title: Next-generation sequencing of the oscillation zone of arf7 and iaa18/potent reveals new biological processes involved in the root clock function in Arabidopsis

first: Miguel

last: Moreno-Risueno

summary: To understand regulation of the in-phase and the antiphase oscillations by IAA18/POTENT and ARF7, we performed next-generation sequencing (NGS). OZ samples were taken at the minimum of DR5::Luciferase expression for the wild type, and simultaneously for arf7-1 and iaa18/potent regardless of DR5::Luciferase expression to avoid bias.

overall_design: A total of nine samples were taken, three independent samples for each of the three cases of study: three samples of Col0 with minimum oscillation, three samples of potent and three samples of arf7.

first: Juan

last: Perianez-Rodriguez

first: Guy

last: Wachsman

first: Laura

last: Serrano-Ron

first: Philip

last: Benfey


title: Genome analysis by next-generation sequencing of potent x Ler descendants

status: Public on Jan 06 2021

city: Pozuelo de Alarcon

middle: Angel

state: Madrid

department: Biotechnology

country: Spain

institute: Center for Plant Biotechnology and Genomics

type: Genome variation profiling by high throughput sequencing

summary: To gain further insight into the root clock mechanism, we performed a mutagenesis screen in plants carrying DR5::Luciferase in combination with markers for subsequent LR organogenesis (pWOX5::ER-YFP and pSCR::ER-GFP12). Out of this screening, we identified a mutant with increased expression of DR5::Luciferase in the OZ and PBS distributed throughout the primary root axis without evident spacing. We further refer to this mutant as potent.

overall_design: Approximately 200 seeds generated in the fifth cross of potent x Ler were sown and seedlings with (mutant) and without (Control) potent phenotype were collected and frozen at -80° C. The SNPtrack pipeline was used to identify single variant/nucleotide polymorphisms associated to Ler ecotype as well as all the heterozygous mutations present in the potent sample.

middle: C

last: del Pozo

middle: A


title: Transcriptome of lung epithelial cells stimulated by hemozoin

status: Public on Jan 05 2021

city: NEW YORK

first: Shivang

last: Shah

state: NY

address: 622 WEST 168TH ST

country: USA

institute: Columbia University Medical Center

type: Expression profiling by high throughput sequencing

summary: We assessed the transcriptome of epithelial cells exposed to hemozoin, using a combination of robust quantitative transcript modeling and informatics-based network analyses. Results revealed a transcriptional program induced by hemozoin that comprised numerous pro-inflammatory and epithelial- and extracellular matrix-associated genes.

overall_design: Examination of response to in vitro stimulation of airway epithelial cells for 24 h with hemozoin at three different concentrations


title: Cell Atlas Of The Chick Retina: Single Cell Profiling Identifies 150 Cell Types

status: Public on Jan 04 2021

city: Cambridge

first: Wenjun

last: Yan

state: MA

address: 52 Oxford Street

department: Department of Molecular  and Cellular Biology

institute: Harvard University

summary: Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. Here, we used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. From ~40000 single cell transcriptomes, we identified ~150 cell types distributed among the six classes conserved across vertebrates – photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and glial cells.  To match molecular profiles to morphology, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral and central-peripheral retinal axes.  We also identified immature photoreceptor, horizontal cell and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mice and primate retinal cell types. Taken together, our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.

overall_design:
We used scRNA-seq to profile cells from the chick retina. From ~40,000 single cell transcriptomes, we identified cells of all 6 classes named above (PR, HC, BC, AC, RGC and glia) and used unsupervised methods to divide them into ~150 clusters. We show that 136 of the groups represent putative cell types, with others corresponding to developmental intermediates. We then devised a method for CRISPR-based somatic cell integration of fluorescent reporters into genes shown by scRNA-seq to be expressed by specific types. Using this technique along with other histological methods, we matched molecular profiles to morphology for many neuronal types. We also found a positional signature in Müller glia, with distinct expression patterns based on their location along the anterior-posterior, dorsal-ventral and central-peripheral retinal axes. Finally, we compared the cell classes and types of chick retina with those of three mammalian species – mouse, macaque and human – demonstrating conserved molecular features of all classes and some types, along with multiple differences between chick and mammals. Together, our results provide new insights into retinal structure and evolution generally, as well as a foundation for anatomical, physiological, and developmental studies of avian retina.

GEO series of mammalian data used for comparison:
Mouse rods: GSE63472
Mouse Bipolar cells: GSE81904
Mouse all other classes: GSE149715
Macaque all classes: GSE118852
Human all classes: GSE148077

first: Masahito

last: Yamagata

first: Joshua

middle: R

last: Sanes


title: Mutant SF3B1 promotes AKT and NF-kB driven mammary tumorigenesis

city: NewYork

first: Zhaoqi

last: Liu

state: New York

address: ICRC Building 8th Floor, Office 803B 1130 St. Nicholas Ave

department: Systems Biology

institute: Columbia university

summary: Mutations in the core RNA splicing factor SF3B1 are prevalent in leukemias and uveal melanoma, but also recurrent in epithelial malignancies such as breast cancer. Whereas hotspot mutations in SF3B1 alter hematopoietic differentiation, whether SF3B1 mutations contribute to epithelial cancer development and progression is unknown. Here, we identify that SF3B1 mutations in mammary epithelial and breast cancer cells induce a recurrent pattern of aberrant splicing leading to activation of AKT and NF-kB, enhanced cell migration, and accelerated tumorigenesis. Transcriptomic analysis of human cancer specimens, MMTV-cre Sf3b1K700E/WT mice, and isogenic mutant cell lines identified hundreds of aberrant 3’ splice sites induced by mutant SF3B1, a portion of which were breast-specific. Across mouse and human tumors, mutant SF3B1 promoted aberrant splicing (dependent on aberrant branchpoints as well as pyrimidines downstream of the aberrant branchpoint) and consequent suppression of PPP2R5A and MAP3K7, critical negative regulators of AKT and NF-kB. Coordinate activation of NF-kB and AKT signaling was observed in the knock-in models, leading to accelerated cell migration and tumor development in combination with mutant PIK3CA but also hypersensitizing cells to AKT kinase inhibitors. These data identify mutations in SF3B1 as drivers of breast tumorigenesis and reveal unique vulnerabilities in cancers harboring them.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 23 samples, including 11 mouse and 12 human samples with varying SF3B1 status

first: Bo


title: Gain-of-Function p53 Protein Transferred via Small Extracellular Vesicles Promotes Conversion of Fibroblasts to a Cancer-Associated Phenotype [microarray]

city: Houston

first: Shaolin

last: Ma

state: Tx

address: 1881 East Road

institute: MD Anderson Cancer Center

summary:
We demonstrated that gain-of-function (GOF) p53 protein can be packaged into small EVs and transferred to fibroblast cells.
We used microarrays to determine the gene expression of NoF 151 fibroblasts after treating with small EVs from HT29-ntsh and -shP53 cells.

