title: Multi-modal single cell analysis reveals gut microbiota reshapes brain immune landscape in aged mouse model

city: Dallas

first: Siyuan

last: Zhang

address: 6001 Forest Park Rd

department: Department of Pathology

institute: UT Southwestern Medical Center

summary: We used CITE-seq (10x Gemoics-based) to profile and compare the transcriptomes and cell surface expression of a set of immune markers of naïve brain myeloid cells from young-adult (12 weeks old) wild type C57Bl/6 mice, aged (72 weeks old) wild type C57Bl/6 mice and young-adult and aged  C57Bl/6  mice with antibiotics induced gut microbiota depletion. In one sequencing round, we sequenced a total of 12 different mouse brains (3 young control, 3 aged control, 3 young depleted gut microbiota (ABX) and 3 aged depleted gut microbiota (ABX)). We created an antibody pool consisting of 31 different antibodies (CCR2/CD192, C117/c-kit, CD11b, CD11c, CD172a/SIRPα, CD25, CD3, CD38, CD4, CD44, CD45, CD45R/B220, CD86, CD8a, CD90.1, Cx3cr1, F4/80, I-A/I-E, Ly6C, Ly6G, NK1.1, PD-1, PD-L1, CD169/Siglec-1, Siglec-H, TMEM119, XCR1, CD24, CD103, CD64, CD83) and stained each brain individually with this antibody pool. Then, we stained each brain with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium. Young Control and Young ABX brains were tagged with HTOs 1-6 (HTO 1-3 for young control and HTO 4-6 for young ABX). Aged control and Aged ABX brains were tagged with HTOs 1-6 (HTO 1-3 for aged control and HTO 4-6 for aged ABX). Young and aged were loaded in separate wells on the 10X Chromium. The samples were tagged with unique nextera index primers to allow for preparation of one sequencing library consisting of all 12 samples and were separated in silico by it's unique hashing antibody and nextera index barcode combinations.

overall_design: Naïve brains were obtained from twelve 12 week old female C57Bl/6 mice and twelve 72 week old female C57Bl/6 mice . Six young-adult and six aged mice received vehicle treatment and had an unperturbed gut microbiota and six young-adult and six aged mice received antibiotics treatment and had a depleted gut microbiota. All 24 brains (6 from each group) were individually processed into cell suspensions and collapsed into 12 final samples (3 young control, 3 aged control, 3 young depleted gut microbiota (ABX) and 3 aged depleted gut microbiota (ABX)). Cell suspensions were prepared by percoll gradient centrifugation enrichment to obtain suspensions enriched for immune cells. We created an antibody pool consisting of 31 different antibodies (CCR2/CD192, C117/c-kit, CD11b, CD11c, CD172a/SIRPα, CD25, CD3, CD38, CD4, CD44, CD45, CD45R/B220, CD86, CD8a, CD90.1, Cx3cr1, F4/80, I-A/I-E, Ly6C, Ly6G, NK1.1, PD-1, PD-L1, CD169/Siglec-1, Siglec-H, TMEM119, XCR1, CD24, CD103, CD64, CD83) and stained each brain individually with this antibody pool. Young Control and Young ABX brains were tagged with HTOs 1-6 (HTO 1-3 for young control and HTO 4-6 for young ABX). Aged control and Aged ABX brains were tagged with HTOs 1-6 (HTO 1-3 for aged control and HTO 4-6 for aged ABX). Young and aged were loaded in separate wells on the 10X Chromium. The samples were tagged with unique nextera index primers to allow for preparation of one sequencing library consisting of all 12 samples and were separated in silico by it's unique hashing antibody and nextera index barcode combinations.

first: Samantha

last: Golomb


title: CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators

status: Public on Nov 04 2020

city: Shenzhen

first: Guipeng

last: Li

address: 1088 Xueyuan Avenue

country: China

institute: Southern University of Science and Technology

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

overall_design: Refer to individual Series


title: miRNA sequencing in L02 cells

city: WUHAN

first: heng

middle: zhi

last: du

address: 1095 Jiefang Dadao, Wuhan City, Hubei Province, China

institute: Wuhan Tongji Hospital

type: Non-coding RNA profiling by high throughput sequencing

summary: miRNA was performed in L02 cells

overall_design: We report the application of sequencing technology for high-throughput profiling of miRNA expressions in L02 cells. In this project, rRNA was removed from the total RNA by ribosomal RNA removal kit, and the way of ion breaking were used to break the RNA into 200-300bp fragments. By using RNA as template, the first strand of cDNA was synthesized with 6 base random primers and reverse transcriptase. The first strand cDNA was used as the template to synthesize the second strand cDNA. When the second strand cDNA is synthesized, the base T is replaced by U to achieve the goal of a chain specific library. All of our RNA-seq projects used strand specific kits to construct the libraries. Finally, we show that  mRNA different expression in  L02 cells. This study provides abundance of miRNA that may predict liver diseases.

first: yinzhong

last: wei

first: zhaoyan

last: ru

first: duheng

last: zhi


title: CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators I

summary: Cellular RNA is decorated with over 160 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that could modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking.  Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq).

overall_design: CIGAR-seq of HAP1 cells

first: Wei

last: Chen


title: TGFb-blockade uncovers stromal plasticity in tumors by revealing the existence of a novel subset of interferon-licensed fibroblasts [human]

status: Public on Nov 03 2020

city: Cambridge

first: Viviana

last: Cremasco

state: MA

address: 250 Massachusetts Avenue

country: USA

institute: Novartis Institutes for BioMedical Research

type: Expression profiling by high throughput sequencing

summary: Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we performed an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFβ in vivo led to a profound remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a novel fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlated with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFβ and PD1 co-blockade in the clinical settings, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.

overall_design: Dissociated human CRC tumor treated by TGFb blockade in vitro

first: Marc

last: Pelletier

first: Beverly

last: Nguyen

first: Yan

last: Zheng

first: Jincheng

last: Wu


title: CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators II

overall_design: mRNA-BisSeq of HAP1 cells


title: Single-cell Transcriptome Analysis Dissects the Replicating Process of Pancreatic Beta Cells in Partial Pancreatectomy Model.

city: Kyoto

first: Akira

last: Watanabe

state: Kyoto

address: Rm504, Miyako Bldg, 233, Isa-cho, kamigyo-ku

country: Japan

institute: CyberomiX Inc

summary: We employed single-cell RNA sequencing of islets in young mice subjected to partial pancreatectomy (PPTx). Four clusters of pancreatic beta cells including a subpopulation of replicating beta cells. Pseudo-time course analysis clarified the gradual changes in gene expression from non-replicating beta cells to replicating ones. Upstream analysis visualized the transcriptional networks associated with beta cell replication by PPTx.

overall_design: Pancreatic islets were isolated from young mice 2 days after PPTx or without PPTx. Cells were dispersed by trypsin and went to single cell RNA-seq. Data were processed by dimensional reduction and clustering. Subpopulations in pancreatic islet cells and differentially expressed genes were analyzed. Cell cycle analysis and expression of cell cycle genes identified one cluster as replocating beta cells. Pseudo time analysis were performed and gradual change of gene expression from non-replicating beta cells to replicating beta cells were visualised. Upstream analysis were conducted using genes differentially expressed in each subpopulation and different timepoint in pseudo-time.

first: Hisato

last: Tatsuoka

first: Satoko

last: Sakamoto

first: Daisuke

last: Yabe

first: Ryotaro

last: Kabai

first: Tatsuya

last: Okumura

first: Nobuya

last: Inagaki


title: RNA sequencing discerns sub-populations of pancreatic β-cells and dynamics of gene expression in partial pancreatectomy model

first: Ainur

last: Botagarova

first: Shinsuke

last: Tokumoto

first: Ryota

last: Usui

first: Masahito

last: Ogura

first: Kazuaki

last: Nagashima

first: Eri

last: Mukai

first: Yoshio

last: Fujitani


title: TGFb-blockade uncovers stromal plasticity in tumors by revealing the existence of a novel subset of interferon-licensed fibroblasts

first: Viviana

last: Cremasco

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Sequencing discerns sub-populations of pancreatic β-cells and dynamics of gene expression in partial pancreatectomy model


title: The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis Study (GRADS)  Alpha-1 study [A1AT]

city: New Haven

first: Jen-hwa

last: Chu

state: CT

department: Pulmonary, Critical Care and Sleep Medicine

institute: Yale University School of Medicine

summary: Whole genome mRNA and microRNA profiling of bronchoalveolar lavage (BAL) and peripheral blood mononuclear cell (PBMC) in Alpha-1 Antitrypsin Deficiency patients with PiZZ or PiMZ alpha-1 antitrypsin genotypes

overall_design: 89 patients affected by AATD were recruited at Alpha-1 Foundation Research Registry at the Medical University of South Carolina (MUSC) with matched BAL and PBMC samples. The study population includes three groups: (1) individuals with PiZZ not receiving augmentation therapy (n=29), (2) individuals with PiZZ receiving augmentation therapy (n=22) and (3) individuals with PiMZ not receiving augmentation therapy (n=38).

first: Xiting

last: Yan

first: Naftali

last: Kaminski


title: Azidonucleoside-incorporated RNA sequencing for new transcripts analysis under heat shock in E.coli

status: Public on Nov 01 2020

city: Beijing

first: Xing

last: Chen

address: Yiheyuan Road

department: Chemical Biology

institute: Peking University

summary: Cellular RNA levels result from the interplay of RNA transcription, processing, and degradation. Multiple complementary methods have been developed to dissect these tightly regulated processes. One of the methods exploits chemically active nucleoside analogs (i.e., noncanonical nucleosides) to metabolically label newly transcribed RNA. The labeled RNA are then chemically conjugated with fluorophores for imaging or affinity tags for enrichment and sequencing, which allow for studying transcription by separation of nascent RNA from the pre-existing populations. Here we used AzG to profile RNA dynamic changes under heat shock in E.coli.

overall_design: E. coli grown under normal condition at 37 ℃ or under mildly heat shocked condition at 42 ℃ were metabolically labeled with AzG for 10 min, followed by extraction of total RNA.  After reacting with DBCO-biotin, the newly transcribed RNA  were isolated by streptavidin beads and subjected to RNA-seq.

first: Liying

last: Meng


title: HP1γ regulates H3K36 methylation and pluripotency in embryonic stem cells (ChIP-Seq)

city: Madison

first: Rupa

last: Sridharan

state: WI

address: 330 N. Orchard Street

department: Wisconsin Institute for Discovery

institute: University of Wisconsin

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The heterochromatin protein 1 (HP1) family members are canonical effectors and propagators of gene repression mediated by histone H3 lysine 9 (H3K9) methylation. HP1γ exhibits an increased interaction with active transcription elongation-associated factors in embryonic stem cells (ESCs) compared to somatic cells. However, whether this association has a functional consequence remains elusive. Here we find that genic HP1γ colocalizes and enhances enrichment of transcription elongation-associated H3K36me3 rather than H3K9me3. Unexpectedly, sustained H3K36me3 deposition is dependent on HP1γ. HP1γ-deleted ESCs display reduced H3K36me3 enrichment, concomitant with decreased expression at shared genes which function to maintain cellular homeostasis. Both the H3K9me3-binding chromodomain and histone binding ability of HP1γ are dispensable for maintaining H3K36me3 levels. Instead, the chromoshadow together with the hinge domain of HP1γ that confer protein and nucleic acid-binding ability are sufficient because they retain the ability to interact with NSD1, an H3K36 methyltransferase. HP1γ-deleted ESCs have a slower self-renewal rate and an impaired ability to differentiate towards cardiac mesoderm.  Our findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency.

