticancer activity available at our laboratory. From these compounds we identified the nucleoside analogue ly101-4B as a strong inhibitor of the E2F activity. We confirmed its activity by studying its effect on MiaPaCa2's cell viability , caspase 3/7 activity  and LDH release, to measure cell growth, apoptosis and necrosis after treatment with LY101-4B 25 µM. Then, to investigate the mechanism of action of ly101-4B we performed a transcriptomic analysis by using an Affymetrix approach (Genechip® Human Gene 1.0 ST Arrays).

overall_design: MiaPaCa2 cells were analyzed after vehicle or LY101-4B 50 µM treatment for 24 h. Two control and two treated biological replicates were analyzed.

first: Wenjun

last: Lan

first: Benjamin

last: Bian

first: Yi

last: Xia

first: Odile

last: Gayet

first: Martin

last: Bigonnet

first: Patricia

last: Santofimia-Castaño

first: Mei

last: Cong

first: Ling

last: Peng

first: Nelson

last: Dusetti

first: Juan

last: Iovanna


title: Quiescence modulates stem cell maintenance and regenerative capacity in the aging brain – SMART-seq2

city: Heidelberg

first: Ana

last: Martin-Villalba

address: Im Neuenheimer Feld 280

department: Molecular Neurobiology

country: Germany

institute: German Cancer Research Center

summary: Single cell RNA-seq of neural stem cell and astrocytes from old mice

first: Georgios

last: Kalamakis

first: Daniel

last: Brüne


title: Restoring PU.1 induces apoptosis and modulates viral transactivation via interferon-stimulated genes in primary effusion lymphoma

city: Kumamoto

first: Seiji

last: Okada

address: 2-2-1 Honjo, Chuo-ku

country: Japan

institute: Kumamoto University

summary: Primary effusion lymphoma (PEL), which is an aggressive lymphoma associated with Kaposi sarcoma-associated herpesvirus/human herpes virus-8 (KSHV/HHV-8), is refractory to standard chemotherapy, and exhibits a poor prognosis. Although PU.1 is down-regulated in PEL among B-cell transcription factors, the potential role of its reduction remains to be elucidated. In this investigation, we analyzed the DNA methylation of PU.1 cis-regulatory elements in PEL and the effect of restoring PU.1 on PEL cells. The expression of PU.1 mRNA was down-regulated in PEL cells. The promoter and enhancer regions of the PU.1 gene were highly methylated. The restoration of PU.1 inhibited cell growth and induced apoptosis in PEL cells. A microarray analysis revealed that interferon-stimulated genes (ISGs) including pro-apoptotic ISGs were strongly increased in BCBL-1 cells after the induction of PU.1. The up-regulation of PU.1 induced the transactivation of pro-apoptotic ISG promoters, such as the XAF1, OAS1, and TRAIL promoters, in reporter assays. Mutations at the PU.1 binding sites suppressed its transactivation. We confirmed the binding of PU.1 to the XAF1, OAS1, and TRAIL promoters in a chromatin immunoprecipitation assay. PU.1 suppressed ORF57 activation by inducing IRF7. The reinduction of PU.1 reduced formation of ascites and lymphoma cell infiltration of distant organs in PEL xenograft model mice. These results suggest that PU.1 plays a role in tumor suppression in PEL and its down-regulation is associated with PEL development. Up-regulation of PU.1 with demethylation agents may be a novel therapeutic strategy for PEL.

overall_design: We generated BCBL-1 cells that conditionally express PU.1 using a tet-on system. We compared control and PU.1-up-regulated BCBL-1 cells with Dox using microarrays

first: Hiroki

last: Goto


title: Aging retains neural stem cells in a dormant state


title: Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity

city: Boston

first: Peter

last: van Galen

state: Massachusetts

address: 4 Blackfan Circle

department: Hematology

institute: Brigham and Women's Hospital / Broad Institute of MIT and Harvard

summary: Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies.

overall_design: Bone marrow aspirates of five healthy control donors, 16 AML patients at diagnosis, and 19 matched samples during treatment were analyzed using Seq-Well single-cell RNA-sequencing (scRNA-seq). Acute myeloid leukemia (AML) patients in this study underwent standard induction chemotherapy between diagnosis (D0) and later time points (D14 and later). The only exception is AML328, who was treated with azacitidine + venetoclax. All 35 AML patient samples were also subjected to targeted amplification of known mutations in AML driver genes. One AML patient (AML921A) was analyzed in technical duplicate.

first: Volker

last: Hovestadt

first: Bradley

middle: E

last: Bernstein


title: Quiescence modulates stem cell maintenance and regenerative capacity in the aging brain – 10X Chromium

summary: Single cell RNA-seq of neural stem cells from young and old mice


title: Field transcriptome revealed a novel relationship between nitrate transport and flowering in Japanese beech

status: Public on Feb 27 2019

city: Fukuoka

first: Akiko

last: Satake

state: Fukuoka

address: 744 Motooka, Nishi-ku

department: Department of Biology Faculty of Sciences

institute: Kyushu University

summary: Recent advances in molecular and genetic studies about flowering time control have been increasingly available to elucidate the physiological mechanism underlying masting, the intermittent and synchronized production of a large amount of flowers and seeds in plant populations. To identify unexplored developmental and physiological processes associated with masting, genome-wide transcriptome analysis is a promising tool, but such analyses have yet to be performed. We established a field transcriptome using a typical masting species, Japanese beech (Fagus crenata Blume), over two years, and analyzed the data using a nonlinear time-series analysis called convergent cross mapping. Our field transcriptome was found to undergo numerous changes depending on the status of floral induction and season. An integrated approach of high-throughput transcriptomics and causal inference was successful at detecting novel causal regulatory relationships between nitrate transport and florigen synthesis/transport in a forest tree species. The synergistic activation of nitrate transport and floral transition could be adaptive to simultaneously satisfy floral transition at the appropriate timing and the nitrogen demand needed for flower formation.

overall_design: Leaf and bud samples were collected from June to October in 2015 and 2016 from three individuals (HG1, HG2, and HG3) of Fagus crenata. Sample title indicates year_month_individual ID.

first: Kazutaka

last: Kawatsu

first: Kosuke

last: Teshima

first: Daisuke

last: Kabeya

first: Qingmin

last: Han


title: Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression

first: Ge

last: Shan

state: Anhui

address: 443 Huangshan Road

department: School of Life Sciences

institute: University of Science and Technology of China

summary: Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 30 -Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR34a.

overall_design: biotin-labelled miR-34a pulldown and RNA sequencing of miR-34a overexpression samples

first: Hassaan

middle: M

last: Awan

first: Shuai

last: Wei


title: MiR-135 suppresses glycolysis and promotes adaptation to metabolic stress in pancreatic cancer cells by targeting phosphofructokinase-1

city: Duarte

first: Haiqing

last: Li

state: CA

address: 1500 East Duarte Road

department: Department of Computational & Quantitative Medicine

institute: Beckman Research Institute of City of Hope, Center for Informatics

summary: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a nutrient-poor environment; however, the mechanisms by which PDAC cells undergo metabolic reprogramming to adapt and survive in metabolic stress are still poorly understood. Here, we show that microRNA-135 is significantly increased in PDAC patient samples compared to adjacent normal tissue and represses aerobic glycolysis. Mechanistically, we found that miR-135 accumulates specifically in response to glutamine deprivation and requires ROS-dependent activation of mutant p53, which directly promotes miR-135 expression. Functionally, we found miR-135 targets phosphofructokinase-1 (PFK1) and inhibits aerobic glycolysis, thereby promoting the utilization of glucose to support the tricarboxylic acid (TCA) cycle. Consistently, miR-135 deficient PDAC cells preferentially use glutamine carbon to replenish the TCA cycle, and miR-135 silencing sensitizes PDAC cells to glutamine deprivation and represses tumour growth in vivo. Consistent with these findings, patient pancreatic cancer tissue displays decreased PFK1 level compared to adjacent normal tissue. Together, these results identify a mechanism used by PDAC cells to survive the nutrient-poor tumour microenvironment, and also provide insight regarding the role of mutant p53 and miRNA in pancreatic cancer cell adaptation to metabolic stresses.

overall_design: Detect the difference of miRNA expression between pancreatic cancer tumor tissue and adjacent normal tissue

first: Ying

last: Yang

first: Mari

middle: B

last: Ishak Gabra

first: Eric

last: Hanse

first: Xazmin

middle: H

last: Lowman

first: Thai

middle: Q

last: Tran

first: Neta

last: Milman

first: Juan

last: Liu

first: Michael

last: Reid

first: Jason

last: Locasale

first: Ziv

last: Gil

first: Mei

last: Kong


title: ALYREF is shared by polyadenylated and nonpolyadenylated mRNA processing and export pathways

city: Shanghai

first: Hong

last: Cheng

address: 320 YueYang Road

department: Shanghai Institute of Biochemistry and Cell Biology

institute: Shanghai Institutes for Biological Sciences


title: ALYREF is shared by polyadenylated and nonpolyadenylated mRNA processing and export pathways [iCLIP-seq]

summary:
we try to investigate the binding of  ALYREF and NXF1 on histone mRNA when the cell treated with indicated siRNAs.
ALYREF plays key roles in nuclear export of polyadenylated mRNAs and also modulates their 3' processing, but whether it is involved in regulating RNAs beyond polyadenylated mRNAs is unknown.  The replication-dependent (RD) histone mRNAs are not polyadenylated, but end in a stem-loop (SL) structure.  Here we demonstrate that ALYREF prevalently binds a region next to the SL on RD histone mRNAs.  SL-binding protein (SLBP) directly interacts with ALYREF and ensures this binding. To examine how SLBP KD impact ALYREF distribution on the histone mRNA, we carried out ALYREF iCLIP in control and SLBP KD cells. To investigate the functional consequence for ALYREF binding on histone mRNAs, we isolated polyA+ and polyA- RNAs from control and ALYREF KD cells, and carried out RNA-seq separately.

overall_design: The ALYREF-iCLIP assay was performed in control and SLBP knock down cells using ALYREF antibody.  The NXF1-iCLIP experiments were carried out in Flag-NXF1 stable expressed cells treated with control, ALYREF and SLBP siRNAs for 72 hr using Flag antibody.

first: Jing

last: Fan

first: Xian

last: Du

first: Yu

last: Zhou


title: Towards precision medicine for pain: diagnostic biomarkers and repurposed drugs

city: Indianapolis

first: Dr. Alexander

middle: B.