overall_design: We isolated small Evs from HT29-ntsh and -shP53 cells using ultracentrifugation method. NoF 151 cells were treated with 10 ug/mL small EVs twice a day for 2 days. The total RNA samples were extracted and proceeded to Affymetrix Clariom D array.

first: Anil

middle: K

last: Sood


title: Spatiotemporal Analysis of Human Intestinal Development at Single Cell Resolution

city: Oxford

first: Alison

last: Simmons

address: John Radcliffe Hospital/Headley Way

department: The MRC Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

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

overall_design: Refer to individual Series


title: Spatiotemporal Analysis of Human Intestinal Development at Single Cell Resolution - scRNA-Seq

summary: Development of the human intestine is not well understood. Here we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including novel epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe new functions for these including as vascular niche cells.  We pinpoint the origins of Peyer’s patches and gut-associated lymphoid tissue (GALT) and describe location specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders.

overall_design: Colon and terminal ileum tissue sections from 8-22 post-conceptual week fetal intestine samples were subject to single cell RNA-seq analysis.

first: David

last: Fawkner-Corbett

first: Agne

last: Antanaviciute

first: Kaushal

last: Parikh

first: Marta

last: Jagielowicz

first: Ana

last: Sousa Gerós

first: Tarun

last: Gupta

first: Neil

last: Ashley

first: Hashem

last: Koohy


title: An epitranscriptomic switch at the 5´-UTR controls genome selection during HIV-1 genomic RNA packaging

status: Public on Jan 02 2021

city: Santiago

first: Daniela

last: Toro Ascuy

state: Region Metropolitana

address: Av. Independencia 1027

department: Virology

country: Chile

institute: University of Chile

summary: During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that an epitranscriptomic switch involving the demethylation of adenosine residues present within HIV-1 5´-UTR regulates full-length RNA packaging. We further identified two conserved adenosines within the 5’-UTR that have a crucial structural and functional role and that are modulated by N6-methylation. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promotes the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus contributing to full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the full-length RNA molecules that will be used as viral genomes.

sample_organism:
Homo sapiens
HIV-1 vector pNL4-3

platform_organism:
Homo sapiens
HIV-1 vector pNL4-3

overall_design: We are providing RNAseq data from inputs (fragments of polyA RNA) and m6A-immunoprecipitated RNA from both HEK293T cells expressing HIV-1 NL4.3 as well as from purified viral particles. Two biological replicates are included. We also provide SHAPE-MaP data obtained using polyA RNA extracted from cell expressing HIV-1 NL4.3 and overexpressing d2EGPF (control) or METTL3/14.

first: Ricardo

last: Soto Rifo

first: Camila

last: Pereira Montecino


title: Effect of emodin on long non-coding RNA-mRNA networks in rats with severe acute pancreatitis-induced acute lung injury

status: Public on Jan 03 2021

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

country: China

institute: ABLife, Inc.

summary: Long non-coding RNAs (lncRNAs) reportedly contribute to disease pathogenesis and drug treatment effects. Both emodin and dexamethasone (DEX) have been used for the treatment of severe acute pancreatitis-associated acute lung injury (SAP-ALI). However, lncRNA regulation networks related to SAP-ALI pathogenesis and drug treatment are unreported. In this study, lncRNAs and mRNAs in the lung tissue of SAP-ALI and control rats, with or without drug treatment (emodin or DEX), were assessed by RNA sequencing. Results showed that both emodin and DEX were therapeutic for SAP-ALI and that mRNA and lncRNA levels differed between untreated and treated SAP-ALI rats. Gene expression profile relationships for emodin treated and control rats were higher than DEX treated and untreated animals. By comparison of control and SAP-ALI animals, more upregulated than downregulated mRNAs and lncRNAs were observed with emodin treatment. For DEX treatment, more downregulated than upregulated mRNAs and lncRNAs were observed. Functional analysis demonstrated both upregulated mRNA and co-expressed genes with upregulated lncRNAs were enriched in inflammatory and immune response pathways. Further, emodin associated lncRNAs and mRNAs co-expressed modules were different from those associated with DEX. Quantitative polymerase chain reaction demonstrates that selected lncRNA and mRNA co-expressed modules were different in the lung tissue of emodin and DEX treated rats. Also, emodin had different effects compared to DEX on the co-expression network of lncRNAs Rn60_7_1164.1 and AABR07062477.2 for the blue lncRNA module and Nrp1 for the green mRNA module. In conclusion, this study provides evidence that emodin may be a suitable alternative or complementary medicine for treatment of SAP-ALI.

overall_design: lncRNAs and mRNAs expression of lung tissues from control and SAP-ALI rats with or without drug treatments (emodin or DEX), were determined by RNA_seq

first: Caiming

last: Xu

first: Hailong


title: Calcitriol induces a distinctive transcriptional program in Chronic Lymphocytic Leukemia (CLL) cells

status: Public on Jan 01 2021

city: Thessaloniki

first: Kostas

last: Stamatopoulos

address: 6th km Charilaou-Thermi Rd

department: Institute of Applied Biosciences (INAB)

country: Greece

institute: Centre for Research and Technology-Hellas (CERTH)