overall_design: H3K36me3 ChIP-Seq of WT, HP1gamma KO, and HP1gamma rescue constructs ESCs

first: Nur

middle: Z

last: Zaidan


title: AzG facilitated RNA dynamic analysis in E.coli


title: The stratum corneum transcriptome in atopic dermatitis can be assessed by tape stripping

status: Public on Oct 31 2020

city: Hellerup

first: Julie

middle: Breinholt Kjær

last: Sølberg

state: Danmark

address: Gentofte Hospitalsvej 20A

department: Department of dermatology and allergy, Gentofte Hospital

country: Denmark

institute: The national allergy research centre

summary:
Background: Skin biopsies represent a gold standard in skin immunology and pathology but can cause pain and induce scarring. Non-invasive techniques will facilitate study recruitment of e.g. patients with pediatric atopic dermatitis (AD), hand eczema or facial dermatitis.
Objective: By RNA sequencing, we examined whether the stratum corneum transcriptome in AD skin can be assessed by tape stripping, as compared to the epidermal transcriptome of AD in skin biopsies. To make the procedure clinically relevant tape strips were stored and shipped at room temperature for up to 3 days.
Methods: Nine adult Caucasian AD patients and three healthy volunteers were included. Tape samples were collected from non-lesional and lesional skin. Biopsies were collected from lesional skin and were split into epidermis and dermis. Total RNA was extracted, and shotgun sequencing was performed.
Results: Shotgun sequencing could be performed on skin cells obtained from two consecutive tape strips which had been stored and shipped at room temperature for up to three days. The most prominent differences between the tape strip and biopsy derived transcriptome were due to structural genes, while established molecular markers of AD, including CCL17, CCL22, IL17A and S100A7-S100A9, were also identified in tape strip samples. Furthermore, the tape strip derived transcriptome showed promise in also analysing the skin microbiome.
Conclusion: Our study shows that the stratum corneum (SC) transcriptome of AD can be assessed by tape stripping the skin, supporting that this method may be central in future skin biomarker research.

overall_design: 9 patients with atopic dermatitis and 3 healthy subjects were included.  Biopsy samples was collected from lesional and healthy skin areas (healthy subjects only). Tape samples was obtained from lesional and non-lesional skin sites and from healthy skin areas (healthy subjects only).

first: Stine

middle: B

last: Jacobsen

first: Jeppe

middle: D

last: Andersen

first: Thomas

last: Litman

first: Nina

middle: H

last: Ulrich

first: Malin

middle: G

last: Ahlström

first: Marie-Louise

last: Kampmann

first: Niels

last: Morling

first: Jacob

middle: P

last: Thyssen

first: Jeanne

last: Johansen


title: HP1γ regulates H3K36 methylation and pluripotency in embryonic stem cells


title: Gene expression analysis of E.coli K12 treated with and without AzG

summary: We developed AzG as a new nucleoside analog to metabolic label eukaryotic RNAs. Here, we analyzed gene expression of E.coli K12 treated with and without AzG to confirm that AzG doesn’t affect transcription at transcriptome level.

overall_design: Three biological replicates of E.coli K12 treated with or without AzG analysed.


title: Azidonucleoside-incorporated RNA sequencing for RNA stability analysis in E.coli.

summary: To study RNA degradation by RNA-seq, global inhibition of transcription has to be performed in order to measure RNA decay with no interference from RNA sysnthesis. However, transcription inhibitors are generally toxic, can affect abundance many transcripts, and cause delay in RNA decay by residual RNA synthesis. We envisioned that AIR-seq, in conjunction with the pulse-chase labeling, would enable genome-wide analysis of RNA degradation in E. coli with no need of transcription inhibition.

overall_design: After pulse labeling with AzG for 2 h, the E. coli cells were chased with guanosine for 0, 5, and 10 min, respectively, followed by extraction of total RNA.  After reacting with DBCO-biotin, the labeled RNA  were isolated by streptavidin beads and subjected to RNA-seq.


title: ATAC-seq analysis of murine thymic iNKT subsets from C57BL/6 mice and Hivep3-deficient mice

city: Aurora

first: Laurent

last: Gapin

state: CO

department: Immunology and Microbiology

institute: University of Colorado Anschutz Medical campus

summary: ATAC-seq analysis of murine thymic iNKT subsets from two independent C57BL/6 mice and two independent Hivep3-deficient mice

overall_design: This subseries consists of biological replicates (Mouse_1 and Mouse_2) of Omni ATAC-seq experiments in wild-type and Hivep3-/- iNKT cell subsets.


title: Small RNA Sequencing of the Plant Growth Promoting Bacterium Azospirillum brasilense Sp245

city: Gurugram

first: Mandira

last: Kochar

state: Haryana

address: Nanobiotechnology Centre, Sustainable Agriculture Division, TERI Gram, Gual Pahari, Gurugram-Faridabad Highway

department: Sustainable Agriculture Division

country: India

institute: The Energy and Resources Institute, New Delhi

summary: Small RNAs are the non-coding RNAs known to regulate various biological functions such as stress adaptation, metabolism, virulence as well as pathogenicity across wide range of bacteria, mainly by controlling mRNA stabilization or regulating translation. Identification and functional characterization of sRNAs that has been carried out in various plant growth promoting bacteria have shown to help the bacterial cell cope up with environmental stress. Till now no study has been carried out to uncover these regulatory molecules in diazotrophic alpha-proteobacterium Azospirillum brasilense Sp245. RNA-Seq is a suitable approach for expression-based sRNA identification in bacteria.

overall_design: Identification of A. brasilense Sp245 sRNAs in control and nitrogen-stress conditions

first: Vatsala

last: Koul


title: HP1γ regulates H3K36 methylation and pluripotency in embryonic stem cells (RNA-seq)

overall_design: RNA-sequencing of WT or HP1gamma KO ESCs


title: Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology.

city: New York

last: Zhang

state: New York

address: 522 First Avenue, Smilow Research Building, 1106

department: Pathology

institute: New York University School of Medicine

summary: Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in inducible Pax7-expressing skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterized at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identified key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that were dependent on Pax7 binding. Remarkably, we found that the majority of super-enhancers were bound by Pax7 and a cadre of associated factors that maintained an epigenetic memory of active enhancers in the absence of Pax7. Lastly, we identified a previously uncharacterized Pax7-bound enhancer hub that simultaneously regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the three-dimensional conformation of chromatin in muscle stem cells.

overall_design: Examination of chromatin structure re-organizaion during myogenic differentiation in the inducible Pax7-expressing (iPax7) skeletal muscle progenitors. (1) in situ Hi-C in both undifferentiated progenitor (doxycyline treated, +Dox) and differentiated (-Dox) iPax7 cells; (2) promoter capture Hi-C (pCHi-C) in both undifferentiated progenitor (+Dox) and differentiated (-Dox) iPax7 cells; (3) ChIP-seq of CTCF and Smc3 in both undifferentiated progenitor (+Dox) and differentiated (-Dox) iPax7 cells; (4) ATAC-seq of iPax7 ESC (-Dox), wild-type (WT) and Myod1 knock-out primary myoblasts.

first: Brian

last: Dynlacht


title: RNA sequencing of CEA-TCB (cibisatamab)-treated and untreated tumors derived from MKN-45 tumor-bearing humanized mice

status: Public on Oct 29 2020

city: Basel

first: Andreas

last: Roller

address: Grenzacherstrasse 124

country: Switzerland

institute: Roche

summary: These experiments were done as part of a larger study that investigated the mode of action of T-cell bispecific (TCB) antibodies (including CEA-TCB [cibisatamab]) by characterizing molecular and cellular features of immune cells and tumors following TCB treatment in vivo in humanized mice and syngeneic tumor models. To characterize the molecular parameters associated with CEA-TCB treatment we performed bulk RNA sequencing of CEA-TCB-treated and untreated tumors derived from MKN-45 tumor-bearing humanized mice. Tumor tissue was analyzed after seven consecutive TCB treatments and compared to matching controls.  RNA sequencing analysis revealed a clear distinction between CEA-TCB-treated and control animals, with several genes being upregulated in tumors treated with CEA-TCB as compared to controls. Among the top 15 upregulated genes in CEA-TCB-treated tumors there were many reflective of a strong T-cell activation, migration, immune cell response (CXCL13, GNLY, GZMB, CXCL10, IDO1, SLA, CD2, TRBC2, TIGIT, IL2RB, CXCL9, CX3CL1, CCL4L2, and CD3E). Among the top downregulated ones we found keratin 6A and keratin 20 (KRT6A and KRT20) suggestive of the reduction of tumor cells as the result of TCB-mediated killing. A Gene Set Enrichment Analysis (GSEA) was conducted that further enabled identification of the main biological pathways upon CEA-TCB treatment and confirmed that the main Gene Ontology families that characterize the TCB response consist of T-cell activation (Response to interferon gamma; Adaptive Immune Response; T-cell activation; Inflammatory Response; Activation of Immune response; Cytokine secretion) and migration (Leukocyte Migration; Regulation of Cell Adhesion). We also noticed upregulation of many major histocompatibility class II molecules that are known to be expressed on antigen presenting cells (HLA-DRA, HLA-DRB5, and HLA-DRB1) along with CX3CL1 (fractalkine, a known monocyte/T-cell attractant molecule) and CSF1 (colony stimulating factor 1, macrophage) suggestive of myeloid cell recruitment and activation at tumor sites post TCB treatment.  The signature appeared to be robust, as components of the signature were confirmed using complementary techniques: RNA expression analysis and protein expression as determined by flow cytometry and multiplex analysis.

overall_design: Profile of differentially expressed genes between CEA-TCB (cibisatamab)-treated hematopoietic stem cell humanized NOG mice bearing human gastric adenocarcinoma cells (MKN-45) and vehicle-treated controls. N=12 mice were treated in each treatment group, of which RNAseq data was obtained from N=7 treated with CEA-TCB (cibisatamab) and N=10 treated with vehicle (Histidine buffer).