last: Niculescu

state: IN

address: 320 W. 15th Street

department: Psychiatry

institute: Indiana University Medical School

summary: We endeavored to identify objective blood biomarkers for pain, a subjective sensation with a biological basis, using a  stepwise discovery, prioritization, validation, and testing in independent cohorts design. We studied psychiatric patients, a  high risk group for co-morbid pain disorders and increased perception of pain. For discovery, we used a powerful withinsubject  longitudinal design. We were successful in identifying blood gene expression biomarkers that were predictive of pain  state, and of future emergency department (ED) visits for pain, more so when personalized by gender and diagnosis.

overall_design: First, we used a powerful longitudinal within-subject design in individuals with psychiatric disorders to discover blood gene expression changes between self-reported low pain and high pain states. Second, we prioritized the list of candidate biomarkers with a Bayesian-like Convergent Functional Genomics approach, comprehensively integrating previous published human and animal model evidence in the field for involvement in pain, and directly citing it. Third, we validated our top biomarkers from discovery and prioritization in an independent cohort of psychiatric subjects with a clinical diagnosis of a pain disorder and with high scores on pain severity and functional impact ratings.  Fourth, we tested if the candidate biomarkers from the first three steps are able to predict high pain state, and future emergency department (ED) visits for pain, in another independent cohort of psychiatric subjects. We tested the biomarkers in all subjects in the independent test cohort, as well as in a more personalized fashion by gender and psychiatric diagnosis, showing increased accuracy with the personalized approach. Fifth, we assessed if our biomarkers have evidence for involvement in other psychiatric and related disorders, as well as analyzed the biological pathways and networks they are involved in. Sixth, we bioinformatically identified which of our individual biomarkers are modulated by existing drugs and thus can be used for pharmacogenomic population stratification and measuring of response to treatment, as well as used the gene expression signatures of the top predictive biomarkers to interrogate the Connectivity Map database from Broad/MIT to identify drugs and natural compounds that could be repurposed for treating pain.

first: Alexander

first: Helen

last: Le-Niculescu


title: GREB1 amplifies androgen receptor output in prostate cancer and contributes to antiandrogen resistance

city: New York

first: Eugine

last: Lee

address: 550 1st avenue

institute: NYU school of medicine

summary: Genomic amplification of the androgen receptor (AR) is an established mechanism of antiandrogen resistance in prostate cancer.  Here we show that the magnitude of AR signaling output, independent of AR genomic alteration or expression level, also contributes to antiandrogen resistance, through upregulation of the coactivator GREB1.  We demonstrate 100-fold heterogeneity in AR output within cell lines and show that cells with high AR output have reduced sensitivity to enzalutamide.  Through transcriptomic and shRNA knockdown studies, together with analysis of clinical datasets, we identify GREB1 as a gene responsible for high AR output.  We show that GREB1 is an AR target gene that amplifies AR output by enhancing AR DNA binding and promoting p300 recruitment.  GREB1 knockdown in high AR output cells restores enzalutamide sensitivity in vivo.  Thus, GREB1 is a candidate driver of enzalutamide resistance through a novel feed forward mechanism.

overall_design: RNA-seq was performed on following samples, LNCaP prostate cancer cells with 1) low or high AR activity (AR-low vs AR-high), 2) high AR activity infected with shRNA against Renilla or GREB1, treated with vehicle or DHT (AR-high shRenilla veh or DHT vs. AR-high shGREB1 veh or DHT)

first: Charles

last: Sawyers


title: Poly(A)-specific ribonuclease sculpts the 3′ ends of microRNAs

city: Seoul

first: Chanseok

last: Shin

address: 1 Gwanak-ro

department: Department of Agricultural Biotechnology

country: South Korea

institute: Seoul National University

summary: The role of 3ʹ-5ʹ exonuclease, PARN, in the 3ʹ end miRNA transcriptome in human cells

overall_design: Wild-type and PARN knockouts from HeLa S3 cells (two biological replicates for the knockouts)

first: June-Hyun

last: Park

first: Dooyoung


title: LncRNA expression profiling of ischemic stroke patients

city: tianjin

first: lili

last: tian

address: NO.154 Anshan Road

institute: tianjin medical university general hospital

summary: Our study reveals that ischemic stroke can influence the expression of LncRNAs and mRNAs in the peripheral blood at both the acute and subacute stages

overall_design: we analyzed the RNA-sequencing  datasets obtained at two time points (24 h and 7 days) from the peripheral blood mononuclear cells of ischemic patients. Vascular risk factor-matched healthy adults were enrolled as controls.

first: Y

last: Yan

first: L

last: Tian


city: Bronx

first: Deyou

last: Zheng

state: NY

address: 1301 Morris Park Ave

institute: Albert Einstein College of Medicine

overall_design: AR ChIP was performed on LNCaP prostate cancer cells with high AR activity infected with shRNA against Renilla or GREB1

first: Ping

last: Wang


title: Using Gjd3-CreEGFP mice to examine atrioventricular node morphology and composition

city: Dallas

first: Gary

last: Hon

address: 5323 Harry Hines Blvd.

department: Department of Obstetrics and Gynecology

institute: UT Southwestern

summary: Gjd3-CreEGFP mice is a novel genetic tool to study the structural and molecular signatures of Atrioventricular Node (AVN) at a high resolution.

overall_design: Focusing on the cardiac conduction system, we developed and rigorously characterized a geentic tool Gjd3-CreEGFP to perform in-depth analysis of AVN structure and composition. Utilizing this AVN-specific mouse model, we performed scRNA-Seq on neonatal Gjd3-CreEGFP mice to guide our single-cell atlas of the Atrio-ventricular conduction system (AVCS).

first: Nikhil

last: Munshi

first: Samadrita

last: Bhattacharyya

first: Jialei

last: Duan


title: GATA3 enhances the neurogenic potential of primary human astrocytes after traumatic injury

first: Caghan

last: Kizil

institute: Columbia University Irving Medical Center

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


title: New and subgroup-specific miRNAs and miRNA-offset RNA unraveled by RNA-sequencing in multiple myeloma

city: MILAN

first: Luca

last: Agnelli

address: Venezian 1

department: Department of Advanced Diagnostics

country: Italy

institute: IRCCS Istituto Nazionale dei Tumori

platform_organism:
Homo sapiens
synthetic construct


title: New and subgroup-specific miRNAs and miRNA-offset RNA unraveled by RNA-sequencing in multiple myeloma [array]

summary: Microarray analysis of the miRNome in multiple myeloma (MM) has unraveled the differential expression of miRNAs in cytogenetic subgroups, their involvement in the tumor biology and their effectiveness in prognostic models. Herein, we provide a view of small RNA transcriptional landscape in MM at a higher definition, exploiting the possibilities offered by small RNA-sequencing (sRNA-seq), including improved detection of known/new mature species and accurate investigation of isomiRs and miRNA-offset RNAs (moRNAs). Matched sRNA-seq data and miRNA microarray profiles in a panel of 30 primary MM tumors were obtained and compared. A good concordance was observed in the detection of the expression level of known species and in the differential analysis between subgroups. The superior discovery power of RNA-seq allowed characterizing 655 known and 17 new mature miRNAs, and describing 74 moRNAs, most of which highly expressed and group-specific. Ectopic expression of miR-135 in t(4;14) patients, undetected by arrays was detected by RNA-seq and validated by qPCR.

overall_design: Total RNA samples from highly purified plasma cells of 30 MM cases at onset

first: Andrea

last: Bisognin

first: Enrico

last: Gaffo

first: Katia

last: Todoerti

first: Martina

last: Manzoni

first: Serena

last: Galletti

first: Stefania

last: Bortoluzzi

first: Antonino

last: Neri


title: Oncogenic Notch promotes long-range regulatory interactions within hyperconnected 3D cliques [HCC1599_ChIP-seq]

city: Philadelphia

first: YEQIAO

last: ZHOU

address: 421 Curie Boulevard

department: Pathology

institute: University of Pennsylvania

summary:
Purpose: To investigate the impact of oncogenic Notch on the 3D genome organization of cancer cells.
Methods: We generated cohesin HiChIP and 1D epigenomic data sets in two different Notch-dependent cancer cell types, triple-negative breast cancer (TNBC) and mantle cell lymphoma (MCL), in the Notch-on and -off states.
Results: We report here that Notch transcription complexes control their direct target genes through two distinct regulatory modes: either through existing loops or by facilitating new long-range regulatory interactions. This combination of pre-existing and Notch-promoted loops coalesce enhancers and promoters to form highly interacting clusters, termed “3D cliques”. Notch preferentially activates enhancers and promotes looping interactions within highly connected 3D cliques that regulate key oncogenes.
Conclusions: These observations suggest a general mechanism that oncogenic transcription factors can exploit to regulate the transcriptional outputs of cancer cells.

overall_design: ChIP-seq, RNA-seq and HiChIP in Notch-on, -off, -recovery conditions in TNBC and MCL cell lines to profile Notch transcriptional complex binding, histone modification, Notch target genes and contact between regulatory elements.

first: Yeqiao

last: Zhou

first: Jelena

last: Petrovic

first: Robert

last: Faryabi


title: Oncogenic Notch promotes long-range regulatory interactions within hyperconnected 3D cliques [Rec1_ChIP-seq]


title: Oncogenic Notch promotes long-range regulatory interactions within hyperconnected 3D cliques [DND41_RNA-seq]


title: Oncogenic Notch promotes long-range regulatory interactions within hyperconnected 3D cliques [HCC1599_RNA-seq]


title: Oncogenic Notch promotes long-range regulatory interactions within hyperconnected 3D cliques [MB157_RNA-seq]


title: Human lineage tracing enabled by mitochondrial mutations and single cell genomics

first: Caleb

last: Lareau

state: New York

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

institute: Memorial Sloan Kettering

overall_design: Refer to individual Series


title: New and subgroup-specific miRNAs and miRNA-offset RNA unraveled by RNA-sequencing in multiple myeloma [miRNA-Seq]


title: Colonic single epithelial cell census reveals goblet cell drivers of barrier breakdown in inflammatory bowel disease

city: Oxford

first: Alison

last: Simmons

address: John Radcliffe Hospital/Headley Way

department: The MRC Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

summary: We report the results of 3 single cell RNA-sequencing (10x, chromium) experiments looking to characterise the composition of the intestinal epithelial cells in health and Ulcerative Colitis in order to identify drivers of intestinal inflammation at the molecular level. The data in this submission comprises of 3 repeat experiments (A, B, C) with biopsy samples taken from colonic biopsies collected from healthy patients (Healthy) and those with UC inflammation from an inflamed area of colon and adjacent non-inflamed area. All biopsies underwent and epithelial cell isolation protocol by digestion and then were loaded onto the 10x single cell RNA sequencing platform with subsequent GEM generation, cDNA isolation, amplification and library construction into one library using illumina barcodes. A single library was then sequenced on a NovaSeq 6000

overall_design: single cell RNA-sequencing on intestinal epitehlial cells from healthy controls and Ulcerative colitis

first: Kaushal

last: Parikh

first: Agne

last: Antanaviciute

first: David

last: Fawkner-Corbett

first: Marta

last: Jagielowicz

first: James

last: Kinchen

first: Anna

last: Aulicino

first: Neil

last: Ashley

first: Errin

last: Johnson

first: Philip

last: Hublitz

first: Leyuan

last: Bao

first: Rajinder

middle: S

last: Andev

first: Elisabet

last: Bjorkland

first: Hashem

last: Koohy


title: Rpd3L Contributes to the DNA Damage Sensitivity of Saccharomyces cerevisiae Checkpoint Mutants

city: Seville

first: Belén

last: Gómez-González

email: gomezb@us.es, belen.gomez@cabimer.es

address: Cabimer/Avda Américo Vespucio, 24

country: Spain

institute: University of Seville

type: Genome variation profiling by high throughput sequencing

summary: DNA replication forks that are stalled by DNA damage activate an S phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the EXO1 gene. Here,we identified that loss of the histone deacetylase complex Rpd3L promotes survival of rad53∆ cells exposed to DNA damaging agents. From epistasis analysis, we show that this suppression operates in a separate pathway from the previously described suppression by deletion of EXO1.