summary:
Purpose: It has been proposed that vitamin D may play a role in prevention and treatment of cancer while epidemiological studies have linked vitamin D insufficiency to adverse disease outcome in chronic lymphocytic leukemia (CLL). However, the underlying mechanisms have not yet been revealed. In this study, we sought to identify key signaling pathways and molecules that are altered after calcitriol, the biologically active form of vitamin D, supplementation of CLL cells in vitro.
Methods: An RNA-Sequencing analysis was performed in primary CLL cells that were treated in vitro with calcitriol. Total RNA was extracted from calcitriol-treated and non-treated CLL cells, while mRNA selection was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module. Library preparation for RNA-Sequencing (RNA-Seq) analysis was conducted with the NEBNext Ultra II Directional RNA Library Prep Kit. The libraries were paired-end sequenced on the NextSeq 500 Illumina platform. Differential expression analysis was performed using DESeq2; genes with log2FC>|1| and P≤0.05 were considered as differentially expressed.
Results: Differential expression analysis revealed 85 differentially expressed genes (DEGs) (log2FC≥|1| and p≤0.05), of which 28 (32.9%) were overexpressed in calcitriol-treated cells versus unstimulated CLL cells, thus, contrasting the remaining 57 (67.1%) which showed the opposite pattern. Supervised hierarchical clustering analysis, based on the differentially expressed genes, was performed and revealed distinct gene expression patterns between calcitriol-treated and control CLL cells. Moreover, pathway enrichment analysis revealed that calcitriol-regulated genes are implicated in signaling pathways known to be deregulated in CLL biology.
Conclusions: Transcriptome analysis highlighted the possible impact of calcitriol on the regulation of immune signaling pathways relevant to CLL pathophysiology.

overall_design: RNA-Sequencing (RNA-Seq) was performed in 6 Chronic Lymphocytic Leukemia (CLL) cases after in vitro treatment with calcitriol for 24 hours. In detail, there were 6 calcitriol-treated and 6 non-treated samples.

first: Marina

last: Gerousi

first: Fotis

last: Psomopoulos

first: Maria

last: Tsagiopoulou

first: Stavroula

last: Ntoufa


title: Oct4 induces embryonic pluripotency via Stat3 signalling and metabolic mechanisms

first: Giuliano

middle: Giuseppe

last: Stirparo

state: Cambridgeshire

address: 418 Cambridge Science Park Milton Rd

institute: Cerevance ltd

summary: OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signalling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, upregulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.

overall_design: Single cell transcriptomics on control and OCT4 null blastocyst inner cell masses at two developmental stages.

first: G

middle: G


title: mRNA Sequencing of control and huntington's disease iPSC-derived medium spiny neuron-like cells and Q175 HET or WT mice. Plus and minus knockdown of PIAS1.

city: Irvine

first: Ryan

middle: Gar-Lok

last: Lim

state: California

address: 845 Health Sciences Rd.

department: MIND

institute: UC Irvine

summary: HD and control patient-derived induced pluripotent stem cells were used to generate medium spiny neuron-like cells. four control in duplicate and  three HD samples in duplicate with CAG repeat length in juvenile onset range  were differentiated as biological growth replicates (separate differentiations) into medium spiny neuron-like cells. Cells were treated with LNPs with siRNA for knockdown of PIAS1 or a luciferase control. Total RNA was isolated using the Qiagen RNeasy Kit and QIAshredders for cell lysis.  For mouse data WT and HET GFP+ micro-dissected flash frozen brain regions (striatum) were homogenized in TRIzol reagent (Invitrogen) and RNA were collected and purified using RNEasy Mini kit (Qiagen). Mice were treated with AAV2/1 expressing either a miRNA against Pias1 (miPias1.3), a control scrambled miRNA (miSafe). 1 µg of RNA with RIN values >8 were used for library preparation using the strand specific Illumina TruSeq mRNA protocol. Libraries were sequenced on the HiSeq 4000 using 100 cycles to obtain paired-end 100 reads.

overall_design: mRNAseq profiles of control and HD patient iPSC-derived medium spiny neuron-like cells and WT and HET Q175 mouse striatal tissue were generated by deep sequencing, using Illumina Hiseq 4000

first: Jie

last: Wu

first: Leslie

middle: M

last: Thompson


title: The physiological role of 3’CBEs in antibody class switching (3C-HTGTS)

city: Boston

first: Frederick

middle: W

last: Alt

address: 1 Blackfan Circle

department: PCMM

institute: Boston Children's Hospital

summary: We performed CSR-HTGTS-Seq, 3C-HTGTS, GRO-Seq and ChIP-Seq in mature splenic B cells with different stimulation  study physiological roles of 3'CBEs in antibody class switching

first: Xuefei

last: Zhang


title: Survival of hypoxia-induced dormancy is not a common feature of all strains of the Mycobacterium tuberculosis complex I

city: Borstel

first: Christian

last: Utpatel

address: Parkallee 1

country: Germany

institute: Forschungszentrum Borstel

summary: While persistence in a dormant state is crucial for the life cycle of Mycobacterium tuberculosis, no investigation regarding dormancy survival of different strains across different lineages was performed so far. We analyzed responses to oxygen starvation and recovery in terms of growth, metabolism and transcription. All different strains belonging to the (Euro-American) lineage (L4) showed similar survival and resuscitation characteristics. Different clinical isolates from the Beijing (L2) , East African-Indian (L3), and Delhi/Central Asian (L1) did not survive oxygen starvation. We show that dormancy survival is lineage-dependent. Recovery from O2 starvation was observed in Euro-American (L4) but no other used strains (L1, L2, L3). Thus, resuscitation from dormancy after oxygen starvation is not a general feature of all M. tuberculosis strains as thought before. Our findings are of key importance to understand infection dynamics of non-Euro American vs Euro American strains and to develop drugs targeting the dormant state.

overall_design: Hypoxia-induced gene expression of M. tuberculosis H37Rv and Beijing was measured after 10 days of oxygen depletion. Three independent experiments were performed for each timepoint.

first: Barbara

last: Tizzano

first: Tobias

last: Dallenga

first: Jochen

last: Behrends

first: Susanne

last: Homolka

first: Thomas

last: Kohl

first: Stefan

last: Niemann


title: The physiological role of 3’CBEs in antibody class switching (GRO-Seq)

overall_design: GRO-Seq


title: Survival of hypoxia-induced dormancy is not a common feature of all strains of the Mycobacterium tuberculosis complex II

overall_design: Hypoxia-induced gene expression of M. tuberculosis H37Rv was measured after 0 and 10 days of oxygen depletion. Three independent experiments were performed for each timepoint.


title: S100A8/S100A9 cytokine functions as transcriptional coactivator during breast cellular transformation

city: Hangzhou

first: Ruisheng

last: Song

state: Zhejiang

address: 866 Yuhangtang Road

institute: Zhejiang University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: In an inducible model of human breast cellular transformation, we map genome-wide chromatin binding of S100A8, S100A9 and Pol II. We show that the calcium-dependent cytokines S100A8 and S100A9 are recruited to numerous promoters and enhancers. This recruitment is associated with multiple DNA sequence motifs recognized by DNA-binding transcription factors that are linked to transcriptional activation and are important for transformation. Nuclear-specific expression of S100A8/A9 promotes oncogenic transcription and leads to enhanced breast transformation phenotype. These results suggest that, in addition to its classical cytokine function, S100A8/A9 can act as a transcriptional co-activator.