first: Bacac

last: Marina

first: Umaña

last: Pablo


title: The next-generation RNA and ATAC sequencing of MLL-ENL transformed cell lines reveals that C/EBP transcription factors coordinate the expression of the MLL-ENL/Hoxa target genes

status: Public on Oct 30 2020

city: Berlin

first: Achim

last: Leutz

address: Robert-Rössle-Str. 10

department: Cancer Research

country: Germany

institute: MDC Berlin

overall_design: Refer to individual Series


title: The next-generation RNA and ATAC sequencing of MLL-ENL transformed cell lines reveals that C/EBP transcription factors coordinate the expression of the MLL-ENL/Hoxa target genes [RNA-seq]

summary: We report the application of next-generation RNA and ATAC sequencing technology for high-throughput profiling of MLL-ENL transformed cell lines. According to our results, the C/EBP transcription factors coordinate the expression of the MLL-ENL/Hoxa target genes. Genetic removal of both Cebpa and Cebpb revealed a surrogate function of C/EBPε for myeloid MLL-ENL transformation.

overall_design: mRNA and ATAC profiles of MLL-ENL transformed cell lines: wild-type, Cebpa/Cebpb dKO clones, and Cebpa/Cebpb dKO clones expressing exogenous rat C/EBP LAP* isoform.

first: Radoslaw

last: Wesolowski

first: Karin

last: Zimmermann

first: Tatiana

middle: A

last: Borodina

first: Alexander

last: Mildner

first: Dorothea

last: Dörr

first: Maria

last: Hofstätter

first: Elisabeth

last: Kowentz-Leutz


title: Identification of Pax7 binding sites in mouse skeletal muscle progenitors

city: Minneapolis

first: Alessandro

last: Magli

state: MN

address: 2231 6th St SE

department: Medicine

institute: University of Minnesota

summary: Using dox-inducible mouse ES cells, we generated myogenic progenitors expressing the transcription factor Pax7. These cells were used for determining the genomic occupancy of the transcription factor Pax7.

overall_design: ChIP-seq was used to determine Pax7 genomic occupancy in mouse ES cell-derived myogenic progenitors. Pax7 binding was analyzed in 6-day dox-treated cultures.


title: The next-generation RNA and ATAC sequencing of MLL-ENL transformed cell lines reveals that C/EBP transcription factors coordinate the expression of the MLL-ENL/Hoxa target genes [ATAC-seq]

overall_design: mRNA profiles of MLL-ENL transformed cell lines: wild-type, Cebpa/Cebpb dKO clones, and Cebpa/Cebpb dKO clones expressing exogenous rat C/EBP LAP* isoform.


title: The stem/progenitor landscape is reshaped in a mouse model of essential thrombocythaemia and causes excess megakaryocyte production

first: Sam

last: Watcham

address: Jeffery Cheah Biomedical Centre

country: United Kingdom

institute: Wellcome - MRC Cambridge Stem Cell Institute

summary: Frameshift mutations in CALR (calreticulin) are associated with essential thrombocythaemia (ET), but the stages at and mechanisms by which mutant CALR drives transformation remain incompletely defined. Here, we use single-cell approaches to examine the haematopoietic stem/progenitor cell (HSPC) landscape in a mouse model of mutant CALR-driven ET. We identify a trajectory linking HSCs with megakaryocytes, and prospectively identify a novel intermediate population that is overrepresented in the disease state. We also show that mutant CALR drives transformation primarily from the earliest stem cell compartment, with some contribution from megakaryocyte progenitors. Finally, relative to wild-type HSCs, mutant CALR HSCs showed increases in JAK-STAT signalling, the unfolded protein response, cell cycle, and a previously undescribed upregulation of cholesterol biosynthesis. Overall, we have identified a novel megakaryocyte-biased cell population that is increased in a mouse model of ET and described transcriptomic changes linking CALR mutations to increased HSC proliferation and megakaryopoiesis.

overall_design: LK and LSK blood progenitor cells from WT and CALR mutant mice were sequenced by 10X Genomics in two separate experiments (SLX18580 and SLX14832) and compared across genotypes

first: Daniel

last: Prins

first: June

last: Park

first: Nicola

last: Wilson

first: Bertie

last: Gottgens

first: Tony

last: Green


title: Cardiomyocytes Infected by SARS-CoV-2 Recruit Cardiotoxic Macrophages [scRNA-seq dataset]

first: Shuibing

last: Chen

state: NY

address: A 827B, 1300 York Ave

department: Surgery

institute: Weill Cornell Medical College

summary: Heart injury has been reported in up to 20% of COVID-19 patients, yet the cause of myocardial histopathology is unknown. We examined heart tissue from autopsies of healthy and COVID-19 patients using RNA-seq and identified a strikingly increased CCL2 expression and macrophage population in COVID-19 heart sample. Transwell assays using hESC-derived cardiomyocytes (CMs) and macrophages revealed that SARS-CoV-2 infected CMs secrete CCL2, which recruits macrophages, and this was validated using adult human CMs. Macrophages recruited by CCL2 from infected CMs secrete IL-6 and TNF-α, which causes increased ROS and cell apoptosis of CMs. Finally, a high content chemical screen using FDA-approved drugs identified ranolozine and tofacitinib, which rescue SARS-CoV-2 infected CMs from macrophages-induced cardiotoxicity.

overall_design:
scRNA-seq of three conditions:
hESC-derived cardiomyocytes
hESC-derived cardiomyocytes+SARS-CoV-2 Pseudo-entry virus
hESC-derived cardiomyocytes+macrophages+SARS-CoV-2 Pseudo-entry virus

first: Liuliu

last: Yang

first: Yuling

last: Han


title: RNA Sequencing Analysis of a Whole Blood Transcriptome in Estonian Families with Osteogenesis Imperfecta

status: Public on Oct 28 2020

city: Tartu

first: Ene

last: Reimann

address: Riia 23b

department: Institute of Genomics

country: Estonia

institute: University of Tartu

summary:
Purpose: The main objective of this pilot study was to compare blood transcriptional landscape of OI patients with COL1A1 pathogenic variants and their healthy relatives, in order to find out different gene expression and dysregulated molecular pathways in OI.
Methods: We performed RNA sequencing analysis of the whole blood in seven individuals affected with different OI severity and their five unaffected relatives from the three families. The data was analyzed using edgeR package of R Bioconductor. Functional profiling and pathway analysis of the identified differently expressed genes was performed with g:GOSt and MinePath web-based tools.
Results: We identified 114 differently expressed genes. The expression of 79 genes was up-regulated, while 35 genes were down-regulated. The functional analysis identified a presence of dysregulated interferon signaling pathways (IFI27, IFITM3, RSAD12, GBP7). Additionally, the expressions of the genes related to extracellular matrix organization, Wnt signaling, vitamin D metabolism and MAPK-ERK 1/2 pathways were also altered.
Conclusions: The current pilot study successfully captured the differential expression of inflammation and bone metabolism pathways in OI patients.

overall_design: RNAseq Ananlysis of a Whole Blood of 7 affected and 5 unaffected members of three Estonian Osteogenesis Imperfecta kindreds with COL1A1 pathogenic variants

first: Sulev

last: Kõks

first: Katre

last: Maasalu

first: Lidiia

last: Zhytnik

first: Aare

last: Märtson


title: The inhibition of LSD1 via sequestration contributes to tau-mediated neurodegeneration

status: Public on Oct 27 2020

city: atlanta

first: David

middle: J

last: Katz

state: ga

address: 615 Michael st

department: Cell Biology

institute: Emory University

summary: In this dataset, we evalute the effect of modulating the levels of LSD1 in the PS19 tauopathy mouse (mouse that expresses the P301S mutated form of the human tau transgene). First we reduced the levels of LSD1 by crossing PS19 Tau mice with mice heterozygous for Lsd1. This results in an exacerbation of the neurodegenerative phenotype as well as an increase in the transcripational changes observed in taupoathy mice. Secondy, we overexprssed LSD1 in neurons of the adult tauopathy mouse through viral injection direclty into the hippocampus. This resulted in a reduce of neuronal cell death as well as the associated transcriptional changes.

overall_design: RNA sequencing of the hippocampus of adult mice. First, samples 1-14 are 9 month old mice expressing the P301S mutated form of tau transgene with that are either wildtype or heterozygous of Lsd1. Wildtype and Lsd1 heterozgyous mice are used as controls. Second, for samples 15- 23, 11 month old mice expressing the P301S tau transgene injected with a neruonal specfic AAV virus directly into the hippocampus expressing either LSD1 or HA (control) virus. Wildtype mice injected with HA virus are used as controls.

first: Amanda

middle: K

last: Engstrom

first: Dexter

last: Myrick


title: A single cell transcriptome atlas of Human thymic stromal compartment

city: London

first: Paola

last: Bonfanti

address: 1 Midland Road

institute: The Francis Crick Institute

summary:
Purpose: The goal of this study is to characterise the large variety of stromal cells populations, particularly cortex (cTECs) and medullary (mTECs) epithelial cells. It has been speculated that mTECs and cTECs are composed of functionally distinct subsets with different clonogenic potential.
Methods: FACS-isolation protocol to separate 4 different epithelial cells populations from total dissociation of human thymus that is negative for hCD45 then loaded into the 10x Genomics Chromium Platform, and sequenced using Illumina HiSeq 4000.
Conclusions: Our study dissected common clusters to human thymic epithelial cells in cortex and medulla containing stem cells markers conserved when expanded in vitro, as well as identified specialised cell clusters in each of the two compartment

overall_design: 4 FACS sorted epithelial populations were used for single-cell RNA-sequencing (10xGenomics Chromium Platform), and sequenced using Illumina HiSeq 4000.

first: Roberta

last: Ragazzini

first: Stefan

last: Boeing


title: A human tissue map of 5-hydroxymethylcytosines exhibits tissue specificity through gene and enhancer modulation

city: Chicago

first: Xiaolong

last: Cui

state: IL

address: 680 N Lake Shore Drive

department: Department of Preventive Medicine

institute: Northwestern University

type: Methylation profiling by high throughput sequencing

summary: DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe herein, the development of a 5hmC tissue map by characterizing 5hmC genomic distributions in 19 human tissues derived from 10 organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separated donor samples by tissue type. Further comparison of 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC was preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, we report herein an extensive 5hmC map across diverse human tissue types and suggest an important role of 5hmC in tissue-specific development by integration with public epigenomic data. This resource will facilitate future studies of DNA demethylation in pathogenesis and development of 5hmC as biomarkers for various human diseases.

overall_design: We generated genomic 5hmC-Seal and RNA-seq profiles of 19 human tissues and compared with other reference epigenomic data.

first: Xiao-Long

first: Ji

last: Nie

first: Jeremy

last: Ku

first: Sam

last: Levy

first: Chuan

last: He


title: Genome-wide mapping of  RNA Pol II

city: Birmingham

first: Anand

middle: Kumar

last: Singh

state: West Midlands

address: Edgbaston

department: School of Biosciences

institute: University of Birmingham

summary: ChIP with Pol II Ser2 or Pol II antibodies in normal and UPF1-RNAi S2 cells.

overall_design: Role of UPF1 in transcription regulation was examined by Pol II Ser2 or Pol II ChIP in control and UPF1 depleted cells. All the experiments were done using Drosophila embryonic S2 cells.