overall_design: The whole sequence of Saccharomyces cerevisiae suppressors of the MMS sensitivity of rad53Δ checkpoint mutants was compared to wild-type controls.

first: Harshil

last: Patel

first: Anne

last: Early

first: John

middle: F

last: Diffley


title: RNA-seq analysis of breast cancer

city: Gunma

first: Reika

last: Kawabata-Iwakawa

address: 3-39-22, Showa-Machi, Maebashi

institute: Gunma University

summary: Background: We searched triple negative breast cancer (TNBC) specifically expressing candidates for target therapy.  Methods: High expressing genes in three TNBC cell lines compared to three non-TNBC cell lines were evaluated by using RNA sequencing (log2 ratio>5, q<0.05). By integrating the shRNA library screening and public gene expression databases, we identifyied candidates for therapeutic targets. The expression significance of the candidates was examined by using immunohistochemistry of 248 clinical breast cancer patients.  Results: One hundred seventy one genes were detected as highly expressed genes in TNBC cell lines than non-TNBC cell lines, and 13 genes were picked up as candidates not expressing in non-cancerous tissues. Integrated analysis with shRNA screening identified three candidates  as TBNC specific molecular targets.  Protein  staining were stronger in breast cancer tissues, especially TNBC, than in the normal tissues.

overall_design: Gene expression profiles of 6 breast cancer cell lines were generated by RNA-seq using Illumina NextSeq 500 system.

first: Tadashi

last: Handa

first: Arito

last: Yamane

last: Kawabata

first: Tetsunari

last: Oyama

first: Masahiko

last: Nishiyama


title: shRNA screening analysis of breast cancer

overall_design: High-thoughput shRNA screening analysis of 6 breast cancer cell lines were generated by shRNA-seq using Illumina NextSeq 500 system.


title: RNA-seq analysis and shRNA screen of breast cancer


title: ALYREF links 3'processing and nuclear export of histone mRNAs

first: Hong

last: Cheng

summary: To examine whether ALYREF can facilitate the 3'processing of RD histone mRNA,we isolated and sequenced the polyadenylated and nonpolyadenylated RNAs from control and ALYREF KD cells

overall_design: Polyadenylated and nonpolyadenylated RNAs isolated from control or ALYREF KD cells were generated by deep sequencing, using Illumina HiSeq 2000.

first: Xudong

last: Wu


title: Conserved mRNA-granule component Scd6 targets Dhh1 to   repress translation initiation and activates Dcp2-mediated mRNA decay in vivo

city: Bethesda

first: Alan

last: Hinnebusch

state: MD

address: 6 Center Drive, Building 6, Room 233

department: NICHD

institute: NIH

summary: We have identified mRNAs whose abundance or translational efficiency is regulated in nutrient-rich medium by the Scd6 or Dhh1 protein in either wild-type cells or dcp2∆ cells lacking the decapping enzyme

overall_design: Ribosome profiling and RNA-seq was conducted to determine changes in mRNA abundance, translation, or translational efficiecies in comparing scd6∆ or dhh1∆ mutants to an isogenic wild-type strain; or scd6∆dcp2∆ and dhh1∆dcp2∆ strains to a dcp2∆ mutant

first: Fan

last: Zhang

first: Quira

last: Zeidan

middle: G


title: Novel Mechanism of Bispecificity in Forkhead Transcription Factors

first: Martha

last: Bulyk

state: MA

address: 77 Avenue Louis Pasteur

department: Medicine

institute: Brigham & Women's Hospital

summary: The forkhead transcription factor (TF) FoxN3 recognizes both the canonical forkhead DNA motif (RYAAAYA), as well as a novel alternate motif (GACGC). These two motifs are more distinct than the typically observed primary and secondary motifs of other TFs, as the two motifs cannot be aligned and are not clearly related to each other. Amino acids at the canonical base-contacting positions in the forkhead DNA binding domain are largely conserved throughout the forkhead family, so the ability of FoxN3 and other bispecific forkhead factors to recognize the alternate site cannot be explained by amino acid substitutions at these positions. Neither the mechanism of this bispecific DNA recognition nor its regulatory function are understood. Here we show that human FoxN3 does recognize both motifs in cells. We also report the crystal structures of the FoxN3 DNA binding domain bound to the canonical forkhead binding site, and separately to the alternate DNA binding site. FoxN3 adopts a remarkably similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is significantly altered between the two motifs, allowing the same domain to recognize two motifs of different lengths. Changes between the two structures in the conformation of segments of the forkhead domain outside of the direct DNA-contacting positions contribute to the ability to bind the alternate site. Swapping these segments of the domain between different forkhead proteins changes the ability of proteins to recognize the alternate site, highlighting the importance of non-base-contacting positions in determining TF-DNA binding specificity. The structures present a new mechanism by which a single DBD can recognize two divergent sequences.

overall_design: ChIP-seq of FoxN3 in HepG2 cells.

first: Julia

last: Rogers

first: Colin

middle: T

last: Waters

first: Thomas

middle: C

last: Seegar

first: Sanchez

last: Jarret

first: Amelia

middle: N

last: Hallworth

first: Stephen

last: Blacklow


title: circRNA profile in hypopharyngeal cancer

city: Kunming

first: Honglei

address: 1076 Yuhua Road, Chenggong District, Kunming, Yunnan Province

institute: Yunnan university of Chinese Medicine

summary: 3 normal samples and 3 tumor samples were sequenced

overall_design: RNA-seq to profile circRNA in normal hypopharynx and hypopharyngeal cancer

first: Chun

last: Feng


title: IKZF1 as a Master Regulator of Immune Infiltrate Recruitment in Solid Tumors

first: James

last: Chen

state: NEW YORK

address: 1130 St Nicholas Ave

department: Systems Biology

institute: Columbia University

summary: Regulatory network analysis of TCGA tumor cohorts to identify MR-based susceptibility to immune infiltration enhancement therapy

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 15 human tumor cell lines, 12 mouse melanoma xenografts (matched RFP and IKZF1)

first: Rolando

last: Perez-Lorenzo

first: Angela

last: Christiano


title: Gut microbiota analysis associated with hepatocellular carcinoma

city: Nanjing City

first: Xingyin

last: Liu

state: Jiangsu Province

address: Long Mian Street No.1

institute: Nanjing Medical University

summary: Here we report 16s rRNA data in gut microbiota of hepatocellular carcinoma (HCC) patients with HBV induced HCC (HBVC) and non-HBV induced HCC (NHBVC) compared with healthy volunteers. A total of 2047 operational taxonomic units (OTUs) were identified in the sequence data. Our data shows that the NHBVC patients harbor lower anti-inflammatory bacteria and more pro-inflammatory bacteria, while the HBVC patients harbor more anti-inflammatory bacteria.

overall_design: Microbial DNA from fecal samples [healthy control (n=33), NHBVC patients (n=26), HBVC (n=35) was amplified by PCR using universal bacterial primers, and 16S rRNA genes were pyrosequenced.

first: Yewei

first: Qisha


title: Pharmacological AMPK activation induces transcriptional responses congruent to exercise in skeletal and cardiac muscle, adipose tissues and liver

city: Boston

first: eric

last: muise

address: 33 Ave Louis Pasteur

department: Genetics and Pharmacogenomics

institute: Merck

summary: Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.

overall_design: Mice were treated with a single dose of vehicle or AMPK activators and 6 hours post dose, 5 tissues were taken for RNA profiling. Tissues were also taken from mice that went through an acute bout of exercise consisting of 1300 m of treadmill running. RNA was profiled on custom Affymetrix microarrays. There were 5 replicates in each group and the sedentary mice treated with vehicle served as baseline for the comparisons.

first: Eric

last: Muise


title: GRAS transcription factors activated in arbuscule-containing cells of Medicago truncatula

city: Hannover

first: Helge

last: Küster

address: Herrenhäuser Str. 2

department: Institut für Pflanzengenetik

institute: Leibniz Universität Hannover

summary: Arbuscular mycorrhiza (AM) interactions between plants and Glomeromycota fungi primarily support phosphate aquisition by the host. To unravel the role of the AM-specific GRAS transcription factors MtGras1 and MtRam1, we performed genome-wide transcriptome profiling in RNAi-mediated MtGras1 knockdown and MtRam1 (ram1-1) mutant roots. Specifically, we used GeneChip Medicago Transcriptome Assays to identify genes differentially regulated in mycorrhizal MtGras1 knockdown or MtRam1 (ram1-1) mutant roots in comparison to control roots.

overall_design: Mycorrhized Medicago truncatula Gaertn ‘Jemalong’ genotype A17 plants were grown in the climate chamber, applying growth conditions reported by Hartmann et al. (2019).