overall_design: ChIP-seq for S100A8-Flag, S100A9-Flag, or Pol II in non-transformed (EtOH-treated) and transformed (TAM-trated) MCF10A cells expressing ER-Src, a fusion between the v-Src oncoprotein and the ligand-binding domain of estrogen receptor

first: Kevin

last: Struhl


title: The physiological role of 3’CBEs in antibody class switching


title: DUX4 induces a homogeneous sequence of molecular changes, culminating in cellular apoptosis

city: Utrecht

first: Niels

last: Geijsen

state: Utrecht

address: Uppsalalaan, 8

country: Netherlands

institute: Hubrecht institute

summary: Single cell RNA sequencing was performed on a CML derived cell line, which contains a TET-ON inducible system for DUX4 inducible expression (DIE). In attempt of finding key players early in the DUX4 induced process, we performed single-cell RNA sequencing (SCS) on induced DIE cells, as early as 2 hours after induction. In a sequential manner, DIE cells induced for 3, 4, and 6 hours were also collected and used for SCS. By analysing these cells at a single cell level, we aimed to find (subtle) changes in the DIE cell transcriptome, that start early after DUX4 induction.

overall_design: The transcriptome of induced DIE cells was compared to uninduced or DIE cells, to identify differentially expressed genes or pathways that change upon DUX4 induction. Samples were sequenced using Illumina Nextseq 500, 2x75 kit, high output. Two biological replicates per samples were send for sequencing.

first: Ator

last: Ashoti


title: Droplet based mRNA sequencing of fixed and permeabilized cells by CLInt-Seq allows for antigen specific TCR cloning

city: Los Angeles

first: Pavlo

last: Nesterenko

address: 675 Charles E Young

department: MIMG

institute: UCLA

summary: T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via Crosslinker regulated Intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low frequency TCRs specific for any antigen. As a proof-of-principle, intracellular staining for TNFα and IFNγ identified Cytomegalovirus and Epstein-Barr virus reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically-cleavable primary amine crosslinker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ staining followed by single-cell sequencing performed similarly to isolation with multimer staining for Epstein-Barr virus reactive TCRs. We anticipate CLInt-Seq will enable droplet based single cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.

overall_design: We developed a new technique for cloning T cell receptors, based on IC staining,  and compare this technique to state of the art methods such as pMHC multimer selection.

first: Matthew

last: Obusan


title: The physiological role of 3’CBEs in antibody class switching (CSR-HTGTS-Seq)

overall_design: CSR-HTGTS-Seq


title: Microtranscriptome changes upon Human Skeletal Myoblasts differentiation

city: Poznan

first: Michał

last: Szcześniak

address: Uniwersytetu Poznanskiego 6

department: Institute of Human Biology and Evolution

country: Poland

institute: Adam Mickiewicz University

type: Non-coding RNA profiling by high throughput sequencing

summary: The main goal of this work was to determine the miRNAs level chnges during differentiation of human skeletal muscle cells. Our results indicated miRNA significantly alternated during the differentiation of skeletal muscle cells.

overall_design: miRNAs libraries from proliferating HSkM cell (n=3) and differentiated for 4 days HSkM cells (n=3) were generated by deep sequencing, using the NovaSeq 6000

first: Krzysztof

last: Sobczak

first: Agnieszka

last: Piasecka

last: Sekrecki


title: MEF2C knock-down in Human Skeletal Myoblasts [HSkM]

summary: The main goal of this work was to determine the connection between MEF2C expression and its role in miRNA biogenesis in the differentiation of human skeletal muscle cells.In summary, our results demonstrated a significant role for MEF2C myogenic factor in sculpting the microtranscriptome during the differentiation of skeletal muscle cells.

overall_design: miRNAs libraries from HSkM cell treated with control siRNA (n=2) and two separate siRNA against MEF2C: siMEF2C_1  (n=2)  and siMEF2C_2 (n=2) were generated by deep sequencing, using the NovaSeq 6000


title: GRO-seq on MCF7 and MDA-MB-231 breast cancer cells

city: Tel Aviv

first: Ran

last: Elkon

address: Ramat Aviv

department: Human Genetics

country: Israel

institute: Tel Aviv University

summary: We profiled in duplicates nascent transcription in two breast cancer cell lines: MCF7 and MDA-MB-231

overall_design: GRO-seq in MCF7 and MDA-MB-231 cell lines

first: Gozde

last: Korkmaz

first: Reuven

last: Agami

first: Rani


title: RNA-seq on MCF10A-YAP5SA cells (overexpressed constrictively activated YAP in MCF10A cells) transduced with sgRNAs vectors targeting the TEAD4 binding motif within the identified enhancers

summary: We performed a CRISPR-based functional genetic screen targeting TEAD4 binding motif  located within the YAP-bound enhancers. The screen identified seven functional enhancers whose targeting resulted in a marked negative effect on the proliferation of MCF10A-YAP-5SA cells (overexpressed constrictively activated YAP) but no significant effect on MCF10A (parental control) cells. We then carried out RNA-seq analysis on MCF10A-YAP-5SA cells transduced with sgRNA vectors targeting these seven enhancers (enhancers A, B, C, D, E, F, G) as well as the non-targeting sgRNA as control (NT).

overall_design: We have two different sgRNAs targeting each enhancer and we included three different non-targeting (NT)  sgRNAs as control. They are NT (27),  NT (28), NT (30).

first: Li

last: Li


title: Exosomal delivery of FTO confers gefitinib resistance to recipient cells through ABCC10 regulation in m6A dependent manner

status: Public on Dec 31 2020

city: Changsha

first: Wen-liang

last: Liu

address: No.139 Renmin middle road

department: Department of thoracic surgery

institute: The second Xiangya hospital, central South university

summary: In our study, RNA-seq on serum-derived exosomes from NSCLC patients with clinically significant differences in gefitinib sensitivity was performed to explore the differences in serum exosomal RNA expression profiles between the two groups. The exosomes were isolated and the saucer shaped round vesicles of 40–200 nm in diameter were observed with TEM and nanoparticle tracing system. Then, RNA-seq was performed on serum-derived exosomal RNAs and bioinformatics analysis was performed to analyze the differentially expressed RNAs between GS and GR group. The results demonstrated that there were 289 mRNAs with significant differences between the two groups, 252 of which were significantly up-regulated and 37 significantly down regulated (GR vs GS) .