first: Saverio

last: Brogna


title: Coagulopathy in Malnourished Mice is Sexually Dimorphic and Regulated by Nutrient-sensing Nuclear Receptors

city: Houston

first: Geoffrey

last: Preidis

state: TX

address: 1102 Bates Avenue

department: Gastroenterology, Hepatology & Nutrition

institute: Baylor College of Medicine

summary: Liver dysfunction including coagulopathy is a prominent feature of proteinenergy malnutrition. To identify mechanisms underlying malnutrition-associated coagulopathy, we administered low-protein low-fat diet to lactating dams and examined hepatic transcription and plasma coagulation parameters in young adult weanlings.  Malnutrition impairs growth and liver synthetic function more severely in males versus females. Malnourished males are coagulopathic and exhibit decreased hepatocyte peroxisomes, FXR agonist bile acids, FXR binding on Fga and F11 gene regulatory elements, and coagulation factor synthesis. These effects are absent in female mice, which have low baseline levels of PPARα, suggesting that nutrient-sensing nuclear receptors regulate coagulation factor synthesis in response to host nutritional status in a sex-specific manner.

overall_design: Wild type C57BL/6J mice (Jackson Laboratory) were maintained in the Baylor College of Medicine Center for Comparative Medicine in a temperature-controlled 14:10 hour light:dark room. Dams with 8-day-old pups were randomized to receive irradiated purified control diet (15% fat, 20% protein, 65% carbohydrate) or isocaloric LPLFD (5% fat, 7% protein, 88% carbohydrate) to induce malnutrition. On day-of-life 21, pups were weaned to their dams’ diet for another 5 weeks. At 8 weeks of life, mice were lightly anesthetized with isoflurane. After euthanization by 2 minutes of CO2 inhalation, plasma was collected by cardiac puncture into syringes pre-filled with 0.9% sodium citrate. Liver lobes then were harvested.

last: Moore


title: Genome-wide mapping sites of Set3-TAP chromatin binding in WT and H2B 4KA mutant

first: Hong-Yeoul

last: Ryu

address: 266 Whitney Ave

department: MB&B

institute: Yale

summary: To map Set3 binding sites on a genome-wide scale, we carried out ChIP experiment combined with sequencing assay

overall_design: Examination of TAP-tagged Set3, duplication

first: Christopher

last: Castaldi

first: Dejian

last: Zhao


title: Gene expression profiles in WT, set3Δ, and H2B 4KA

summary: To compare gene expression profiles among WT, set3Δ, and H2B 4KA, we carried out RNA sequencing assay

overall_design: Examination of RNA levels, duplication


title: Cardiomyocytes Infected by SARS-CoV-2 Recruit Cardiotoxic Macrophages

first: Shuibing

last: Chen

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


title: Cardiomyocytes Infected by SARS-CoV-2 Recruit Cardiotoxic Macrophages [bulk RNA-seq dataset)

overall_design: Bulk RNA-seq of hESC-derived cardiomyocytes, adult human cardiomyocytes, and macrophages with mock or SARS-CoV-2 infection.

first: Liuliu

last: Yang

first: Yuling

last: Han


title: Characterization of Atlantic salmon head kidney extracellular vesicles

status: Public on Oct 26 2020

city: St. John's

first: Sherri

middle: L

last: Christian

state: Newfoundland

address: 232 Elizabeth Ave

department: Biochemistry

country: Canada

institute: Memorial University of Newfoundland

summary: We report the miRNA profile of extracellular vesicles released from Atlantic salmon head kidney white blood cells that have been cultured for 1 day or 5 days.

overall_design: Extracellular vesicles released from Atlantic salmon head kidney cells that were cultured for 1 day or for 5 days were analyzed using RNA seq, transmission electron microscopy, nanoparticle tracking and western blot

first: Smith

middle: C

last: Nicole

first: Wajnberg

last: Gabriel

first: Chacko

last: Simi

first: Lacroix

last: Jacynthe

first: Crapoulet

last: Nicolas

first: Lewis

middle: M

last: Stephen

first: Rise

last: Matthew

first: Christian

last: Sherri

first: Woldemariam

middle: T

last: Nardos

first: Andreassen

last: Rune

first: Ayre

last: D. Craig


title: Kinetic modeling of stem cell transcriptome dynamics to identify regulatory modules of normal and disturbed neuroectodermal differentiation

first: Johannes

last: Meisig

address: Chariteplatz 1

department: Institut für Pathologie

institute: Charite Universitätsmedizin

summary: Thousands of transcriptome data sets are available, but approaches for their use in dynamic cell response modelling are sparse, especially for processes affected simultaneously by two orthogonal influence variables. We approached this problem for neuroepithelial development of human pluripotent stem cell (differentiation variable), in the presence or absence of valproic acid (signaling variable). Using few basic assumptions (sequential differentiation states of cells; discrete on/off states for individual genes in these states), and time-resolved transcriptome data, a comprehensive model of spontaneous and perturbed gene expression dynamics was developed. The model made reliable predictions (average correlation of 0.86 between predicted and subsequently tested expression values). Even regulations predicted to be non-monotonic were successfully validated by PCR in new sets of experiments. Transient patterns of gene regulation were identified from model predictions. They pointed towards an activation of wnt signaling as candidate pathway leading to a re-direction of differentiation away from neuroepithelial cells towards neural crest. Experimental intervention experiments, using a wnt/beta-catenin antagonist, led to a phenotypic rescue of this disturbed differentiation. Thus, our broadly applicable model allows the analysis of transcriptome changes in complex time/perturbation matrices..

overall_design: We applied a test system (referred as the STOP-tox(UKN) or UKN1 test system) of developmental toxicity, related to the generation of neuroectoderm from human embryonic stem cells (hESCs), and (i) followed undisturbed differentation in a tightly measured time course (0h-144h; samples were taken at  0h, 6h, 10h, 16h, 24h, 36h, 48h, 60h, 72h, 96h, 120h, 144 h). (ii) Disturbed differentiation was followed by exposing cells to 600 µM valproic acid (VPA; sampling at 24h, 48h, 72h, 96h, 144h) and (iiI) cells were exposed for six days to 9 VPA concentrations (0.025, 0.15, 0.35, 0.45, 0.55, 0.6, 0.65, 0.8 and 1 mM) and samples were taken only at 144h. Time windows of VPA exposure (24-72h, 24-96h, 72-144h and 96-144h) were harvested at 144h. At the respective time points, total RNA was isolated and gene expression studies were performed applying the human Genome U133 plus 2.0 arrays. Expression changes are always shown relative to an untreated control. At least three independent experiments have been performed.

first: Nils

last: Blüthgen

first: Marcel

last: Leist


title: Genome binding/occupancy profiling of methylated Pontin under glucose starvation

city: Seoul

first: Young Suk

last: Yu

address: 1-Gwanak-ro, Gwanak-gu

country: South Korea

institute: Seoul National University

summary: To determine where methylated Pontin is recruited, we performed ChIP-sequencing (ChIP-seq) in WT MEFs after glucose starvation.

overall_design: Genome binding/occupancy profiling of WT MEF cells with normal media or glucose starvation media

first: Young-Suk

first: Hijai

middle: R

last: Shin

first: Kyoung-Jae

last: Won

first: Sung-Hee

last: Baek


title: Set1 and Set3

city: New Haven

first: Hong-Yeoul

last: Ryu

overall_design: Refer to individual Series


title: Radiation-induced damage to prepubertal Pax7+ skeletal muscle stem cells drives lifelong deficits in myofiber size and nuclear number

city: Rochester

first: Joe

last: Chakkalakal

address: 601 Elmwood Avenue, Box 711

institute: University of Rochester

summary: During prepubertal development, muscle stem cells (satellite cells, SCs) actively contribute to myofiber growth. Because some SCs are active during this time, they may be particularly susceptible to damage. Using a Small Animal Radiation Research Platform (SARRP), we investigated the effects of local fractionated radiation treatment on prepubertal SCs. Immediately after this regimen, there was a reduction in SC number. Although surviving SCs had deficiencies in function, some myogenic potential remained.  Indeed, some muscle regenerative capacity persisted immediately after irradiation. Lastly, we assessed the long-term consequences of radiation-induced SC loss during prepuberty. We observed a reduction of myofiber size and corresponding loss of nuclei in both fast- and slow-contracting muscles 14 months post-irradiation. Notably, prepubertal SC depletion mimicked these lifelong deficits. This work highlights the susceptibility of prepubertal SCs to radiation exposure. We also reveal the importance of prepubertal SC contribution to the lifelong maintenance of skeletal muscle.

overall_design: RNA was isolated from gastrocnemius muscles of CTL (0 Gy), CL (contralateral limb ), and RL (irradiated limb) 3 weeks post irradiation (8.2 Gy 3X MWF). Mice were irradiated at 4 weeks of age and sacrificed at 8 weeks. Total RNA Seq was performed to assess radiation-induced changes in gene expression.

first: J

middle: F

last: Bachman

middle: V


title: Analytical validation of SKY92 for the identification of high-risk Multiple Myeloma

city: Rotterdam

first: Martin

middle: van

last: Vliet

address: Lichtenauerlaan 40

department: Bioinformatics

country: Netherlands

institute: SkylineDx

summary: Multiple myeloma is an incurable plasma cell cancer with a large variability in survival. Multiple myeloma patients classified high risk by the SKY92 gene expression classifier are at high risk of relapse and short survival. We performed analytical validation of the SKY92 assay with primary bone marrow specimens from 12 multiple myeloma patients and 7 reference cell line specimens. The SKY92 results were 100% concordant - with the reference and/or their expected result - for sensitivity, specificity, microarray stability and RLT buffer stability; The SKY92 results were 90% concordant for primary specimen stability, 96.4% for intermediate precision and 80%-100% for RNA stability. For the cell-line reproducibility the concordance was at least 92.9%, except for one near-cut point specimen. For the clinical specimen reproducibility, the concordance was 100%, except for two near-cut point specimens. Three independent laboratories were concordant in ≥ 77.8% and ≥ 92.9% of experiments (respectively patient specimens and cell lines). Statistical acceptance thresholds were developed as Delta ≤ 1.48 (change in SKY92 score) and standard deviation ≤ 0.45 (SD across SKY92 scores). Using the CLSI method of choice (EP05-A2/A3), restricted maximum likelihood or REML, the observed Deltas (0 - 1.14) and standard deviations (0.22-0.31) passed acceptance criteria. The analytical validation for the SKY92 assay as a prognostic molecular test for individual multiple myeloma patients is therefore successfully proven.

overall_design:
The following Analytical Validation studies were designed and performed according to CLSI standards: Analytical Sensitivity, Precision, Repeatability, Intermediate Precision, Reproducibility, Microarray Stability, Sample Stability.
SkylineDx, Rotterdam, The Netherlands

last: van Vliet


title: Genome-wide mapping sites of H2B-SUMO chromatin binding in WT and set1 mutant

summary: To map H2B-SUMO binding sites on a genome-wide scale, we carried out ChDIP experiment combined with sequencing assay

overall_design: Examination of H2B-SUMO, duplication

first: Christopher

last: Castaldi

first: Dejian

last: Zhao


title: mRNA profiling of Pontin WT and RA under glucose starvation

summary: To determine which genes are affected by methylated Pontin, we performed RNA-sequencing (RNA-seq) in Pontin WT and RA MEFs after glucose starvation.