first: Rico

last: Hartmann

first: Jörg

middle: D

last: Becker

first: Natalija

last: Hohnjec


title: Single cell RNAseq analysis of fetal growth plate-derived bone marrow mesenchymal stromal cells

status: Public on Feb 26 2019

city: Houston

first: Noriaki

last: Ono

state: Texas

department: School of Dentistry

institute: University of Texas Health Science Center at Houston

summary: We revealed a single cell transcriptional landscape of bone marrow mesenchymal stromal cells derived from the fetal growth plate cartilage

overall_design: GFP+ and tdTomato+ cells were FACS-isolated from postnatal day 21 (P21) femurs of Cxcl12-GFP; Fgfr3-iCreER; R26R-tdTomato mice, with tamoxifen injection at embryonic day (E12.5). 18,000 cells were loaded onto 10X Genomic Chromium Single Cell Expression Solution version 2. platform. cDNA was sequenced by Illumina HiSeq4000.


title: Comparative bulk RNAseq analysis of CXCL12-abundant reticular (CAR) cells, fetal perichondrium-derived CAR cells and chondrocyte-derived CAR cells.

summary: We revealed the unique molecular signature characterizing fetal perichondrium-derived and chondrocyte-derived CAR cells

overall_design: GFP+tdTomato+ cells were FACS-isolated from postnatal day 21 (P21) femurs of Cxcl12-GFP; Hes1-creER; R26R-tdTomato and Cxcl12-GFP; Fgfr3-iCreER; R26R-tdTomato mice, both pulsed with a single dose of tamoxifen at embryonic day 12.5 (E12.5).  mRNA were amplifed by SMARTer mRNA-seq kit (Clonetech/Takara) before deep-sequenced by Illumina HiSeq4000.


title: Single cell RNAseq analysis of fetal perichondrium-derived bone marrow mesenchymal stromal cells

summary: We revealed a single cell transcriptional landscape of bone marrow mesenchymal stromal cells derived from the fetal perichondrium.

overall_design: GFP+ and tdTomato+ cells were FACS-isolated from postnatal day 21 (P21) femurs of Cxcl12-GFP; Hes1-creER; R26R-tdTomato mice, with tamoxifen injection at embryonic day 12.5 (E12.5). 18,000 cells were loaded onto 10X Genomic Chromium Single Cell Expression Solution version 2. platform. cDNA was sequenced by Illumina HiSeq4000.


title: RNA-sequencing of bystander-activated memory-like CD4+ T cells

first: Hong-Gyun

last: Lee

address: #527, Natural Science building, Hanyang Univ, Wangsimni-ro 222, Seondong-gu

institute: Hanyang University

summary: We report the differential mRNA expression of memory-like CD4+ T cells stimulated with iL-7, IL-1β, and IL-23. We found memory-like CD4+ T cells stimulated with IL-1β, or with IL-1β and IL-23, in the presence of IL-7, displayed a unique gene expression signature.

overall_design: Comparison of RNA expression of memory-like CD4+ T cells stimulated with iL-7, IL-1β, and IL-23.

first: Je-Min

last: Choi


title: iNKT cells RNA-Seq (WT vs SFR KO)

city: Montreal

first: André

last: Veillette

state: Québec

address: 110 avenue des pins ouest

institute: Institut de Recherches Cliniques de Montréal

summary:
RNA transcriptome difference between WT and SFR KO iNKT cells
To understand how SLAM family receptors (SFRs) contribute to iNKT cell development, a mouse lacking all SFRs in addition to the ligand of 2B4, CD48, was generated, and the transcriptional profiles of thymic iNKT cells from wild-type and SFR KO mice were compared, using RNA sequencing.

overall_design:
Examine RNA expression in WT and SFR KO iNKT cells
Thymocytes were isolated from WT and SFR KO mice, and iNKT cells were enriched by negative selection.  Unwanted cells (CD11b+ CD11c+ Gr-1+ Ter-119+ CD19+ CD8a+ cells) were targeted for removal with biotinylated antibodies (BioLegend), streptavidin-coated magnetic particles (RapidSpheres) and EasySep magnet (STEMCELL), and followed by staining with mCD1d/PBS-57 and anti-TCR.  Then, iNKT cells were sorted with BD FACSAria III (BD Biosciences), and total RNA was isolated from sorted cells according to the manufacturer’s instructions using the RNeasy plus micro kit (Qiagen). RNA-Seq library preparation was performed using the Illumina TruSeq Stranded mRNA Kit, according to manufacturer’s instructions, and sequenced with Illumina HiSeq 2000 Sequencer.  Read quality was confirmed using FastQC v0.10.1 before alignment using TopHat v2.0.10 on the mouse GRCm38/mm10 genome.

first: Yan

last: Lu


title: RNA-Seq transcriptome analysis of whole newborn lenses from FVB/N, Le-Cre and P0-3.9GFPCre mice

first: Michael

last: Robinson

state: Ohio

address: 700 E High St

department: Biology

institute: Miami University

summary: Here we report RNA-Seq data from RNA isolated from newborn lenses from FVB/N control mice as well as from newborn lenses of two different transgenic mouse lines (Le-Cre and P0-3.9GFPCre).  Sequence reads of 51 bp were obtained from an illumine HiSeq 2000 system and mapped to the C57BL/6 reference genome (assembly GRCm38 (mm10)) using GSNAP software. Adapters and poor-quality regions were trimmed using Trimmomatic-0.36 software. Gene and isoform abundance was quantified using RSEM-1.3.0 software. Differential expression analysis was completed using DESeq2-1.10.1 software. For differential expression we used a cutoff value of equal to or greater than 1.5-fold change with an adjusted p value ≤ 0.05. The transcriptomes of both Le-Cre and P0-3.9GFPCre lenses closely matched the FVB/N control lenses. However, Le-Cre lenses exhibited deregulation of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre lenses only deregulated two murine genes.

overall_design: Lens mRNA profiles of newborn wild type FVB/N strain, Le-Cre and P0-3.9GFPCre mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

first: Phuon

last: Lam

first: Stephanie

last: Padula

first: Thanh

middle: V

last: Hoang

first: Lin

first: Chun

last: Liang


title: Characterization of molecular changes at meiotic initiation in mice

city: Cambridge

first: Mina

last: Kojima

address: 455 Main Street

institute: Whitehead Institute

summary: Characterization of the molecular changes occurring at murine meiotic initiation: identification of gene expression changes in male germ cells undergoing meiotic initiation; identification of STRA8-bound sites in mouse testes enriched for germ cells initiating meiosis

overall_design: Identification of gene expression changes in male germ cells (preleptotene stage) undergoing meiotic initiation by RNA-seq; Identification of STRA8's binding sites in mouse testes enriched for cells initiating meiosis using ChIP-seq

first: David

last: Page


title: Chromatin accessibility landscape upon induction of Msgn1, Pax3 and Myf5 in mesodermal cells and identification of conserved Pax3 binding sites and target genes during skeletal myogenesis

city: Minneapolis

first: Alessandro

last: Magli

state: MN

address: 2231 6th St SE

institute: University of Minnesota

summary: The transcriptional mechanisms driving lineage specification during development are still largely unknown as the interplay of multiple transcription factors makes it difficult to dissect these molecular events. Using a cell-based differentiation platform to probe transcription function, we investigated the role of the key paraxial mesoderm and skeletal myogenic commitment factors, Msgn1, Tbx6, Foxc1, Pax3, Paraxis, Meox1, Six1 and Myf5, in paraxial mesoderm and skeletal myogenesis. From this study, we define a genetic hierarchy, with Pax3 emerging as the gatekeeper between the presomitic mesoderm and the myogenic lineage. By assaying chromatin accessibility, genomic binding and transcription profiling in mesodermal cells from mouse and human Pax3-induced embryonic stem cells and Pax3-null E9.5 mouse embryos, we identified conserved Pax3 functions in the activation of the skeletal myogenic lineage through modulation of Hedgehog, Notch, and BMP signaling pathways. In addition, we demonstrate that Pax3 molecular function involves chromatin remodeling of its bound elements through an increase in chromatin accessibility and cooperation with Six4 and Tead2 factors. Together, our data provide the first integrated analysis of Pax3 function, demonstrating its ability to remodel chromatin in mesodermal cells from developing embryos and proving a mechanistic footing for the transcriptional hierarchy driving myogenesis.

overall_design: Pax3, Msgn1 and Myf5 function during mesoderm specification was investigated using doxycycline-inducible embryonic stem (ES) cells. ATAC-seq was used to identify changes in chromatin accessibility induced by Msgn1, Pax3 and Myf5 upon 1-day induction in serum and serum-free differentiation media. In addition, Pax3-induced changes in chromatin accessibility were investigated also when Pax3 was expressed in NIH3T3 fibroblasts. ChIP-seq was used to determine Pax3 genomic occupancy in mouse ES cells undergoing myogenic specification (1-day and 6-day induction), human differentiating ES cells (1-day induction) and in mouse NIH3T3 fibroblasts (1-day induction). Gene expression changes induced by Pax3 in mouse (1-day and 6-day induction) and human (1-day induction) differentiating ES cells were assessed by RNA-seq.

first: Rita

last: Perlingeiro

first: Scott

middle: A

last: Swanson


title: Transcriptome profiling of blood leukocytes from FtH LysM-/- and FtH fl/fl mice following sham or CLP surgery

city: Birmingham

first: Sooryanarayana

last: Varambally

state: Alabama

address: 1824 6th Avenue S

institute: University of Alabama

summary: Despite the prevalence and recognition of its detrimental impact, clinical complications of sepsis remain a major challenge. Here, we investigated the effects of myeloid ferritin heavy chain (FtH) in regulating the pathogenic sequelae of sepsis. We demonstrate that deletion of myeloid FtH leads to tolerance towards sepsis as evidenced by reduced serum cytokine levels, multi-organ dysfunction and subsequent mortality. We identified that such tolerance is predominantly mediated by the compensatory increase in circulating ferritin (ferritin light chain; FtL) in the absence of myeloid FtH. Our in vitro and in vivo studies indicate that prior exposure to ferritin provides significant tolerance to the septic process by restraining an otherwise dysregulated response to infection. These findings are mediated by an inhibitory action of ferritin on NF-κB activation and its downstream effects. Taken together, our findings suggest an essential immunomodulatory function for circulating ferritin and enhances our understanding of this acute phase reactant.

overall_design: Total RNA were isolated from blood leukocytes of wild type FtH mice and Myeloid deficient FtH mice following sham and CLP surgery. Three biological replicates were considered for each genotype and surgery type.

first: Subhashini

last: Bolisetty

first: Darshan

last: Chandrashekar


title: Ablation of adipocyte BCL6 enhances subcutaneous adiposity and insulin sensitivity [ChIP-seq]

city: Chicago

first: Grant

middle: D.