overall_design: Serum exosomal RNA expression profiles of gefitinib resistant-Exo (GR) and gefitinib sensitive-Exo (GS) groups.

first: Peng

last: Xiao

first: Yu-kang

first: Wei

last: Han

first: Yan

last: Hu

first: Bo-you

last: Zhang


title: Identification of Lead Drug Candidates for COVID-19 based on Drug Screening Using Human Pluripotent Stem Cell-Derived Cells/Organoids

city: New York

first: David

last: Redmond

address: 1300 York Avenue

institute: Weill Cornell Medicine

summary: We systematically probed which cell types are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection of hPSC-derived lung organoids revealed upregulation of chemokines but not type I/III interferon signaling, similar to what was seen in primary human COVID-19 pulmonary infection.

overall_design: Transcriptomic analysis of stem cell derived lung organoids

first: Robert

last: Schwartz

first: Shuibing

last: Chen

first: Huanhuan

middle: J

first: Benjamin

last: tenOever


title: Expression data from infant nasal airway epithelial cells treated with IFN-lambda, poly(I:C), or both

city: Washington

first: Karima

last: Abutaleb

state: District of Columbia

address: 111 Michigan Ave NW

department: Center for Genetic Medicine Research

institute: Children's National Hospital

summary: Type III interferon, also known as interferon lambda (IFN-λ), is the newest addition to the IFN family. IFN-λ is primarily produced by the airway epithelium and curbs viral infections without triggering systemic pro-inflammatory cytokine responses1. Of the current IFN COVID-19 trials (as of 6/27/2020), four propose the use of IFN-λ. However, it remains to be determined whether IFN-λ induces ACE2 expression in the human airway epithelium.

overall_design: Using primary human nasal airway epithelium of young children, we employed RNA extraction and hybridization on Affymetrix microarrays to examine for the first time the effects of IFN-λ and a viral mimic (poly I:C) on the expression of ACE2 and the global antiviral transcriptomic program.

first: Kyle

last: Salka

first: Elizabeth

last: Chorvinsky

first: Maria

last: Arroyo

first: Jose

last: Gomez

last: Gutierrez

first: Dinesh

last: Pillai

first: Jyoti

last: Jaiswal

first: Gustavo

last: Nino


title: CRISPR-Cas9-engineered PIGV341E mouse model mirrors human phenotype, shows hippocampal synaptic dysfunctions and suggests a major role of impaired Eph/Abl1 signaling in GPI anchor deficiencies

city: Bonn

first: Peter

last: Krawitz

address: Venusberg-Campus 1

institute: University Hospital Bonn

summary: Pathogenic germline mutations in PIGV result in a glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the GPI pathway show cognitive impairments, a motor delay and often epilepsies. Thus far, the pathophysiology underlying the disease remains unclear and suitable rodent models are lacking that mimic human pathophysiology. We therefore generated a mouse model using CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, at a site that is also conserved in mice, Pigv:c.1022C>A (p.A341E). Pigv341E mice showed deficits in motor coordination and cognitive impairment with poorer long-term spatial memory. Furthermore, Pigv341E mice showed alterations in sociability and sleep patterns reflecting the human phenotype. Immunohistochemistry showed decreased synaptophysin-immunoreactivity and electrophysiology recordings demonstrated reduced hippocampal synaptic transmission in Pigv341E mice that may explain impaired memory formation. Moreover, we performed single-cell RNA sequencing of hippocampal cells from brains of adult Pigv341E mice that exhibit changes of gene expression, most prominently in a microglia subtype and subicular neurons. A significant reduction of Abl1 transcripts in several cell clusters suggest a link to the signaling pathway of GPI-anchored ephrins. We also identified increased levels of Hdc that might affect histamine metabolism with consequences for circadian rhythm and neuronal excitability. In summary, we present here the first mouse model with a patient-specific hypomorphic mutation that mirrors the human phenotype and will allow us to explore the pathophysiology in GPIBDs in future studies.

overall_design: For single-cell RNA sequencing we collected both left and right hippocampi from Pigv341E mice (n=4) and wild-type mice (n=4). Isolated hippocampus cells were pooled by genotype, resulting in a total of 2 samples from which sequencing libraries were generated using the Chromium Single-cell Gene Expression Solution v3 (10X Genomics, Inc.) and sequenced with the Illumina NovaSeq 6000. Both the pooled Pigv341E and wild-type sample were sequenced on the same lane.

first: Miguel

last: Rodriguez de los Santos

first: Marion

last: Rivalan

first: Friederike

middle: S

last: David

first: Alexej

last: Knaus

first: Alexander

last: Stumpf

first: Laura

last: Moreno Velasquez

first: Anne

last: Voigt

first: Daniele

last: Mattei

first: Melissa

last: Long

first: Guido

last: Vogt

first: Lars

last: Wittler

first: Bernd

last: Timmermann

first: Denise

last: Horn

first: Stefan

last: Mundlos

first: Uwe

last: Kornak

first: Dietmar

last: Schmitz

first: York

last: Winter


title: Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer [RNA-seq]

first: Hideo

last: Watanabe

address: One Gustave L. Levy Place

department: Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine

institute: Icahn School of Medicine at Mount Sinai

summary: Small cell lung cancer (SCLC) frequently harbors mutually exclusive genomic amplification of MYC family members. Therefore, it has been long suggested that they are functionally equivalent; however, more recently, their expression has been associated with specific neuroendocrine markers and distinct histopathology. Integrated transcriptomic and epigenomic analyses showed that c-Myc and L-Myc impart neuronal and non-neuroendocrine associated transcriptional programs, respectively, both associated with distinct SCLC lineage. Genetic replacement of c-Myc with L-Myc in c-Myc-SCLC induced a neuronal state but was insufficient to induce ASCL1 lineage. In contrast, c-Myc induced transition from ASCL1-SCLC to NeuroD1-SCLC characterized by distinct LCNEC-like histopathology. Collectively, we characterize an undescribed role of historically defined general oncogenes, c-Myc and L-Myc, for regulating lineage plasticity across molecular subtypes as well as histological subclasses.