overall_design: mRNA expression profiles of Pontin WT and Pontin RA MEF cells with normal media or glucose starvation media were examined by Illumina Hiseq4000.


title: TGFb-blockade uncovers stromal plasticity in tumors by revealing the existence of a novel subset of interferon-licensed fibroblasts [mouse]

status: Public on Oct 25 2020

city: Cambridge

first: Viviana

last: Cremasco

address: 250 Massachusetts Avenue

institute: Novartis Institutes for BioMedical Research

summary: Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we performed an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFβ in vivo led to a profound remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a novel fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlated with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFβ and PD1 co-blockade in the clinical settings, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy.

overall_design: 4T1 mice treated with hIgG2 or TGFb blockade

first: Angelo

last: Grauel

first: Beverly

last: Nguyen

first: David

last: Ruddy


title: Functional Dissection of Transcription Factor Networks Governing Haematopoietic Progenitor States

first: Bertie

last: Gottgens

address: Jeffrey Cheah Biomedical Centre

institute: University of Cambridge

summary: Blood is generated by a constant stream of differentiating haematopoietic progenitor cells. The process is controlled by an immensely complex gene regulatory networks. These have been difficult to comprehend using correlative evidence and limited systematic functional data. Hoxb8-FL cell line is a model system of lympho-myeloid progenitors, which self-renews in vitro and is amenable to genetic perturbations. To construct a functionally defined transcription factor (TF) network we targeted 39 transcription factors using CRISPR/Cas9 gene targeting in Hoxb8-FL cells. We measured the resulting transcriptional changes by small scale RNA-Seq within 2-4 d of each perturbation. Our network analysis revealed >17,000 TF-target interactions across >7,000 target genes, established new interactions among TFs and shed new light on the mechanisms maintaining self-renewal and multipotency.

overall_design: RNA-Seq profiles of Hoxb8-FL cells with CRISPR/Cas9 perturbations against 39 TFs. Samples are organised in plates as indicated, each plate contains samples corresponding to 3 targeted TFs, each with 3 sgRNAs and 2-3 sets of controls (either non-targeting sgRNA or sgRNA targeting the Rosa26 locus). Each condition was performed in 8 replicates. Additionally, 8 replicates were added for Hoxb8-FL cells 16 h after switching off Hoxb8 expression. Additionally, we provide ChIP data for 16 TFs and H3K27Ac histome modification.

first: Iwo

last: Kucinski

first: Nicola

last: Wilson

first: Sarah

last: Kinston

first: Rebecca

last: Hannah

first: Berthold


title: Sis1 potentiates the stress response to protein aggregation  and elevated temperature

city: Martinsried

first: Courtney

middle: Lee

last: Klaips

address: Am Klopferspitz 18

department: Department of Cellular Biochemistry

institute: Max Planck Institute for Biochemistry

summary: Cells adapt to conditions that compromise protein conformational stability by activating various stress response pathways, but the mechanisms used in sensing misfolded proteins remain unclear. Moreover, aggregates of disease proteins often fail to induce a productive stress response. Here, using a yeast model of polyQ protein aggregation, we identified Sis1, an essential Hsp40 co-chaperone of Hsp70, as a critical sensor of proteotoxic stress. At elevated levels, Sis1 prevented the formation of dense polyQ inclusions and directed soluble polyQ oligomers towards the formation of permeable condensates. Hsp70 accumulated in a liquid-like state within this polyQ meshwork, resulting in a potent activation of the HSF1 dependent stress response. Sis1, and the homologous DnaJB6 in mammalian cells, also regulated the magnitude of the cellular heat stress response, suggesting a general role in sensing protein misfolding. Sis1/DnaJB6 functions as a limiting regulator to enable a dynamic stress response and avoid hypersensitivity to environmental changes.

overall_design: Transcriptome analysis via mRNA sequencing of WT yeast (YPH499) and yeast overexpressing Sis1, Htt97Q, or Sis1 and Htt97Q, in triplicate.

first: F

middle: U

last: Hartl


title: Expression data from RCM-1 human colorectal cancer cells treated with methylthioacetic acid

status: Public on Oct 23 2020

city: Kagoshima

first: Shigehisa

last: Okamoto

address: 1-21-24 Korimoto

department: Food Science and Biotechnology

institute: Kagoshima University

summary: Methylthioacetic acid (MTA) is thought to potentially have an anticancer activity because it promotes the differentiation of unorganized cell mass into dome tissues in RCM-1 human colorectal cancer cell line. To elucidate a molecular mechanism underlying MTA-mediated dome formation, we used microarrays to disclose the global changes in gene expression caused by MTA and define the candidate genes involved in this phenomenon.

overall_design: RCM-1 human colorectal cancer cells at near confluency were treated with 1 mM MTA for 0, 6, 12, and 18 h, followed by a further culture in the absence of the chemical for up to 24 h after MTA addition, at which dome tissues were not apparently observed. The RNA extracted was subjected to microarrays to analyze global changes in gene expression in the course of dome formation.

first: Miyu

last: Kamimura

first: Azusa

last: Sasaki

first: Shimpei

last: Watanabe

first: Shiho

last: Tanaka

first: Akiko

last: Fukukawa

first: Kazuya

last: Takeda

first: Yasushi

last: Nakamura

first: Takako

first: Kouji

last: Kuramochi

first: Yui

last: Otani

first: Fumio

last: Hashimoto

first: Kanji

last: Ishimaru

first: Tomoaki

last: Matsuo


title: Cold and b3Adr-agonism lead to distinct immunological shifts during beige remodeling of white adipose tissue

status: Public on Oct 24 2020

city: Boston

first: Nabil

last: Rabhi

state: Massachusetts

address: Silvio Conte Building, 72 E. Concord St

institute: Boston University

summary: White adipose tissue (WAT) is a dynamic tissue, which responds to environmental stimuli and dietary cues by changing its morphology and metabolic capacity. The ability of WAT to undergo a beige remodeling has become an appealing strategy to combat obesity and its comorbidities. Here, by using single cell RNA sequencing, we provide a comprehensive atlas of the cellular dynamics during beige remodeling. We reveal drastic changes both in the overall cellular composition and transcriptional states of individual cell subtypes between Adrb3- and cold-induced beiging. Moreover, we demonstrate that cold induces a myeloid to lymphoid shift of the immune compartment compared to Adrb3 activation. Further analysis, showed that Adrb3 stimulation leads to activation of the interferon/Stat1 pathways favoring infiltration of myeloid immune cells, while repression of this pathway by cold promotes lymphoid immune cells recruitment. These findings highlight that pharmacological mimetics may not provide the same beneficial results as physiological stimuli.

overall_design: Single cell of the stromal vascular fraction from mice inguinal adipose tissue foloowing 3 days beta 3 adrenergic or cold exposure.

first: Anna

last: Belkina

first: Kathleen

last: Desevin

first: Briana

middle: N

last: Cortez

first: Stephen

last: Farmer


title: GmNAC81 involved in leaf senecence and drought stress

city: Viçosa

first: Otavio Jose

middle: Bernardes

last: Brustolini

state: MG

address: Av. PHRolfs, S/N

department: Bioquimica e Biologia Molecular

country: Brazil

institute: Universidade Federal de Viçosa

summary: We examined the transcriptome induced by GmNAC81 overexpression and leaf senescence and showed that GmNAC81 further modulates leaf senescence by regulating an extensive repertoire of functionally characterized senescence-associated genes (SAGs). GmNAC81 overexpression also uncovered the regulation of typical drought-responsive genes. Key regulators and effectors of ABA signaling were suppressed by GmNAC81 overexpression.

overall_design: RNA was extracted from leaves collected in two developmental stages of the plant, vegetative stage three (V3; 20 days after germination, DAG) and reproductive stage six (R6; 80 DAG). Each developmental stage comprised a set of nine libraries.

first: Elizabeth

last: Pacheco Batista Fontes

first: Dalton

last: de Oliveira Ferreira

first: Otavio

last: Bernardes Brustolini


title: RNA-Seq expression profiling of srta mutant and WT strains of Chlamydomonas reinhardtii

city: Berkeley

first: Sean

middle: D.

last: Gallaher

state: California

address: 176 Stanley Hall, room 408

department: California Institute for Quantitative Biosciences

institute: UC Berkeley

summary: Two strains of the green alga, Chlamydomonas reinhardtii, were selected for high expression of transgenes. These strains, called UVM4 and UVM11, were found to both have unique mutations in the SRTA gene (Cre10.g462200). In this study, the basal transcriptome of these two strains was compared to that of their WT parental strain, Elow47, to identify what effects, if any, the srta mutation has on the transcriptome. Additionally, two SRTA complemented strains in a UVM11 background (UVM11-C1 and UVM11-C9) were included for comparison.

overall_design: Two different srta mutant strains (UVM4 and UVM11), the SRTA WT parental strain (Elow47), and two SRTA complemented strains (UVM11-C1 and UVM11-C9) were used for an RNA-Seq analysis to identify  differentially expressed genes. The analysis was conducted in two batches. In batch 1, RNA was purified and analyzed from four separate cultures of UVM4, UVM11, and Elow47. In batch 2, RNA was purified and analyzed from three separate cultures of UVM11 (repeated to identify batch effects), UVM11-C1, and UVM11-C9.

middle: D

first: Daniela

last: Strenkert

first: Juliane

last: Neupert

first: Yinghong

last: Lu

first: Rouhollah

last: Barahimipour

first: Sorel

last: Fitz-Gibbon

first: Na'ama

last: Segal

first: Michael

last: Schroda

first: Sabeeha

middle: S

last: Merchant

first: Ralph

last: Bock


title: A comprehensive library of human transcription factors for cell fate engineering (Lentiviral Screen CRTD5)

status: Public on Oct 22 2020

first: Alex

last: Ng

state: MA

address: 77 Avenue Louis Pasteur Rm 233

department: Genetics

institute: Harvard Medical School

summary: Human pluripotent stem cell (hPSC)-derived cells offer an unprecedented opportunity to model diverse human tissue functions. However, the field lacks a generalized approach to rapidly and efficiently generate individual cell types of interest and to incorporate them into complex but defined tissues. To systematically explore the transcription factor (TF)-mediated hPSC programming landscape, we constructed the Human TFome: a comprehensive human TF library containing 1,564 TF (1,732 splice-isoforms). We discovered 241 previously unreported factors that individually converted hPSCs into diverse lineages with up to 99% efficiency in four days without alteration of external factors. Among these, we programmed neurons, fibroblasts, oligodendrocytes and endothelial cells that have molecular, transcriptomic and functional similarity to primary cells. Splice-isoform-specificity doubled endothelial conversion efficiency into in vivo-functional cells. Our cell-autonomous approach enabled parallel and orthogonal hPSCs programming into multiple cell types simultaneously without altering any microenvironmental cues. We generated in vivo-engraftable induced oligodendrocytes that expedited myelination within synthetically accelerated cerebral organoids.