last: Barish

state: IL

address: 303 E Superior Street

institute: Northwestern University

summary: In adipocyte-specific knockout mice (Bcl6AKO), we found that Bcl6 deletion results in strikingly increased inguinal but not perigonadal adipocyte size and tissue mass in addition to marked insulin sensitivity. Genome-wide DNA binding and RNA expression analyses revealed that BCL6 controls gene networks involved in cell growth and fatty acid biosynthesis. Thus, our studies identify BCL6 as a negative regulator of subcutaneous adipose tissue expansion and metabolic health.

overall_design: Identification of adipocyte BCL6-regulated genes


title: RNA seq comparison between scrambled and shGRP78 cells

city: Miami

first: Patricia

last: Dauer

state: FL

address: 1550 NW 10th Ave

department: Surgery

institute: University of Miami

summary: Pancreatic cancer cells transduced with sh knockdown of GRP78

overall_design: Pancreatic cancer mRNA profiles of scrambled control versus shGRP78 cell line, in triplicate, using Illumina Truseq Stranded Total-RNA library


title: COX-2 mediates tumor-stromal Prolactin signaling to initiate tumorigenesis [DF]

status: Public on Feb 25 2019

first: Ben

middle: S.

last: Wittner

address: 149 13th Street

department: Center for Cancer Research

institute: Massachusetts General Hospital

summary: Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single cell RNA-sequencing in an orthotopic mouse prostate cancer model, we find upregulation of Prolactin receptor as cancer cells that have disseminated to the lung expand into micrometastases. Secretion of the ligand Prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E-2 (PGE-2). PGE-2 treatment of fibroblasts activates the nuclear orphan receptor NR4A (Nur77), with Prolactin as a major transcriptional target for the NR4A-Retinoid X receptor (RXR) heterodimer. Ectopic expression of Prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor Celecoxib abrogates Prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, Prolactin, and Prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine crosstalk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.

overall_design: We performed RNA-seq on the human dermal fibroblast cell line DF treated for six hours with PGE-2 or untreated.

first: Yu

last: Zheng

first: Valentine

last: Comaills

first: Risa

last: Burr

first: Gaylor

last: Boulay

last: Miyamoto

first: Erin

last: Emmons

first: Srinjoy

last: Sil

middle: W

last: Koulopoulos

first: Katherine

last: Broderick

first: Eric

last: Tai

first: Shruthi

last: Rengarajan

first: Anupriya

last: Kulkarni

first: Toshi

last: Shioda

first: Chin-Lee

last: Wu

first: Sridhar

last: Ramaswamy

last: Ting

first: Mehmet

last: Toner

first: Miguel

last: Rivera

first: Shyamala

last: Maheswaran

first: Daniel

last: Haber


title: COX-2 mediates tumor-stromal Prolactin signaling to initiate tumorigenesis [CE1-4]

first: Ben

last: Wittner

overall_design: We performed RNA-seq on the mouse prostate cancer cell line CE1-4 treated for six hours with PGE-2 or untreated.

first: Yu

last: Zheng

last: Comaills

last: Burr

last: Boulay

first: David

first: Michael


title: RNA-seq datasets for "A neuron-optimized CRISPR/dCas9 activation system for robust and specific gene regulation"

first: Jeremy

middle: Jason

last: Day

state: AL

address: 1825 University Blvd

department: Neurobiology

institute: UAB

summary: This dataset contains whole-genome RNA sequencing results from rat embryonic hippocampal neuronal cultures and serves as the basis for characterization of CRISPR/dCas9 gene activation in neuronal systems.

overall_design: This experiment contains 9 biological samples, each of which underwent directional, paired-end PolyA+ RNA-seq on an Illumina Next-seq 500. Samples were treated with Lacz sgRNA (LZ2, LZ4, & LZ5), Bdnf-I sgRNA (B16, B17, B18), or Bdnf-IV sgRNA (BIV11, BIV14, BIV15), in addition to a dCas9-VPR fusion. Datasets were obtained using RNA-seq from PolyA+ fractions fractions of RNA. Each sample has multiple files, corresponding to different sequencing lanes (e.g., L001, L002, etc) or different reads (e.g., R1, R2).

middle: E

last: Savell

first: Svitlana

last: Bach

first: Morgan

last: Zipperly

first: Jasmin

last: Revanna

first: Nicholas

last: Goska

first: Jennifer

middle: J

last: Tuscher

first: Corey

last: Duke

first: Faraz

last: Sultan

first: Julia

last: Burke

first: Derek

last: Williams

first: Lara

last: Ianov


title: Assembly and functionality of the ribosome with tethered subunits

city: Austin

first: Adam

middle: John

last: Hockenberry

state: TX

department: Department of Integrative Biology

institute: The University of Texas at Austin

summary: Ribo-T is an engineered ribosome whose small and large subunits are tethered together by insertion of circularly permutated 23S rRNA into 16S rRNA [Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A.S. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015)]. Whether the growth defects are related to problems with Ribo-T functionality is unclear. The translation in Ribo-T cells was examined by ribosome profiling (Ribo-seq) and RNA sequencing (RNA-seq). Besides the control cells carrying dissociable WT ribosome (SQ171),  RIBO-T cells are the original SQ171 strain transformed with pRibo-T plasmid and cured of the plasmid carrying wt rRNA that was evolved to have improved growth characteristics. The Ribo-T strain carries two mutations: a nonsense mutation in the Leu22 codon of the ybeX gene and a missense mutation in codon 549 of the rpsA gene encoding ribosomal protein S1. Analysis of Ribo-T performance by ribosome profiling showed a notably higher ribosome occupancy of the start codons and somewhat increased occupancy at the stop codons of many genes, suggesting that subunit tethering specifically impairs the initiation and termination stages of translation.

overall_design: Ribosome profiling and RNA-seq was performed on Escherichia coli Ribo-T (a derivative of the strain SQ171, 2 biological replicates) and compared to a SQ171 control cells carrying WT ribosomes (2 biological replicates).

first: Nikolay

last: Aleksashin

first: Margus

last: Leppik

first: Dorota

last: Klepacki

first: Nora

last: Vázquez-Laslop

last: Jewett

first: Jaanus

last: Remme

first: Alexander

last: Mankin


title: Ablation of adipocyte BCL6 enhances subcutaneous adiposity and insulin sensitivity [RNA-seq]

first: Grant

last: Barish


title: Cxcr3 expressing leukocytes are necessary for neurofibroma formation in mice

status: Public on Feb 24 2019

city: Cincinnati

first: Nathan

last: Salomonis

state: OH

address: 3333 Burnet Avenue

department: Biomedical Informatics

institute: Cincinnati Children's Hospital

summary: Plexiform neurofibroma is a major contributor to morbidity in Neurofibromatosis type I (NF1) patients. Macrophages and mast cells infiltrate neurofibroma, and data from mouse models implicate these leukocytes in neurofibroma development. Anti-inflammatory therapy targeting these cell populations has been suggested as a means to prevent neurofibroma development. Here, we compare gene expression in inflamed nerves from NF1 models which invariably form neurofibroma to those with inflammation driven by EGFR overexpression which rarely progresses to neurofibroma. We find that the chemokine Cxcl10 is uniquely up-regulated in NF1 mice that invariably develop neurofibroma. Global deletion of the CXCL10 receptor, Cxcr3, prevented neurofibroma development in these neurofibroma-prone mice. Cxcr3 expression localized to T cells and dendritic cells (DCs) in both inflamed nerves and neurofibromas. These data support a heretofore unappreciated role for T cells/DCs in neurofibroma initiation.

overall_design: To identify cell populations associated with Cxcl10 expression, we utilized a single-cell RNA-Seq (scRNA-Seq) data set collected from 2-month Dhh-Cre;Nf1 fl/fl nerve/DRG using the 10x Genomics Chromium platform.

first: Jonathan

last: Fletcher

first: Jianqiang

last: Wu

first: Walter

last: Jessen

first: Jay

last: Pundavela

first: Jacob

last: Miller

first: Eva

last: Dombi

first: Mi-Ok

last: Kim

first: Tilat

last: Rizvi

first: Kashish

last: Chetal

first: Nancy

last: Ratner


title: A diffusion-based microfluidic device for single-cell RNA-seq

city: Blacksburg

first: Chang

last: Lu

state: VA

address: 235 Goodwin Hall, 635 Prices Fork Road, Virginia Tech

department: Chemical Engineering

institute: Virginia Tech

summary: Microfluidic devices provide a low-input and efficient platform for single-cell RNA-seq (scRNA-Seq). Here we present microfluidic diffusion-based RNA-seq (MID-RNA-seq) for conducting scRNA-seq with a diffusion-based reagent swapping scheme. This device incorporates cell trapping, lysis, reverse transcription and PCR amplification all in one microfluidic chamber. MID-RNA-Seq provides high data quality that is comparable to existing scRNA-seq methods while implementing a simple device design that permits multiplexing. The robustness and scalability of MID-RNA-Seq device will be important for transcriptomic studies of scarce cell samples.

overall_design: We performed single-cell RNA-seq from human and mouse cell lines using MID-RNA-seq devices. We produced 6 scRNA-seq data sets using single GM12878 cells and 6 data sets using single MEF cells using three 2-unit devices in each case. We also generated 4 data sets using a 4-unit MID-RNA-Seq device using 10 pg RNA in each unit. In addition, we produced 29 data sets on single GM12878 cells in 5 runs of the 6-unit device. Furthermore, 2 bulk RNA-seq samples using 1000 cells each were prepared using a commercial kit. In the file names, "SC" indicates single cell RNA-seq data set, followed by cell line name (either "GM12878" or "MEF"), the type and number of device used (e.g. "2UnitDevice1" or "6UnitDevice1"), and the number of the unit with a multi-unit device (e.g. "U1" or "U2").

first: Mimosa

last: Sarma


title: Targeting an RNA-binding Protein Splicing Network in Acute Myeloid Leukemia

city: New York

first: Omar

last: Abdel-Wahab

state: NY

address: 1275 York Ave

department: HOPP

institute: MSKCC

summary: RNA-binding proteins (RBPs) are essential modulators of transcription and translation and are often dysregulated in cancer. Here we systematically interrogated RBP vulnerabilities in acute myeloid leukemia (AML) by performing a comprehensive CRISPR/Cas9 screen, targeting the RNA-binding domains of all classical RBPs. Our screen revealed RBPs that are exclusively required for leukemia survival, including the splicing factor RBM39. Proteomics analysis identified a network of RBP’s interacting with RBM39 crucial for maintaining RNA splicing and survival in AML. Mechanistically, RBM39 suppression led to altered splicing of genes involved in essential oncogenic pathways. Selective targeting of RBM39 via ubiquitin-mediated pharmacologic degradation induced broad anti-leukemic effects in vitro and potent single agent activity in vivo. The effects of RBM39 loss on alteration of splicing further resulted in preferential lethality of AML cells bearing spliceosomal gene mutations, thereby providing a strategy for treating AML patients bearing RBP splicing mutations.