overall_design: Transcriptomic profile of NCI-H82 cells genetically engineered to overexpress L-Myc and ablate the expression of c-Myc with control and of NCI-H1963 genetically engineered cells to overexpress c-Myc with control.

first: Ayushi

last: Patel


title: The effects of codonopsis pilosula on RNA expression profile in digestive system of D-galactose-induced aging mice

city: Lanzhou

first: Jie

last: Meng

state: Gansu

address: 35 Dingxi dong lu, Chengguan district, Lanzhou city, Gansu province, China

institute: Gansu University of Chinese Medicene

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: To investigate the underlying mechanism that Codonopsis Pilosula anti-aging in gastrointestinal tract of aged mice induced by D-galactose.we integrated stomach and intestine tissues, and aimed to explore the underlying mechanism that Codonopsis Pilosula participates in protecting gastrointestinal structure and regulating gastrointestinal function by microarray and real-time quantitative polymerase chain reaction (qRT-PCR).

overall_design: Gene expression was measured in gastric and intestinal tissues of normal, aging, and high-dose codonopsis mice.Each experiment used a different donor for three separate experiments.

first: Jiajia

last: Liu

first: Dongmei

last: Chen

first: Dandan

last: Li

first: Jing

last: Wang


title: Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer [ATAC-seq]

overall_design: Accessible chromatin and Myc accessible profile of six SCLC cell lines, NCI-H82 cells genetically engineered to overexpress L-Myc and ablate the expression of c-Myc with control and of NCI-H1963 genetically engineered cells to overexpress c-Myc with control.


title: RNA-Seq of major populations present in Ter119NegCD45Neg compartment of mouse fetal liver

city: Paris

first: Francisca

last: Soares da Silva

address: 25 Rue du Dr Roux

country: France

institute: Institut Pasteur

summary: The goal of this study was to characterize the non-hematopoietic compartment (Ter119NegCD45Neg) of the mouse embryonic liver. For that, we sorted the 3 major populations at embryonic day 14.5 and CD54 expressing cells at embryonic day 18.5 for whole genome analysis.

overall_design: mRNA sequencing data of enriched cells from E14.5 or E18.5 mouse fetal livers, according to expression of CD54, CD31 and Kit. For each population, 3 biological replicates were analyzed.

first: Odile

last: Burlen-Defranoux

last: Sismeiro

last: Soares-da-Silva

first: Ana

last: Cumano


title: Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer

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

overall_design: Refer to individual Series


title: Setd1a and Setd1b loss-of-function in murine macrophages

city: Freising

first: Franziska

last: Greulich

address: Gregor-Mendel-Str. 2

department: Metabolic Programming

institute: TU München

summary: Setd1bKO primary murine bone marrow-derived macrophages (BMDMs) were treated with lipopolysaccharide (LPS) or dexamethasone and LPS (Dex+LPS) and gene expression differences in response to treatment analysed by PolyA RNA-Sequencing. No Dex-treatment dependent gene expression differences were identified. Setd1aDel/+ Raw264.7 cells with reduced Setd1a expression were analyzed with regards to their reponse to Dex when inflammatorily challenged with LPS by mRNA-Seq. We observed reduced GR-dependent gene acivation in Setd1a hypermorphic Raw264.7 cells. Wild type and Setd1aDel/+ Raw264.7 cells were treated with LPS or LPS and interferon beta (IFNB1) to show the IFNB1-dependent loss of gene expression in LPS-stimulated Setd1aDel/+ cells.

overall_design: We profiled mRNA expression by RNA-Seq in form wild-type or Setd1bKO BMDMs treated with either vehicle (0.1% EtOH) for 16h, 6h LPS (0.1% EtOH, 100ng/ml LPS) or 16h Dex+6h LPS (1 uM Dex, 100ng/ml LPS) in order to study the function of Setd1b as GR?s nuclear interaction parner. For the analysis of Setd1a-dependent gene expression, we performed mRNA-Seq from wild-type or Setd1aDel/+ Raw264.7 cells. Raw264.7 cells were treated with Dex for either 16h (vehicle: 16h 0.1% EtOH; LPS: 6h 100 ng/ul LPS, Dex+LPS: 16h 1 uM Dex, 6h 100 ng/ul LPS) or 6h (vehicle: 6h 0.1% EtOH; LPS: 6h 100 ng/ul LPS, Dex+LPS: 6h 1 uM Dex, 6h 100 ng/ul LPS; Dex: 6h 1 uM Dex). Furthermore, we profiled wild type and Setd1aDel/+ RAW264.7 cells after 6h of LPS stimulation (100 ng/ml) and compared the effect of Setd1a deletion on gene expression to LPS+IFNB1 (6h 100ng/ml LPS + 10 ng/ml IFNB1) stimulated cells of both genotypes. Three biological replicates were sequenced per treatment and genotype.

first: Nina

middle: H

last: Uhlenhaut


title: Epigenetic modification of GR enhancers by SETD1A/COMPASS complex in murine macrophages

summary: Nuclear interaction studies by ChIP coupled with mass spectrometry identified the COMPASS/SETD1A complex as interaction partner of the glucocorticoid receptor (GR) in murine bone marrow-derived macrophages (BMDMs). Here, we profiled the occupancy of SETD1A and CXXC1, two subunits of the COMPASS/SETD1A complex in murine macrophages after LPS and Dex+LPS stimulation. We show, that GR recuits SETD1A and CXXC1 to GR enhancer and that SETD1A recruitment does correlate with changes in H3K4methylation at some of those enhancers.

overall_design: We profiled changes in SETD1A and CXXC1 occupany as well as changes in H3K4methylation and H3K27Ac in murine bone marrow-derived macrophages after challenge with LPS or Dex+LPS. All ChIP-Seq experiments were done in duplicates or triplicates, partially including Drosophila melanogaster chromatin as spike-in control.


title: Transcriptomic profiling of primed and naive primate pluripotent stem cells by RNA-sequencing

city: Lyon

first: Irene

last: Aksoy

address: 18 avenue du Doyen Lepine

institute: Stem Cell and Brain Research Institute

summary: Analysis of rhesus monkey ESC cells that have been reprogrammed to naïve pluripotency using 7 different protocols including, TL2i,  4i/L/b, E-NHSM, NHSM-v, t2iLGöY, LCDM (EPS), TL-CDK8/19i and compared to primed cells.