overall_design: This dataset contains the CRTD5 hiPSC portion of the paper. This paper describes three hiPSC lines that were transduced with lentiviruses containing the Human TFome. TFs were induced for four days and cells sorted for TRA-1-60 signal. PCR using universal primers amplified TFs from genomic DNA, and PCR products were sequenced.

first: Parastoo

last: Khoshakhlagh

first: Jesus Eduardo

last: Rojo Arias

first: Giovanni

last: Pasquini

first: Anka

last: Swiersy

first: Volker

last: Busskamp

first: George

last: Church


title: Consequences of aneuploidy in human fibroblasts with trisomy 21

city: worcester

first: Eduardo

last: Torres

address: 364 Plantation Street, LRB-523

department: Department of Molecular, Cell and Cancer Biology

institute: University of Massachusetts Medical School

summary: RNA-Seq of primary human fibroblasts

overall_design: Compare gene expression of trisomy fibroblasts to euploid controls

first: Sunyoung

last: Hwang

first: Paola

last: Cavaliere

first: Rui

last: Li

first: Lihua

middle: J

last: Zhu

first: Noah

last: Dephoure


title: Transcriptome-wide identification of nuclear and cytoplasmic RNA-binding sites of FUS RBD-only constructs

status: Public on Oct 21 2020

first: Marc-David

last: Ruepp

address: 5 Cutcombe Road

department: Dementia Research Institute

institute: King's College

summary: We report the binding sites of three FUS constructs: (1) The full-length protein (FL); (2) A deletion mutant consisting of amino acids 242-526 (CT); and (3) A aa 242-526 deletion mutant with ALS-associated P525L mutation and heterologous nuclear export signal (CTstar) with the aim to assess the role of protein-protein interactions on RNA-binding and to identify nuclear and cytoplasmic RNA-binding sites of Fused in Sarcoma (FUS).

overall_design: Three HITS-CLIP experiments were performed in triplicate each. Input RNA was sequenced after depletion of ribosomal RNA.

first: Daniel

last: Jutzi


title: Analysis of control and svp RNAi adult Drosophila female fat bodies

city: Bloomington

first: Lesley

last: Weaver

state: IN

address: 1001 E 3rd Street

institute: Indiana University

summary: We took an RNA sequencing approach to identify candidate Svp targets specifically in the adult Drosophila female fat body

overall_design: mRNA profile of fat bodies from RNAi control and svp RNAi adult females after 5 days of transgene expression

first: Daniela

last: Drummond-Barbosa


title: microRNA expression in subcerebral and callosal projection neurons in the mouse at postnatal day 1

city: Palo Alto

first: Suzanne

last: Tharin

state: CA

address: 453 Quarry Road

department: Neurosurgery

institute: Stanford University School of Medicine

summary:
subcerebral and callosal projection neurons were purified by retrograde labeling and FACS, total microRNA was isolated, and expression analysed via TaqMan qPCR expression profiling
three independent samples each of subcerebral and callosal projection neurons were analysed

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: TaqMan Low Density Arrays (TLDA rodent miRNA v2.0, Applied Biosystems, Carlsbad, CA) performed at the Dana Farber Cancer Institute Molecular Diagnostics Laboratory (Boston, MA). The TLDA card corresponds to Sanger miRbase version 10.


title: SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling

status: Public on Oct 20 2020

city: New York

first: Adolfo

last: Garcia-Sastre

address: 1 Gustave Levy Pl

department: Microbiology and Immunology

institute: Icahn School of Medicine at Mount Sinai

summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health threat. Evasion of interferon-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of interferon (IFN) stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) where it directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor complexes and disrupt nuclear import functions. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a new mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

overall_design: Single cell RNA sequencing of mock and SARS-CoV-2 infected Vero E6 African green monkey cells, that were stimulated with Type I, II and III interferons

first: Lisa

last: Miorin

first: Thomas

last: Kehrer

first: Maria

last: Sanchez-Aparicio


title: Maternal n-3 PUFAs deficiency regulates gene expression profile in microglia at P21 in offspring

city: Bordeaux

first: Charlotte

last: Madore

address: 146 rue Leo Saignat

department: Alimentation Humaine

country: France

institute: INRAE

summary:
Maternal n-3 polyunsaturated fatty acids (PUFAs) deficiency modulates microglia transcriptional phenotypes and functions. We identified a specific n-3 PUFA-dependent signature of microglia at weaning during mouse brain development.
MG550 custom-design NanoString chip was used to identify n-3 PUFAs deficient microglia signature in a maternal model of n-3 PUFAs dietary deficiency at weaning in offspring.

overall_design: NanoString MG550 chip profile of FCRLS+ sorted microglia. Note: data show mRNA transcriptomes/100ng RNA.

first: C

first: O

last: Butovsky


title: Dormancy-to-death transition in yeast spores occurs due to gradual loss of gene-expressing ability

city: Delft

first: Theo

last: Maire

address: Van der Maasweg 9

department: Bionanoscience

institute: Delft University of Technology

summary: Dormancy is colloquially considered as extending lifespan by being still. Starved yeasts  form dormant spores that wake-up (germinate) when nutrients reappear but cannot  germinate (die) after some time. What sets their lifespans and how they age are open  questions because what processes occur - and by how much - within each dormant spore  remains unclear. With single-cell-level measurements, we discovered how dormant yeast  spores age and die: spores have a quantifiable gene-expressing ability during dormancy  that decreases over days to months until it vanishes, causing death. Specifically, each  spore has a different probability of germinating that decreases because its ability to -  without nutrients - express genes decreases, as revealed by a synthetic circuit that forces  GFP expression during dormancy. Decreasing amounts of molecules required for gene  expression - including RNA polymerases - decreases gene-expressing ability which then  decreases chances of germinating. Spores gradually lose these molecules because they  are produced too slowly compared to their degradations, causing gene-expressing ability  to eventually vanish and, thus, death. Our work provides a systems-level view of  dormancy-to-death transition.

overall_design: Transcriptome analysis of non-germinated wild-type yeast spores, after exposure to different glucose concentration (0.002% or 0%) in minimal media (SC) at 30C for different amount of time (0h, 16h, 48h, 96h). For each of the 8 different conditions we used 3 biological replicates.

first: Hyun

last: Youk


title: Bladder resident macrophage subsets differently shape the response to urinary tract infection

city: Paris

first: Rachel

last: Legendre

address: 28, rue du docteur Roux

department: Research and Resource Center for Scientific Informatics

institute: Institut Pasteur

summary: Resident macrophages are highly abundant in the bladder, playing key roles in directing immunity to uropathogens. Yet, whether they are heterogeneous, where they come from, and precisely how they respond to infection remain largely unknown. We identified two macrophage subsets in mouse bladders with distinct localization, protein expression, and transcriptomes. Using a model of urinary tract infection, we validated our transcriptomics analyses finding that one macrophage subset phagocytosed more bacteria and polarized to a more anti-inflammatory profile, whereas the other subset died rapidly after infection. After resolution of infection, tissue-resident macrophage subsets were partially replaced by monocyte-derived cells with distinct transcriptional profiles. Elimination of these macrophages led to a type 1 biased immune response to challenge infection. Our study brings considerably more knowledge about the biology of bladder resident macrophages and their response to primary and recurrent infection, which may have broader implications for macrophage subsets in other mucosal tissues.

overall_design: Samples were obtained from the whole bladders of naïve and 6 week post-infected (uropathogenic E coli) female C57BL/6J mice.  Four separate fluorescence-activated cell sorts (FACS) were performed to generate biological replicates, each sort was a pool of 10 mouse bladders. Total RNA was extracted using the RNAeasy micro kit following the manufacturer’s instructions. Directional libraries were prepared using the Smarter Stranded Total RNA-Seq kit Pico Input Mammalian following manufacturer’s instructions. The quality of libraries was assessed with the DNA-1000 kit on a 2100 Bioanalyzer and quantification was performed with Quant-It assays on a Qubit 3.0 fluorometer. Clusters were generated for the resulting libraries with Illumina HiSeq SR Cluster Kit v4 reagents. Sequencing was performed with the Illumina HiSeq 2500 system and HiSeq SBS kit v4 reagents.

first: Livia

last: Lacerda Mariano

first: Matthieu

last: Rousseau

first: Hugo

last: Varet

first: Rebecca

last: Gentek

first: Javier

last: Saenz Coronilla

first: Marc

last: Bajenoff

first: Elisa

last: Gomez Perdiguero

first: Molly

middle: A

last: Ingersoll


title: RNA polymerase II CTD S2P is dispensable during embryogenesis but regulates the developmental diapause in C. elegans

city: Namur

first: Carlo

last: Yague-Sanz

address: 61 rue de Bruxelles

department: Medecine

country: Belgium

institute: Namur University

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

overall_design: Refer to individual Series


title: RNA polymerase II CTD S2P is dispensable during embryogenesis but regulates the developmental diapause in C. elegans [RNA-seq]

summary: We show that inactivating CDK-12 or expressing a full-length CTD S2A mutant in Caenorhabditis elegans results in developmental arrest at the L1 stage,which mimicks the diapause induced when hatching occurs in the absence of food. Transcriptomic analyses indicate that when CDK-12 is inhibited, only a subset of growth-related genes is not properly expressed. These genes correspond to SL2 trans-spliced mRNAs located in position 2 and over within operons. We show that CDK-12 is required for maximal occupancy of CstF (cleavage stimulatory factor) required for SL2 trans-splicing. The addition of food to developmentally arrested worms leads to an increase of both CTD S2P and SL2 trans-splicing. We propose that CTD S2P functions as a gene-specific signaling mark ensuring the nutritional control of C. elegans development.

overall_design: RNA-seq in wild-type and CDK12-as strain, in presence of absence of CDK12-as inhibitor, for L1-arrested C.elegans cells either starved or fed during 4h post-starvation.

first: Clement

last: Cassart

first: Maxime

last: Wéry

first: Antonin

last: Morillon

first: Damien

last: Hermand


title: RNA polymerase II CTD S2P is dispensable during embryogenesis but regulates the developmental diapause in C. elegans [ChIP-seq]

overall_design: ChIP-seq in CDK12-as strain, in presence of absence of CDK12-as inhibitor, for L1-arrested C.elegans cells recovering from starvation

first: Fanélie

last: Bauer


title: Transcriptome reprogramming of CD11b+ bone marrow cells by pancreatic cancer extracellular vesicles

status: Public on Oct 19 2020

city: Lisbon

first: Bruno

last: Costa-Silva

address: Av Brasilia

country: Portugal

institute: Champalimaud Foundation

summary: Pancreatic cancers (PC) are highly metastatic with poor prognosis, mainly due to delayed detection. We previously showed that PC-derived extracellular vesicles (EVs) act on macrophages residing in the liver, preparing pre-metastatic niches that support metastasis to this organ. This process involves the binding of PC-EVs to Kupffer cells, followed by TGFβ secretion and upregulation of fibronectin production by hepatic stellate cells, which supports hepatic accumulation of CD11b+ bone marrow (BM) cells and PC liver metastasis. We here show that PC-EVs preferentially bind to CD11b+ BM cells and induce the expansion of this cell population. Transcriptomic characterization shows that PC-EVs upregulate IgG and IgA genes, which have been linked to the presence of monocytes/macrophages in tumor microenvironments. We also report here the transcription downregulation of genes linked to monocytes/macrophages activation, trafficking and expression of inflammatory molecules. Together, these results demonstrate the existence of a PC-BM communication axis mediated by EVs with a potential role in PC tumor microenvironments.