overall_design: The goals of this project are to investigaet the role of RBM39 degradation in AML

first: Eric

last: Wang

first: Alessandro

last: Pastore

first: Sydney

middle: X

first: Xufeng

last: Chen

first: Jochen

last: Imig

first: Yohana

last: Ghebrechristos

first: Akihide

last: Yoshimi

first: Lillian

last: Bitner

first: Andreas

last: Kloetgen

first: Kuan-Ting

last: Lin

first: Taisuke

last: Uehara

first: Takashi

last: Owa

first: Raoul

last: Tibes

first: Adrian

middle: R

last: Krainer

first: Iannis

last: Aifantis


title: Ablation of adipocyte BCL6 enhances subcutaneous adiposity and insulin sensitivity

first: Grant

last: Barish

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

overall_design: Refer to individual Series


title: Integrative analysis of mRNA and lncRNA profiles identified pathogenetic  lncRNAs  in esophageal squamous cell carcinoma

status: Public on Feb 23 2019

first: Chengguo

last: Wei

address: 1428 Madison Ave1468 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

summary: Systems biology approaches can help understand pathogenesis of complex human diseases like cancers for identification of potential new therapeutic targets. Here in this study, we performed genome-wide screening for mRNA and lncRNA profiles in esophageal cancer to identify the novel cancer-related mRNA and lncRNA in esophageal squamous cell carcinoma (ESCC). We identified 3000 up-regulated/2517 down-regulated mRNAs and 402 up-regulated/741 down-regulated lncRNAs. Further functional analysis revealed differentially expressed genes (DEGs) of mRNA and lncRNA are related to different pathways. mRNA pathways are mainly involved in cell cycles while lncRNA pathways are for regulation and metabolic procession. Differentially expressed mRNAs/lncRNAs were validated with qPCR. At last, mRNA and lncRNA co-expression network were built and highly-connected networks were identified, which may provide a mechanism of mRNA expression regulation by lncRNA. To our knowledge, this is the first study to explore the co-expression networks and the lncRNA-mRNA networks could be therapeutic targets for ESCC treatments.

overall_design: we performed genome-wide screening for mRNA and lncRNA profiles in 7 pairs (S1 nomal S2 Tumor, S1 and S2 paired, S3 normal S4 tumor, S3 and S4 paired, etc.) of esophageal cancer and normal tissues to identify the novel cancer-related mRNA and lncRNA in ESCC. Then we build a co-expression network for all of these mRNA and lncRNA to build the relationship between mRNA and lncRNA.

first: Weiwei


title: Membrane Metallo-Endopeptidase (Neprilysin) Regulates Inflammatory Response and Insulin Signaling in White Preadipocytes (RNA-Seq)

first: Alfred

last: Ramirez

address: One Joslin Place

institute: Joslin Diabetes Center

summary: Accumulation of subcutaneous white adipose tissue (WAT) is associated with increased insulin sensitivity, low levels of inflammation and a generally metabolically-healthy state, whereas accumulation of visceral adipose tissue is associated with insulin resistance, adipose tissue inflammation and metabolic syndrome. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. Here, we show that MME in white preadipocytes is differently expressed in subcutaneous vs visceral WAT, and favors insulin signaling and a low inflammatory response. Thus, knockdown of MME in preadipocytes increases the inflammatory response to substance P and amyloidβ aggregates. This is associated with increased basal insulin signaling and decreased insulin-stimulated signaling. Moreover, MME differentially regulates the internalization and turnover of the α/β subunits of the insulin receptor. Thus, MME is a novel regulator of the insulin receptor in adipose tissue and may serve as a therapeutic target to increase insulin sensitivity and decrease inflammatory susceptibility.

overall_design: RNA-seq of preadipocytes derived from the abomindal subcutaneous, omental, and mesenteric adipose tissue depot

middle: K

first: Simon

last: Dankel

first: Weikang

last: Cai

first: Masaji

last: Sakaguchi

last: Kasif

first: C

last: Kahn


title: The splicing factor U2AF1 contributes to cancer progression through a non-canonical role in translation regulation

first: Markus

last: Hafner

address: 50 South Drive

department: NIAMS

institute: NIH

summary: Somatic mutations in the spliceosome have emerged in recent years as oncogenes in human cancer. These mutations are in the factors involved in splice site selection, including a missense mutation (Ser34Phe) in a conserved nucleic acid binding domain of the spicing factor U2AF1. This protein plays a critical role in recognition of the 3'-splice site and assembly of the pre-spliceosomal complex. However, the role that this mutation plays in oncogenesis is still unknown. Here, we have uncovered a non-canonical function of U2AF1. PAR-CLIP and RIP data show that U2AF1 directly binds mature mRNA in the cytoplasm and that binding on or near the start codon results in translational repression. This splicing-independent translational regulatory role of U2AF1 is altered by the S34F mutation, leading to elevated translation of hundreds of mRNA, as revealed by polysome profiling.

overall_design:
PAR-CLIP on Chromatin and Cytoplasmic fractions of Flp-In-293 cells expressing FLAG-HA tagged WT or S34F mutant U2AF1. RIP-Seq was performed from U2AF1 WT,S34F and FS HBECs. To estimate translation efficiency, Ribosomal profiling followed by RNA sequencing of monosome and polysome- associated mRNA was performed from the same cells.
Please note that the PAR-CLIP processed data was generated from both replicates (i.e. A and B) and is linked to the corresponding A sample.

first: Murali

last: Palangat

first: Dimitrios

middle: G

last: Anastasakis

first: Daniel

last: Larson


title: Membrane Metallo-Endopeptidase (Neprilysin) Regulates Inflammatory Response and Insulin Signaling in White Preadipocytes

summary: Accumulation of visceral white adipose tissue (WAT) associates with insulin resistance, adipose tissue inflammation, and metabolic syndrome, whereas accumulation of subcutaneous WAT may be protective. We aimed to identify molecular mechanisms that might provide mechanistic insights underlying the phenotypic differences in these tissues. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. We hypothesized that MME contributes to adipose depot-specific metabolic properties.

overall_design: We performed RNA sequencing on human subcutaneous and visceral preadipocytes and array gene expression profiling in murine subcutaneous and visceral preadipocytes. We conducted several insulin signaling and inflammatory response experiments on different cellular states of MME expression.


title: Analysis of RNA synthesis in MCMV infected Wild type and Tyk2-/- bone marrow-derived macrophages  2 macrophage interferon antiviral cascade

status: Public on Feb 22 2019

city: Cardiff

first: Widad

last: Dantoft

address: Heath Park

institute: Cardiff University

summary: Murine Cytomegalovirus infection of macrophages results in hundreds of alterations in cellular gene expression. Many of these alterations are due to the autocrine and paracrine effects of virus-elicited type I interferons released by infected cells. Key to the function of type I interferons is the JAK-STAT signalling pathway. The extracellular binding of Type I IFN to the type 1 interferon receptor (consisting of IFNAR1 and IFNAR2) results in cytoplasmic activation of Tyrosine Kinase 2 (TYK2) and Janus Kinase 1 (JAK1) and the subsequent phosphorylation of STAT1 and STAT2. Phosphorylated STAT1 and 2 bind IRF9 forming a complex known as ISGF3. ISGF3 is translocated to the nucleus where it directly interacts with Interferon-sensitive regulatory elements in the genomic DNA. This binding results in direct and indirect alterations in the transcription of hundreds of genes. Whilst the functions of Tyk2 and changes in transcript abundance following MCMV infection are well characterised, little is known about changes in RNA synthesis at very early times after infection in intact cells or cells with a defective IFN signalling pathway. Microarrays were used to analyse alterations in transcript synthesis 30 to 60 mins and 360 to 390 minutes after MCMV infection of Wild Type or Tyk2-/- BMDM.

overall_design: Wild-type (WT) or Tyk2-/- Bone Marrow derived macrophages (BMDM) were grown using L929 conditioned medium. On day 7, BMDM were mock-infected (2x 15cm dishes WT BMDM and 2x 15cm dishes Tyk2-/- BMDM) or infected with MCMV (MOI = 1, 2x 15cm dishes WT BMDM and 2x 15cm dishes Tyk2-/- BMDM) for 1h. After 1h, virus and medium were aspirated and fresh medium added to all dishes – designated time=0. After 30 or 360 minutes post infection, 4-thiouridine (4sU) was used to label newly transcribed RNA in 4 dishes of mock or infected WT and Tyk2-/- BMDM (Mock WT, MCMV WT, Mock Tyk2-/-, MCMV Tyk2-/-) for a period of 30 minutes. Newly transcribed RNA was then isolated from each total RNA sample using a protocol described in Dolken et al (PMID: 18658122). For analysis, all 8 newly transcribed RNA samples were hybridized to Affymetrix GeneST v1.0 arrays.

first: Kevin

last: Robertson

first: W

last: Watkins

first: Birgit

last: Strobl

first: Peter

last: Ghazal


title: Membrane Metallo-Endopeptidase (Neprilysin) Regulates Inflammatory Response and Insulin Signaling in White Preadipocytes (Microarray Expression)

overall_design: FACS Isolated preaadipocytes from subcutaneous and perigonadal (intraabdominal) adipose tissue of mice were used for RNA extraction and hybridization on Affymetrix microarrays.


title: RNA Sequencing Facilitates Quantitative Analysis of Albumin-Cre (Control, Ctl) and Liver-specific Klf15 Knockout (Li-KO) Mouse Liver Transcriptomes

city: Cleveland

first: Ernest

last: Chan

address: 10900 Euclid Ave, Robbins E211

department: PQHS

institute: CWRU

summary: The goals of this study are to compare control and liver Klf15 deficient mouse liver transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis

overall_design: Liver mRNA profiles of 10-week old Albumin-Cre and Klf15 Li-KO mice were generated by deep sequencing, in triplicate, using Illumina GAIIx.