overall_design: Primed cells vs Naïve cells

first: Pierre

last: Savatier


title: Genome-wide expression of PATZ1 knockdown in HepG2 cells

city: Singapore

first: Zhen Long

last: Ng

address: 8 Medical Drive

department: Biochemistry

country: Singapore

institute: National University of Singapore

summary: Liver cancer is the third most common cause of cancer death in the world. POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) is a transcription factor associated with various cancers. However, the role of PATZ1 in cancer progression remains controversial. Here we report that PATZ1 regulates cell proliferation by directly regulating CDKN1B (p27) in hepatocellular carcinoma HepG2 cells. PATZ1 level was found to be ectopically expressed in hepatocellular carcinoma cells compared to normal primary human hepatocytes, thus affirming its relevance in liver cancer. Our gene expression microarray and PATZ1 ChIP-seq analysis further revealed strong enrichment in gene ontology terms related to cellular proliferation. Importantly, siRNA-mediated PATZ1 knockdown in HepG2 cells led to an increased rate of colony formation, elevated Ki-67 expression and greater S phase entry. Furthermore, the increased cancer cell proliferation was accompanied with suppressed expression of the cyclin-dependent kinase inhibitor CDKN1B. Consistently, PATZ1 binds to the genomic loci flanking the transcriptional start site of CDKN1B and positively regulates its promoter activity. Additionally, we found that PATZ1 associates with p53 and the absence of p53 abrogated the PATZ1-mediated regulation of CDKN1B expression. In conclusion, our study provides novel mechanistic insights into the role of PATZ1 in liver cancer progression, thereby providing a promising therapeutic intervention to alleviate tumor burden in liver cancer.

overall_design: Total RNA was extracted from HepG2 cells treated with siRNA against PATZ1 and hybridized onto Illumina HT-12 v4 Expression BeadChip. The results were compared with the corresponding control siRNA-treated HepG2 microarray.

first: Z

middle: L

first: Qiang

last: Wu


title: PATZ1-knockdown expression array and PATZ1 ChIP-seq in HepG2 cells


title: RNA-Seq in LPS and Dex+LPS treated bone marrow-derived macrophages

summary: Wild-type bone marrow-derived macrophages (BMDMs) were treated with vehicle (0.1% EtOH/D-PBS), 6h 100 ng/ml lipopolysaccharide (LPS) or 16h 1uM dexamethasone (Dex) and 6h 100 ng/ml LPS (Dex+LPS) and mRNA expression analysed by RNA-Seq.

overall_design: We profiled mRNA expression by RNA-Seq in BMDMs form wild-type C57BL/6 mice treated with either vehicle (0.1% EtOH) for 16h, 6h LPS (0.1% EtOH, 100ng/ml LPS) or 16h Dex+6h LPS (1 uM Dex, 100ng/ml LPS).Three biological replicates were sequenced per treatment.


title: PATZ1 ChIP-Seq

overall_design: Examination of PATZ1 binding sites in HepG2 cells


title: Gene signature profiles in lung tisses of wild type mice or Nrf2 deficient mice  infected with my nontuberculous mycobacteria

status: Public on Dec 30 2020

city: TSUKUBA

first: Masashi

last: Matsuyama

state: IBARAKI

address: 1-1-1 AMAKUBO

department: respiratory medicine

country: Japan

institute: Tsukuba university

summary: Background.  Nuclear erythroid 2 p45-related factor 2 (Nrf2) regulates the expression of antioxidant and detoxification genes and are thought to be important in protection against intracellular pathogens. The aim of this study was to determine the role of Nrf2 in the host defense against Mycobacterium avium complex (MAC) infection in mice.  Methods.  Wild-type mice and Nrf2-deficient mice were infected with MAC via intranasal inoculation with 1x107 CFU of Mycobacterium avium subsp. hominissuis. At 2 months after infection, the bronchoalveolar lavage fluid, lung tissues were collected, then, comprehensive transcriptome analysis in the lungs was performed by RNA-seq.  Results.  Nrf2-deficient mice were highly susceptible to MAC, compared with wild-type mice. There was no significant changes between genotypes in the level of oxidative stress. In addition, the expression of genes related to Th1 immunity and the proportion of Th1 cells were not changed between genotypes. Comprehensive transcriptome analysis showed that the expression of Nramp1 was much lower in the infected lungs of Nrf2-deficient mice than those of Wild-type mice. The expression of Nramp1 was also much lower in the infected alveolar macrophages of Nrf2-deficient mice than those of Wild-type mice. The result of electron microscopy showed that many infected alveolar macrophages from Nrf2-deficient mice were found to contain a large number of intracellular MAC bacteria compare to alveolar macrophages from wild-type mice, due to inhibition of phagolysosome fusion.   Conclusions.  This study identified that the Nramp1 regulated by Nrf2 is essential in defining the outcome of MAC disease. Nrf2 is a critical determinant for host resistance to MAC infection as a regulator for the expression of Nramp1.

overall_design: 12 strand-specific RNA libraries for high-throughput sequencing were prepared (3 from the lungs of the wild type mice treated with saline, 3 from the lungs of the Nrf2-/- mice treated with saline, 3 from the lungs of the wild type mice infected with M. avium for 2 months, and 3 from the lungs of the Nrf2-/- mice infected with M. avium for 2 months) using the Illumina Stranded mRNA Sample Preparation Kit with 500ng of total RNA according to manufacturer’s instructions.

first: Masayuki

last: Nakajima

first: Masafumi

last: Muratani

first: Nobuyuki

last: Hizawa


title: RNA-Seq analysis of broiler intestinal transcriptome reveals new insight on the interaction between antibiotic growth promoters (AGPs) and the host

city: Tulln

first: Silvia

last: Fibi-Smetana

address: Technopark 1A

department: R&D

country: Austria

institute: BIOMIN Holding GmbH

summary:
Purpose: Identify at the transcriptome level genes that could be involved in the mode of action of avilamycin, an AGP, when administered at non-therapeutic concentrations, and use this information in regard to development of alternatives to AGPs.
Methods: Out of 120 broiler chickens fed either CONTROL feed or AGP feed (supplemented with avilamycin; Surmax® 100, 10 g/1000 kg of feed, Elanco), the RNA from the ileum tissue of five birds per group was isolated from day 35, profiled by RNA-sequencing, and results were verified via RT-qPCR.
Results: Overall, 66 differentially expressed genes (DEGs; FDR ≤ 0.05 and fold change ≥ 2 or ≤ -2) were found in the ileum of chicken fed avilamycin in comparison to the control group. Only partial agreement was found on the DEGs (64%) when the reads were initially mapped to the former genome assembly Galgal5 instead of GRCg6a. Functional enrichment analysis showed that the down-regulated DEGs were associated with signaling by interleukins, and that IL-22 and certain antimicrobial peptides could play a central role in the intestinal host response to avilamycin at day 35. This was further corroborated on sixteen chickens per group via RT-qPCR. In addition, higher activity was detected in module of genes related to lipid metabolism and transport in the avilamycin group, as revealed by the co-expression network analysis.
Conclusions: Improved feed efficiency was correlated with reduced immunological stress in the intestinal mucosa. The RNA-Seq approach allowed generating novel knowledge that could contribute to the development of AGP alternatives, with IL-22 identified as a potential marker.