overall_design: Expression profiles of cd11b+ cells from PC-Evs educated mice and naive mice.

first: Joana

last: Maia

first: Strano

last: Moraes

first: Maria

last: Carolina


title: RNA sequencing in E18 whole brains in PAE and SAC mice

city: Albuquerque

first: Nikolaos

last: Mellios

state: NM

address: 915 Camino De Salud NE

department: Neuroscience

institute: University of New Mexico

summary: These are RNA sequencing data from embryonic day 18 whole brains from embryos whose mother's were exposed to alcohol (Prenatal alchohol exposure or PAE) or sacharin control (SAC) through mating and gestation.

overall_design: Examination of mRNA expression in 6 PAE and 6 SAC samples

first: Mellios

last: Nikolaos


title: Astrocyte Deletion of alpha2-Na/K ATPase Triggers Episodic Motor Paralysis in Mice via a Metabolic Pathway

city: St Louis

first: Sarah

last: Smith

address: 660 S. Euclid Ave

institute: Washington University in St Louis School of Medicine

summary: Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump alpha2-Na/K ATPase represent a key genetic cause of familial hemiplegic migraine, but the mechanisms by which alpha2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we unexpectedly find that mice in which alpha2-Na/K ATPase is conditionally deleted in astrocytes display episodic transient motor paralysis. Functional neuroimaging reveals that conditional knockout of alpha2-Na/K ATPase triggers spontaneous cortical spreading depression events that are associated with low voltage activity events upon EEG monitoring, which in turn correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that loss of alpha2-Na/K ATPase alters metabolic gene expression in astrocytes in vivo with consequent elevation of serine and glycine in the brain. Strikingly, feeding alpha2-Na/K ATPase knockout mice a serine- and glycine-free diet reverses the phenotype of transient motor impairment. Together, our findings define a novel metabolic mechanism regulated by astrocytic alpha2-Na/K ATPase that triggers episodic transient motor paralysis in mice, laying the foundation for potential new treatment strategies for patients with familial hemiplegic migraine.

overall_design: RNA-Seq and TRAP-Seq from Cortex

middle: E

first: Xiaoying

last: Chen

first: Lindsey

last: Brier

first: Jonathan

last: Bumstead

first: Nicholas

last: Rensing

first: Alison

last: Ringel

first: Haewon

last: Shin

first: Anna

last: Oldenborg

first: Jan

last: Crowley

first: Annie

last: Bice

first: Krikor

last: Dikrainian

first: Joseph

last: Ippolito

first: Marcia

last: Haigis

first: Thomas

last: Papouin

first: Guoyan

last: Zhao

first: Michael

last: Wong

middle: P

last: Culver

first: Azad

last: Bonni


title: HIF1A signaling selectively supports proliferation of breast cancer in the brain [brain vs mammary fat pad]

status: Public on Oct 17 2020

first: Ben

middle: S.

last: Wittner

address: 149 13th Street

department: Center for Cancer Research

institute: Massachusetts General Hospital

summary: Blood-borne metastasis to the brain is a major complication of breast cancer, but cellular pathways that enable cancer cells to selectively grow in the brain microenvironment are poorly understood. We find that cultured circulating tumor cells (CTCs), derived from blood samples of women with advanced breast cancer and directly inoculated into the mouse frontal lobe, exhibit striking differences in proliferative potential in the brain. Derivative cell lines generated by serial intracranial injections acquire selectively increased proliferative competency in the brain, with reduced orthotopic tumor growth. Increased Hypoxia Inducible Factor 1A (HIF1A)-associated signaling correlates with enhanced proliferation in the brain, and shRNA-mediated suppression of HIF1A or drug inhibition of HIF-associated glycolytic pathways selectively impairs brain tumor growth while minimally impacting mammary tumor growth. In clinical specimens, brain metastases have elevated HIF1A protein expression, compared with matched primary breast tumors, and, in patients with brain metastases, hypoxic signaling in CTCs predicts decreased overall survival. The selective activation of hypoxic signaling by metastatic cancers in the brain may be an opportunity for therapeutic intervention.

overall_design: We used RNA-Seq to compare CTC-derived tumors growing in the brain and the mammary gland.

first: Richard

middle: Y

last: Ebright

first: Marcus

last: Zachariah

first: Douglas

last: Micalizzi

first: Kira

middle: L

last: Niederhoffer

first: Linda

last: Nieman

first: Brian

last: Chirn

first: Devon

last: Wiley

first: Benjamin

last: Wesley

last: Shaw

first: Edwin

last: Nieblas-Bedolla

first: Lian

last: Atlas

first: Annamaria

last: Szabolcs

first: Johannes

last: Kreuzer

first: Robert

last: Morris

first: Anthony

last: Iafrate

first: Mehmet

last: Toner

first: Wilhelm

last: Haas

first: David

last: Ting

first: Priscilla

middle: K

last: Brastianos

first: Daniel

last: Haber

first: Shyamala

last: Maheswaran


title: Anti-tumor immunity induces the presentation of aberrant peptides in melanoma

status: Public on Oct 18 2020

city: Amsterdam

first: Pierre-Rene

last: Körner

address: Plesmanlaan 121

department: Oncogenomics

institute: Netherlands Cancer Institute (NKI)

summary: Extensive tumor inflammation, reflected by high levels of infiltrating T-cells and Interferon gamma (IFNγ) signaling, is thought to improve checkpoint immunotherapy response. However, many tumors escape by activating multiple cellular pathways that induce immunosuppression. One pivotal immune-suppressive mechanism is the production of Tryptophan metabolites along the kynurenine pathway by IFNγ-induced IDO1 enzyme production. However, phase III clinical trials using chemical inhibition of IDO1 in combination with PD1 pathway blockade failed to improve melanoma treatment, suggesting incomplete understanding of the role of IDO1 and the consequent Tryptophan degradation on mRNA translation and cancer progression. Here, we investigated the effects of prolonged IFNγ treatment on mRNA translation in melanoma cells by ribosome profiling. Our results suggest that IFNγ-induced IDO1-mediated Tryptophan depletion may play a key role in the immune recognition of melanoma cells, by inducing the production and presentation of aberrant peptides.

overall_design: RNA sequencing, ribosome profiling, and mass spectrometry to measure mRNA levels, mRNA translation, and protein levels.

first: Osnat

last: Bertok

first: Abhijeet

last: Pataskar

first: Remco

last: Nagel

first: Ronen

last: Levy

first: Eden

last: Goldfarb

first: Onno

middle: B

last: Bleijerveld

first: Yishai

last: Levin

first: Maarten

last: Altelaar

first: Yardena

last: Samuels

first: Reuven

last: Agami


title: HIF1A signaling selectively supports proliferation of breast cancer in the brain [HIF1A KD]

first: Ben

last: Wittner

overall_design: We infected CTC-derived cell lines from two patients with lentiviral shRNA constructs targeting HIF1A (shHIF1A) or scrambled control (shCtrl). The resulting four cell lines were analyzed by RNA-Seq.

first: Richard

last: Ebright

first: Marcus

last: Zachariah

first: Douglas

last: Micalizzi

first: Kira

last: Niederhoffer

first: Linda

middle: T

last: Nieman

first: Brian

last: Chirn

first: Devon

middle: F

last: Wiley

first: Benjamin

last: Wesley

last: Shaw

first: Edwin

last: Nieblas-Bedolla

first: Lian

last: Atlas

first: Annamaria

last: Szabolcs

first: Johannes

last: Kreuzer

first: Robert

last: Morris

first: Anthony

last: Iafrate

first: Mehmet

last: Toner


title: HIF1A signaling selectively supports proliferation of breast cancer in the brain [parental CTCs inVitro]

last: Wittner

overall_design: We analyzed RNA-seq data from the seven parental CTC lines to determine whether increasing levels of hypoxic signaling correlates with their differential growth rates in the brain microenvironment.

first: Wilhelm

last: Haas

first: David

last: Ting

first: Priscilla

last: Brastianos

first: Daniel

last: Haber

first: Shyamala

last: Maheswaran


title: Expression analysis of knockdown of USP7 in HEK293T cells.

city: Baltimore

first: Jiou

last: Wang

state: MD

address: 615 N. Wolfe Street

department: Biochemistry and Molecular Biology

institute: The Johns Hopkins University

summary: Ubiquitin Specific Peptidase 7 (USP7) is a deubiquitinase of several important regulatory proteins, and important regulator of TGFb pathway. To investigate their role in cell signaling, we analyzed global mRNA levels in HEK293T cells that were knocked down with shRNAs against USP7 and non-targeting control (CTRL).

overall_design: HEK293T cells were transfected with knockdown shRNA plasmids to reduce protein levels of USP7, or none (control knockdown with non-targeting shRNA plasmid). After 78h of knockdown, we isolated total RNA. We pairwise compared mRNA levels from three control samples to three USP7 knockdown samples, six samples total.

first: Goran

last: Periz


title: HIF1A signaling selectively supports proliferation of breast cancer in the brain [F1 vs parental]

first: Ben

overall_design: We injected cells from CTC-derived cell lines (i.e., parental cell lines) from two patients into mice intracranially. We harvested the resulting tumors and sorted for GFP expression to remove mouse cells. We expanded the sorted cells in vitro under anchorage-independent conditions for less than four weeks. We then performed RNA-seq on the resulting F1 cell lines.

first: Richard

last: Ebright

first: Marcus

last: Zachariah

first: Douglas

last: Micalizzi

first: Kira

last: Niederhoffer

first: Linda

last: Nieman

first: Brian

last: Chirn

last: Wiley

last: Wesley


title: A novel cardiomyogenic role for Isl1+ neural crest cells in the inflow tract

status: Public on Oct 16 2020

city: Miami

first: Michael

last: Durante

state: FL

department: Ophthalmology

institute: University of Miami Miller School of Medicine

summary: The goal of this study was to analyze the cellular compsitions of the arterial pole and venous pole regions of the neonatal mouse heart using single-cell RNA sequencing.