first: Shuxin

last: Han

first: Mukesh

last: Jain


title: Loss of androgen receptor signaling in prostate cancer‐associated fibroblasts (CAFs) promotes CCL2‐ and CXCL8‐mediated cancer cell migration

city: Nijmegen

first: Suzan

last: Stelloo

address: Geert Grooteplein Zuid 28

department: Molecular Biology

country: Netherlands

institute: Radboud University

summary: Fibroblasts are abundantly present in the prostate tumor microenvironment (TME), including cancer‐associated fibroblasts (CAFs) which play a key role in cancer development. Androgen receptor (AR) signaling is the main driver of prostate cancer (PCa) progression, and stromal cells in the TME also express AR. High‐grade tumor and poor clinical outcome are associated with low AR expression in the TME, which suggests a protective role of AR signaling in the stroma against PCa development. However, the mechanism of this relation is not clear. In this study, we isolated AR‐expressing CAF‐like cells. Testosterone (R1881) exposure did not affect CAF‐like cell morphology, proliferation, or motility. PCa cell growth was not affected by culturing in medium from R1881‐exposed CAF‐like cells; however, migration of PCa cells was inhibited. AR chromatin immune precipitation sequencing (ChIP‐seq) was performed and motif search suggested that AR in CAF‐like cells bound the chromatin through AP‐1‐elements upon R1881 exposure, inducing enhancer‐mediated AR chromatin interactions. The vast majority of chromatin binding sites in CAF‐like cells were unique and not shared with AR sites observed in PCa cell lines or tumors. AR signaling in CAF‐like cells decreased expression of multiple cytokines; most notably CCL2 and CXCL8 and both cytokines increased migration of PCa cells. These results suggest direct paracrine regulation of PCa cell migration by CAFs through AR signaling.

overall_design: Biopsies were taken directly after prostatectomy from locations of prostate cancer identified by multiparametric MRI and palpation of the tumor and immediately transferred to the laboratory. Primary fibroblast cultures were established as previously described (Villegas and McPhaul, 2005).   Prostate cancer-derived fibroblast cells were hormone‐deprived for 3 days and subsequently treated for 8 hours with R1881 (synthetic testosterone) or DMSO control. Then, cells were fixed for 10 minutes with 1% formaldehyde, quenched with glycine and lysed in LB1, LB2 and LB3 (Schmidt et al, 2009). Lysates were incubated with antibodies for AR or H3K27ac.

first: Wilbert

last: Zwart

first: Andre

last: Bergman


city: Luzhou

first: xu

last: liu

address: 25th Taiping Street

institute: The Affiliated Hospital of Southwest Medical University

summary: HOXC6 is a member of the HOX family, and its aberrant expression has been verified in a variety of cancers, such as prostate, breast,nasopharyngeal carcinoma,gastric, and ovarian cancers.Some studies suggest that HOXC6 might be involved in tumor initiation and progression.However, the role of HOXC6 in esophageal squamous cell carcinoma cells has not been fully investigated.Here we study how HOXC6 affects the malignant phenotype of esophageal squamous cell carcinoma cells

overall_design: Thirty-two paired samples from patients that underwent ESCC resection were used to verify the expression of HOXC6 by IHC, qRT-PCR and western blotting.HOXC6 was stably transfected into esophageal cancer cell lines using a lentivirus-mediated method.Wound healing and Matrigel assays were performed to assess the effect of HOXC6 on the migration and invasion of cancer cells. The growth curving, CCK8, and colony formation assays were utilized to access the proliferation capacities. RNA-seq was performed to evaluate the downstream targets of HOXC6

first: Li

last: Tang

first: Yong

last: Cao

first: Xueqin

last: Song

first: Xiaoyan

last: Wang

first: Yan

last: Li

first: Minglan

last: Yu

first: Mingying

first: Xu

last: Liu

first: Fang

last: Huang

first: Feng

last: Chen

first: Haisu

last: Wan


title: Expression data from the mouse liver with constitutively active FoxO3

city: Ulm

first: Karlheinz

last: Holzmann

address: Helmholtzstreet 8/1

department: Medical Faculty

institute: Ulm University

summary:
Activation of hepatic FoxO3 signaling induces hyperglycemia and hyperinsulinemia. Metformin administration reverses FoxO3-induced phenotype development.
To investigate the underlying mechanism, we used microarrays to detail the global programme of gene expression which is regulated by hepatic FoxO3 signaling.

overall_design: We generated transgenic mice which express a constitutively active FoxO3 controlled under the LAP promotor. The liver samples were collected from transgenic mice as well as from control animals at the age of 7 weeks. RNA extraction was performed from these liver samples and hybridized on Affymetrix microarrays.

first: Sarah

last: Gul

first: Pavel

last: Strnad

first: Yoshiaki

last: Sunami

first: Thomas

last: Wirth


title: Single cell RNA sequencing of aortic CX3CR1-derived monocyte/macrophage from atherosclerotic aorta during progression and regression

status: Public on Feb 21 2019

first: Emily

last: Brown

address: 435 E 30th St

department: Medicine

institute: NYU Langone

summary: In atherosclerosis progression and regression, monocytes or monocyte-derived macrophages are the major immune cells in the plaque. It is important to understand the fate and characteristics of monocyte/macrophage during the plaque progression and regression. To characterize the fate of monocytes/macrophages, we performed single cell RNA sequencing of fate-mapped aortic CX3CR1-derived  monocytes/macrophages from Cx3cr1CreERT2-IRES-YFP/+Rosa26floxed-tdTomato/+ mice with AAV-PCSK9 injection and fed a Western Diet. The single cell RNA-seq analyses revealed the heterogeneity of aortic macrophages and identified a stem-like cell cluster in atherosclerotic aorta.

overall_design: Single cell RNA sequencing of aortic sorted TdTomato+CD11b+Dump- cells (Dump- are excluding CD3+, CD19+, CD49b+, Siglec-F+, Ly6G+ cells) from atherosclerotic Cx3cr1CreERT2-IRES-YFP/+Rosa26floxed-tdTomato/+ mice (n=8, 1012 AAV-PCSK9 at 8wk old mice, then WD for 18 wks). Then 4 mice continue on WD for further 2wks as progression group, 3157 cells identified, Using Illumina HiSeq4000.  The other 4 mice were switched into chow diet for further 2wks and also i.p. treated with ApoB-ASO (50 mg/kg, 2 doses/wk, 2wks) as regression group, 2198 cells identified, Using Illumina HiSeq4000.

first: Jian-Da

last: Lin

first: Hitoo

last: Nishi

first: Jordan

last: Poles

first: Xiang

last: Niu

first: Stephen

last: Ramsey

first: Edward

last: Fisher

first: P'ng

last: Loke


title: Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI

city: Columbus

middle: Rose

last: Theisen

address: 700 Childrens Drive

department: Center For Childhood Cancer and Blood Diseases

institute: Nationwide Children's Hospital

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

The data represent functional transcriptomic profiles of the oncogenic fusion transcription factor EWS/FLI.

overall_design:
Refer to individual Series.

Briefly, EWS/FLI was depleted from A673 cells using RNAi and its function was rescued with the following mutants:
wildtype EWS/FLI - positive control for rescue
delta22 - negative control for rescue; the functional EWS domain is deleted
DAF - contains 17 tyrosine to alanine mutations in the N-terminal portion of the EWS domain
mut9 - contains a deletion of residues 83-246 of the EWS domain
DAF-mut9 - contains a deletion of residues 83-246 of the EWS domain and 9 tyrosine to alanine mutations in residues 1-83.

Experiment 1 includes: iLuc/empty vector rescue (No EWS/FLI depletion), iEF/empty vector rescue (EWS/FLI depletion with no rescue, iEF/wtEF (rescue with wildtype EWS/FLI), iEF/delta22 (rescue with delta22), and iEF/DAF (rescue with DAF)

Experiment 1 includes: iLuc/empty vector rescue (No EWS/FLI depletion), iEF/empty vector rescue (EWS/FLI depletion with no rescue, iEF/wtEF (rescue with wildtype EWS/FLI), iEF/delta22 (rescue with delta22), iEF/DAF (rescue with DAF), iEF/mut9 (rescue with mut9), and iEF/DAF-mut9 (rescue with DAF-mut9).


title: Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI [Experiment 2]

summary: The purpose of this study was to define unique classes of EWS/FLI target genes and the distinct set of structural features present in the EWS domain required for target gene regulation at different EWS/FLI response elements. This study reports the first genome-wide transcriptomic description of a partially-functional EWS/FLI mutant . We report new structure-function dependencies across different classes of response element and tie these dependencies to the ability of EWS/FLI to transform cells.

overall_design: For knockdown of wildtype EWS/FLI and rescue with differing cDNAs in A673 Ewing sarcoma cell lines, shRNAs were retrovirally delivered on day 1. Selection in 2 µg/mL puromycin (Sigma P8833) occurred on days 2-5. Cells were infected with retrovirus containing the cDNA in the pMSCV-hygro backbone on day 5. Double selection in puromycin and 400 µg/mL hygromycin B (Thermo Fisher 10687010) began on day 6 and continued until day 16. Cells were subsequently harvested for RNA, protein, and seeded for soft agar assays.

middle: L

last: Lessnick


title: Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI [Experiment 1]


title: Quantitative Analysis of Wild Type and Neat1 -/- Cerebral Frontal Cortex Transcriptomes

last: Katsel

address: 1 Gustave L Levy Pl

department: Psychiatry

institute: Icahn School of Medicine At Mount Sinai

summary:
Purpose: The goals of this study are to elucidate dowstream effects of  lnc RNA, Neat1 deletion in cerebral frontal cortex of adult mice by comparing Next-generation sequencing -derived  cortical transcriptome profiles (RNA-seq) between wild type and Neat1 knockout mice.
Methods: Brain mRNA profiles of 2-4 moths-old wild-type (WT) and lnc RNA, Neat1 knockout (Neat1−/−) mice were generated by deep sequencing, using Illumina. Reads were mapped to mm10 reference genome using TopHat (version 2.0.9) and Bowtie (version 2.1.0), with the default parameters. Known iGenomes Ensembl mm10 were quantified by HTSeq (version 0.6.0) in intersection-strict mode. A sample-by-gene read count matrix was generated for all samples by the Ensembl genes. Scaling normalization to remove composition biases in sequencing data was applied to log(CPM) (read Counts Per Million total reads) using the trimmed mean of M-values (TMM) method.
Results: RNA-seq showed near-complete depletion of Neat1 RNA levels. 1359 genes were differentially expressed in the frontal cortex of Neat1-/- mice. 25 of these differentially expressed genes withstood multiple testing corrections. Examination of RNA-seq data by principle component analysis showed two principle components that were mutually uncorrelated and orthogonal. Hierarchical cluster tree analysis showed that joined nodes from Neat1-/- samples were distanced from control subset cluster confirming the results of the PCA.
Conclusions: Analyses of differentially expressed gene signature from NEAT1-/- mice revealed a significant impact on processes related to oligodendrocyte differentiation and RNA post-transcriptional modification with the underlying mechanisms involving Wnt signaling, cell contact interactions, and regulation of cholesterol/lipid metabolism.