overall_design: The dorsal and ventral domains of the cardiac base were dissected from 3-day old C57Bl6 male and female neonatal mice (n=15) and enzymatically dissociated into single-cell suspensions. The ventral domain included the proximal aorta, pulmonary artery, and outflow tract base. The dorsal domain included the sinoatrial node, cardiac ganglia and pulmonary veins. Single-cell preparations from 15 neonatal hearts were combined to generate two pools of dorsal and ventral cells, and cultured in vitro for 5 days to allow recovery from enzymatic and mechanical stress. Subsequently, cells were  processed for 3' single cell RNA-seq using the 10X Genomics Chromium platform and sequenced on Illumina Novaseq 6000.

first: Kostantinos

last: Chatzistergos


title: Sperm histone H3 lysine 4 trimethylation is altered in a genetic mouse model of transgenerational epigenetic inheritance

status: Public on Oct 15 2020

city: Sainte-Anne-de-Bellevue

first: Sarah

last: Kimmins

address: 21111 Lakeshore Road

institute: McGill University

summary: Advancing the molecular knowledge surrounding fertility and inheritance has become critical given the halving of sperm counts in the last 40 years, and the rise in complex disease which cannot be explained by genetics alone. The connection between both these trends may lie in alterations to the sperm epigenome and occur through environmental exposures. Changes to the sperm epigenome are also associated with health risks across generations such as metabolic disorders and cancer. Thus it is imperative to identify the epigenetic modifications that escape reprogramming during spermatogenesis. Here, we aimed to identify the chromatin signature(s) associated with transgenerational phenotypes in our genetic mouse model of epigenetic inheritance that overexpresses the histone demethylase KDM1A in their germ cells. We used sperm-specific chromatin immunoprecipitation followed by in depth sequencing (ChIP-seq), and computational analysis to identify whether differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3), and histone H3 lysine 27 trimethylation (H3K27me3) serve as mechanisms for transgenerational epigenetic inheritance through the paternal germline. Our analysis on the sperm of KDM1A transgenic males revealed specific changes in H3K4me3 enrichment that occurred independently from non-bivalent H3K4me3/H3K27me3 regions. Many regions with altered H3K4me3 enrichment in sperm were identified on the paternal allele of the pre-implantation embryo. These findings suggest that sperm H3K4me3 functions in the transmission of non-genetic phenotypes transgenerationally.

overall_design: ChIP-Seq for H3K4me3 and H3K27me3 was performed on the sperm of heterozygous transgenic mice that overexpress the histone demehtylase KDM1A in their germ cells (TG), non transgenic mice that do not overexpress KDM1A in their germ cells but are the offspring of TG males that do (nonTG), and wildtype mice (CRwt)

first: Ariane

last: Lismer


title: RNA sequencing keloid transcriptome associates keloids with Th2, Th1, Th17/Th22 and JAK3-skewing

first: Ana

last: Pavel

address: One Gustave L. Levy Place

department: Dermatology

summary: In this study, we aimed to elucidate the profile of lesional and non-lesional keloid skin compared to normal skin. We performed gene (RNAseq, qRT-PCR) and protein (immunohistochemistry) expression analyses on biopsy specimens obtained from lesional and non-lesional skin of African American (AA) keloid patients compared to healthy skin from AA controls. We found that lesional versus normal skin showed significant up-regulation of markers of T-cell activation/migration (ICOS, CCR7), Th2- (IL-4R, CCL11, TNFSF4/OX40L), Th1- (CXCL9/CXCL10/CXCL11), Th17/Th22- (CCL20, S100As) pathways, and JAK/STAT-signaling (JAK3) (false-discovery rate [FDR]<0.05).

overall_design: African American patients and healthy controls were recruited under institutional review board-approved protocols. Biopsy specimens (6 mm) were collected from lesional and non-lesional skin of keloid patients, and from healthy control skin.

first: Jianni

last: Wu

first: Ester

last: DelDuca

last: Espino

first: Alyssa

last: Gontzes

first: Inna

last: Cueto

first: Ning

last: Zhang

first: James

middle: G

last: Krueger

first: Emma

last: Guttman-Yassky


title: Analysis of differential gene expression in a Streptococcus sanguinis ssaACB deletion mutant before and after addition of EDTA to deplete cellular manganese

city: Richmond

first: Todd

last: Kitten

state: VA

address: 521 N. 11th Street

department: Philips Institute for Oral Health Research

institute: Virginia Commonwealth University

summary: The purpose of this study was to determine why manganese is required for the growth of Streptococcus sanguinis. The study employed a mutant derivative of S. sanguinis strain SK36 deleted for the genes encoding the manganese transporter SsaACB. The mutant strain, JFP169, was grown in a fermenter with constant aeration in BHI broth. RNA-seq was used to compare gene expression from samples taken 20 minutes prior to addition of 100 micromolar EDTA to samples taken 10, 25, and 50 min after addition. Samples were treated with xx to deplete ribosomal RNA, then sequenced on a MiSeq sequencer, producing paired-end 150-base reads. Sequences were trimmed and filtered and expression levels compared using the DE-Seq module in the Geneious Prime program.

overall_design: Expression was compared at four time points, one 20 min prior to EDTA addition and the other three at 10, 25, and 50 min after EDTA addition. Four biological replicates were analyzed for each time point.

first: Tanya

last: Puccio


title: A highly scalable method for joint whole genome sequencing and gene expression profiling of single cells

city: Solna

first: Vasilios

last: Zachariadis

address: Visionsgatan 4

department: Oncology-Pathology

country: Sweden

institute: Karolinska Institutet

summary: To address how genetic variation alters gene expression in complex cell mixtures, we developed Direct Nuclear Tagmentation and RNA-sequencing (DNTR-seq), which enables whole genome and mRNA sequencing jointly in single cells. DNTR-seq readily identified minor subclones within leukemia patients. In a large-scale DNA damage screen, DNTR-seq was used to detect regions under purifying selection, and identified genes where mRNA abundance was resistant to copy number alteration, suggesting strong genetic compensation. mRNA-seq quality equals RNA-only methods, and the low positional bias of genomic libraries allowed detection of sub-megabase aberrations at ultra-low coverage. Each cell library is individually addressable and can be re-sequenced at increased depth, allowing multi-tiered study designs. Additionally, the direct tagmentation protocol enables coverage-independent estimation of ploidy, which can be used to identify cell singlets. Thus, DNTR-seq directly links each cell?s state to its corresponding genome at scale, enabling routine analysis of heterogeneous tumors and other complex tissues.

overall_design: Joint whole genome and mRNA sequencing in single cells. Includes cells treated with increasing doses of X-ray irradiation and etoposide at different time points.

first: Huaitao

last: Cheng

first: Nathanael

last: Andrews

first: Martin

last: Enge


title: Prrx1 fibroblasts represent a pro-fibrotic lineage in the mouse ventral dermis

status: Public on Oct 13 2020

city: Stanford

first: Michael

last: Januszyk

address: 257 Campus Dr

department: Surgery

institute: Stanford University

summary: Skin scarring following dermal injury causes extreme pain and pschological trauma for patients. Currently, we do not have effective treatments to prevent or reverse skin scarring. Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using RNA-sequencing and single cell RNA sequencing, we demonstrate that Prrx1-expressing mouse fibroblasts are responsible for acute and chronic scaring in the ventral mouse dermis. In summary, we have identified and characterized a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

overall_design: Mouse wound tissue transcriptomes were profiles using RNA bulk sequencing and single cell RNA-sequencing. RNA was obtained from FACS-isolated mouse fibroblast subpopulations from Prrx1+ (PPF) and Prrx1- (PNF)  mice.  For RNA bulk sequencing RNA from PPF and PNF fibroblasts was collected at four timepoints (Embryonic 16 [E16], E18, Postnatal 1 [P1] and P30), giving a total of eight samples for sequencing. For single cell sequencing RNA from PPF and PNFs mice was collected from caudal and ventral skin and from unwounded and wounded skin,   giving a total of eight samples. The samples were pooled, hashtagged and sequenced into two tubes.

first: Michelle

last: Griffin

first: Mimi

middle: R

last: Borrelli


title: Prenatal Androgenization Alters the Development of GnRH Neuron and Preoptic Area RNA Transcripts in Female Mice (I)

city: Ann Arbor

first: Bioinformatics

last: Core

state: MI

address: BRCF Bioinformatics Core, 2800 Plymouth Rd

institute: University of Michigan

summary:
Polycystic ovary syndrome (PCOS) is the most common form of infertility in women. The causes of PCOS are not yet understood and both genetics and early-life exposure have been considered as candidates. With regard to the latter, circulating androgens are elevated in mid-late gestation in women with PCOS, potentially exposing offspring to elevated androgens in utero; daughters of women with PCOS are at increased risk for developing this disorder. Consistent with these clinical observations, prenatal androgenization (PNA) of several species recapitulates many phenotypes observed in PCOS.
There is increasing evidence that symptoms associated with PCOS, including elevated luteinizing hormone (LH) (and presumably gonadotropin-releasing hormone (GnRH)) pulse frequency emerge during the pubertal transition. We utilized translating ribosomal affinity purification coupled with RNA sequencing to examine GnRH neuron mRNAs from prepubertal (3wk) and adult female control and PNA mice. Prominent in GnRH neurons were transcripts associated with protein synthesis and cellular energetics, in particular oxidative phosphorylation.
The GnRH neuron transcript profile was affected more by the transition from prepuberty to adulthood than by PNA treatment, however PNA did change the developmental trajectory of GnRH neurons. This included families of transcripts related to both protein synthesis and oxidative phosphorylation, which were more prevalent in adults than in prepubertal mice but were blunted in PNA adults. These findings suggest that prenatal androgen exposure can program alterations in the translatome of GnRH neurons, providing a mechanism independent of changes in the genetic code for altered expression.

overall_design: Examination of transcripts enriched in GnRH neurons in control and prenatally androgenized adult and prepubertal female. There are 48 samples including 2 negative controls. GnRH/Total, young/adult, vehicle/DHT (5-6 replicates per group).

first: Laura

last: Burger

first: Sue

last: Moenter

first: Rebecca

last: Tagett


title: Gene expression in the femur hip joint articular cartilage of 12-week-old Gnptg-KO mice

status: Public on Oct 14 2020

city: Hamburg

first: Thorsten

last: Schinke

address: Martinistr. 52

department: Department of Osteology and Biomechanics

institute: University Medical Center Hamburg-Eppendorf

summary:
Mucolipidosis type III is a rare disease caused by mutations in the GNPTG gene, encoding the gamma-subunit of GlcNAc-1-phosphotransferase. Since osteoarthritis and joint stiffness are typical symptoms of the disease, we aimed to investigate the role of the gamma-subunit on cartilage homeostasis.
We used microarray analysis to compare the global gene expression in the articular cartilage of Gnptg-KO and wild-type mice.

overall_design: 2 samples generated from pools of 6 mice per genotype were analyzed. 1st sample: Hip joint articular cartilage of 12-week-old WT mice. 2nd sample: Hip joint articular cartilage of 12-week-old Gnptg-KO mice.

first: Timur

last: Yorgan

first: Anke

last: Baranowsky

first: Lana

last: Rosenthal

first: Lukas

last: Sandoval Flores

first: Sandra

last: Pohl