overall_design: Cerebral frontal cortex mRNA profiles of 2-4 months old wild type (WT) and Neat1 -/- mice (all females) were generated by deep sequencing (N=5 controls; N=4 Neat1 knockout).

first: Panos

last: Roussos


title: RNA stability profiling in SETD2 and METTL14 knockdown HepG2 cells

city: Monrovia

first: Jianjun

last: Chen

state: CA

department: Systems Biology

institute: Beckman Research Institute of City of Hope

summary: To evaluate the effect of SETD2 and METTL14 on mRNA stability, we conducted RNA-seq in SETD2 or METTL14 knockdown HepG2 cells as well as control cells with or without actinomycin D treatment. Our RNA stability profiling revealed that depletion of SETD2 and METTL14 resulted in global reduction of RNA stability, and the changes were correlated between SETD2 and METTL14 knockdown cells.

overall_design: HepG2 cells were infected with lentiviral shRNAs against SETD2, METTL14 and non-specific control (shCtrl), and selected by puromycin to generate stable knockdown lines. We treated HepG2 cells with actinomycin D to inhibit transcription and collected cells at indicated time points (i.e., 0h, 1h, 3h, 6h). The total RNA was extracted by miRNeasy Kit (Qiagen) and sequenced by Illumina.  For RNA stability profiling, RNA half-life was calculated by comparing the gene expression at 1, 3, 6 hours with actinomycin treatment to that in un-treated samples, with two biological replicates for each group.

first: Huilin

last: Huang

first: Hengyou

last: Weng


title: De novo Gene Signature Identification from Single-Cell RNA-Seq with Hierarchical Poisson Factorization

first: Hanna

middle: Mendes

last: Levitin

address: Department of Systems Biology 3960 Broadway, Lasker 203A

department: Department of Systems Biology

institute: Columbia University Medical Center

summary: Common approaches to gene signature discovery in single cell RNA-sequencing depend upon predefined structures like clustering or pseudo-temporal orderings, do not account for the sparsity of single cell data, or require prior normalization.  We present single cell Hierarchical Poisson Factorization (scHPF), a Bayesian factorization method that adapts Hierarchical Poisson Factorization for de novo discovery of both continuous and discrete expression patterns in complex tissues. scHPF does not require prior normalization and outperforms other methods in benchmark datasets.  Applied to single cell RNA-sequencing of the core and margin of a high-grade glioma, scHPF uncovers subtle regional expression biases within glioma subpopulations and an expression signature associated with inferior survival in glioblastoma.

overall_design: Performed single cell RNA-seq on radiographically-localized tissue samples from a high-grade glioma and a glioma tumor-sphere.

middle: M

first: Jinzhou

last: Yuan

first: Yim

last: Cheng

first: Peter

last: Sims


title: HEPATIC TRANSCRIPTOME SIGNATURES IN PATIENTS WITH VARYING DEGREES OF NON-ALCOHOLIC FATTY LIVER DISEASE COMPARED TO HEALTHY NORMAL-WEIGHT INDIVIDUALS

city: Hørsholm

first: Kristoffer

last: Rigbolt

address: Hørsholm Kongevej 11B

country: Denmark

institute: Gubra

summary: Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of conditions ranging from simple steatosis (NAFL), over non-alcoholic steatohepatitis (NASH) with or without fibrosis, to cirrhosis with end-stage disease. The hepatic molecular events underlying the development of NAFLD and transition to NASH are poorly understood. The present study aimed to determine hepatic transcriptome dynamics in patients with NAFL or NASH compared to healthy normal-weight and obese individuals. RNA sequencing and quantitative histomorphometry of liver fat, inflammation and fibrosis was performed on liver biopsies obtained from healthy normal weight (n=14) and obese (n=12) individuals, NAFL (n=15) and NASH (n=16) patients. Normal weight and obese subjects showed normal liver histology and comparable gene expression profiles. Liver transcriptome signatures were largely overlapping in NAFL and NASH patients, however, clearly separated from healthy normal-weight and obese controls. Most marked pathway perturbations identified in both NAFL and NASH were associated with markers of lipid metabolism, immunomodulation, extracellular matrix remodeling and cell cycle control. Interestingly, NASH patients with positive Sonic hedgehog hepatocyte staining showed distinct transcriptome and histomorphometric changes compared to NAFL. In conclusion, application of immunohistochemical markers of hepatocyte injury may serve as a more objective tool for distinguishing NASH from NAFL, facilitating improved resolution of hepatic molecular changes associated with progression of NAFLD.

overall_design: Liver biopsies from healthy, obese, NAFLD and NASH subjects

first: Jens

middle: C

last: Nielsen


title: Lymphoid Biased Hematopoietic Stem Cells are Maintained with Age and Retain Normal Developmental Potential in the Absence of Inflammatory Stimuli

city: Los Angeles

first: Kenneth

last: Dorshkind

state: California

address: 10833 Le Conte Avenue

department: Pathology and Laboratory Medicine

institute: David Geffen School of Medicine at UCLA

summary: The hematopoietic stem cell (HSC) compartment includes lymphoid biased (Ly-HSCs) and myeloid biased (My-HSCs) subsets. Current models propose that the number of Ly-HSCs declines with age and this contributes to the diminution of lymphopoiesis that is a feature of aging. This view is based on a reduction in the frequency of Ly-HSCs in old bone marrow, but we now show that when total HSCs are taken into account, the number of Ly-HSCs is not reduced in old mice. We further demonstrate that old Ly-HSCs exhibit a normal capacity to produce lymphoid progenitors when removed from the bone marrow. However, the production of inflammatory cytokines is known to increase with age, and when Ly-HSCs are exposed to these mediators, lymphocyte production is blocked. We also demonstrate that old Ly-HSCs generate myeloid cells that phenotypically resemble those produced by old My-HSCs, a finding consistent with the myeloid biased gene expression pattern of old Ly-HSCs. These observations indicate that current models of how aging impacts the lymphopoietic potential of HSCs need to be revised and point to changes in the in vivo environment, rather than intrinsic changes in HSCs, as the principal cause of the reduced lymphopoiesis and increased myelopoiesis during aging.

overall_design: We isolated Ly-HSCs (lineage negative, Sca-1+ CD117(c-kit)+ CD48- CD135- CD150low) and My-HSCs (lineage negative, Sca-1+ CD117(c-kit)+ (LSK) CD48- CD135- CD150high) from three independent groups of young (8-12 week-old) and old (18 months) mice and performed RNA-Seq.

first: Ying

last: Kong

first: Encarnacion

last: Montecino-Rodriguez

first: David

last: Casero

middle: D

last: Pioli


title: Transcriptomic profiles of retinal ganglion cells are defined by the magnitude of intraocular pressure elevation in adult mice

city: Houston

first: Edwin

middle: Justin

last: Ostrin

address: 1515 Holcombe Blvd, Unit 1465

department: Pulmonary Medicine

institute: MD Anderson Cancer Center

summary: Elevated intraocular pressure (IOP) is the major risk factor for glaucoma, a sight threatening disease of retinal ganglion cells (RGCs) and their axons.  Despite the central importance of IOP, details of the impact of IOP elevation on RGC gene expression remain elusive.  We developed a novel 4-step immunopanning protocol to extract adult mouse RGCs with high fidelity and used it to isolate RGCs from wild type mice exposed to 2 weeks of IOP elevation generated by the microbead model.  IOP was elevated to 2 distinct levels which were defined as Mild (IOP increase >1mmHg and <4mmHg) and Moderate (IOP increase > 4mmHg).  RNA sequencing was used to compare the transcriptional environment at each IOP level.  Differentially expressed genes were markedly different between the 2 groups, and pathway analysis revealed frequently opposed responses between the IOP levels.  These results suggest that the magnitude of IOP elevation has a critical impact on RGC transcriptional changes.  Furthermore, it is possible that an IOP-based set point exists within RGCs to impact the direction of transcriptional change.  It is possible that this improved understanding of changes in RGC gene expression can ultimately lead to novel diagnostics and therapeutics for glaucoma.

overall_design: RGC mRNA profiles of 6 week old C57BL/6J mice injected with two levels of elevated IOP pressure induced by microbeads.    Samples were pooled and split into tripicates.  Trancriptomes were generated by deep sequencing, using Illumina HiSeq2500.

first: Yong

middle: H

last: Park

first: Rui

first: Benjamin

last: Frankfort


title: Targeting MRTF/SRF in CAP2-dependent dilated cardiomyopathy delays disease onset

city: Philadelphia

first: Jeffrey

last: Field

state: PA

address: 1313 BRB II/III U

department: Pharmacology

institute: University of Pennsylvania

summary: About one third of dilated cardiomyopathy (DCM) cases are caused by mutations in sarcomere or cytoskeletal proteins.  Yet treating the cytoskeleton directly is not possible because drugs that bind to actin are not well tolerated. Mutations in the actin binding protein CAP2 can cause DCM and knockout mice, either whole body (CAP2 KO) or cardiomyocyte specific knockouts (CAP2 CKO), develop DCM with cardiac conduction disease.  RNA-seq analysis of CAP2 KO hearts and isolated cardiomyocytes revealed over-activation of fetal genes including serum response factor (SRF) regulated genes such as Myl9 and Acta2 prior to the emergence of cardiac disease.  To test if we could treat CAP2 KO mice, we synthesized and tested the SRF inhibitor CCG-1423-8u.  CCG-1423-8u reduced expression of the SRF targets Myl9 and Acta2, as well as the biomarker of heart failure, NPPA. The median survival of CAP2 CKO mice was 98 days, while CCG-1423-8u treated CKO mice survived for 116 days and also maintain normal cardiac function longer.   These results suggest that some forms of sudden cardiac death and cardiac conduction disease are under cytoskeletal stress and that inhibiting signaling through SRF may benefit DCM by reducing cytoskeletal stress.

overall_design: Heart mRNA profiles of 86-week old wild type (WT), CAP2-/- mice 3 replicates each for experiment A, and in experiment B, 5 replicates of 13-week old loxp mice and 4 replicates of L/L,Myh6-cre+ mice, were generated by deep sequencing, in triplicate, using Illumina GAIIx. A 5th L/L,Myh6-cre+ had originally failed sequencing and was excluded from the study, but has since been resequenced and is included (sample  KO_m_1623). The processed data of experiment B normalized read counts does not include this last sample as it was not part of the study.

first: Yao

last: Xiong

first: Thomas

last: Brooks