overall_design: Ileum mRNA profiles from 35 day old chickens. For each tissue, 5 biological replicates (= 5 chickens) from Control and 5 from Avilamycin group were used for RNA-extraction. Two technical replicates of each of the biological replicates were used for library preparation. The final multiplexed (n=20) libraries were sequenced in two independant runs on a Illumina HiSeq 4000 machine (run1: Sample 1-9 in one lane; run2: Sample 10-20 in two lanes).

first: Candida

last: Vaz

first: Suzana

last: Ilic

first: Roger

last: Berrios

first: Gerd

last: Schatzmayr

first: Vivek

last: Tanavde

first: Bertrand

last: Grenier

first: Jeremy

last: Le Coz


title: Long non-coding RNA screening for type 2 diabetes

city: Urumqi

first: Qi

last: Ma

address: No.91, Liyushan Road

institute: First Affiliated Hospital of Xinjiang Medical University

summary: Improving the early diagnosis and treatment of type 2 diabetes (T2D) can effectively control blood glucose. To investigate new long non-coding RNAs (lncRNAs) as molecular markers we used microarrays to identify differentially expressed lncRNAs and mRNAs in peripheral blood mononuclear cells from T2D patients and controls.

overall_design: RNA samples from five patients with T2D and five healthy controls. Each sample was tested five times.


title: Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with Multiple Myeloma

city: Würzburg

first: Antoine-Emmanuel

last: Saliba

address: Josef-Schneider-Straße 2 / D15

institute: Helmholtz Institute for RNA-based Infection Research

summary: Chimeric antigen receptor T-cells (CAR-T) targeting B-cell-maturating-antigen (TNRFSF17, BCMA) show unprecedented overall response rates of up to 100% in heavily pretreated relapsed-refractory Multiple Myeloma (RRMM).  However, the efficiency of the treatment is hindered by the rapid emergence of tumor-intrinsic mechanisms of resistance. We herein describe a patient with RRMM and extramedullary disease (EMD) who underwent Idecabtagene vicleucel BCMA CAR-T therapy. The patient rapidly achieved very good partial response including full resolution of EMD, but aggressively relapsed 5 months after CAR T cell infusion. Single cell RNA sequencing and immunohistochemistry on the re-emerging tumor cells demonstrated loss of target expression on mRNA and protein levels, and whole genome sequencing revealed the homozygous deletion of Chr 16p1313, including the BCMA locus. Clonal heterogeneity of BCMA could be observed in 32 RRMM patients from our institution. In three of them heterozygous deletion of BCMA could be confirmed, with none of them having underwent prior anti-BCMA therapy. Thus, our report not only provides first evidence of BCMA loss as the underlying mechanism of disease relapse following BCMA targeted immunotherapy in MM. It furthermore identifies a subset of patients at risk to develop biallelic BCMA inactivation thus linking genomic instability in MM to relapse from targeted immunotherapies.

overall_design: Here, we uncovered a patient with RRMM and extramedullary disease (EMD) who underwent Idecabtagene vicleucel BCMA CAR-T therapy but aggressively relapsed 5 months after CAR T cell infusion due to the homozygous deletion of Chr 16p1313, including the BCMA locus and the subsequent lost of BCMA expression on the protein and mRNA levels. We could furthermore discover a group of RRMM patients (3 out of 32) that showed heterozygous deletion of BCMA before anti-BCMA therapy.

first: Matteo

last: Da Via

first: Panagiota

last: Arampatzi

first: Oliver

last: Dietrich

first: Antoine

middle: E

first: Leo

last: Rasche


title: Genome-wide prediction of topoisomerase IIB binding by architectural factors and chromatin accessibility

city: Madrid

first: Felipe

last: Cortés Ledesma

address: Melchor Fernández Almagro 3

department: Molecular Oncology

institute: CNIO

summary: Aberrant activity of type II topoisomerases (TOP2) often causes blocked double-strand breaks (DSBs), whose inefficient repair can seriously compromise genomic stability. One of the two TOP2 paralogs encoded in vertebrates is TOP2B, which has been linked to essential processes such as transcription or genome organization. Few TOP2B genome-wide maps have been profiled, and a comprehensive study of the mechanisms involved in TOP2B-DNA binding is still lacking. Here, we conduct an in silico approach for the prediction of TOP2B binding sites using publicly available sequencing data. We achieve highly accurate predictions and find that open chromatin and architectural factors are the most informative features. We also validate our predictions on experimental data and generate predicted TOP2B tracks that mirror experimental ones with high precision.

overall_design: TOP2B ChIPseq in MCF7

first: P

last: Martinez-Garcia

first: M

last: Garcia-Torres

first: F

last: Divina

first: J

last: Terron-Bautista

last: Delgado-Sainz

last: Gomez-Vela

last: Cortes-Ledesma


title: Tissue-autonomous immune response regulates stress signalling during hypertrophy

city: Stockholm

first: Ulrich

last: Theopold

state: Stockholm

address: Svante Arrhenius väg 20C

department: Department of Molecular Biosciences, The Wenner-Gren Institute

country: Sweden

institute: Stockholm University

summary: Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via RasV12-overexpression in the postmitotic salivary glands of Drosophila larvae, we overrode the glands adaptability to growth signals, induced hypertrophy and stress accumulation. This allowed us to decipher a novel, spatio-temporally regulated interaction between the JNK-stress response and a genuine tissue-autonomous immune response. Central to this interaction is the direct inhibition of JNK-signalling by the antimicrobial peptide Drosomycin, which blocks programmed cell death and prevents recognition of the stressed tissue by the systemic immune response. While this mechanism might allow growing salivary glands to cope with temporary stress, continuous expression of Drosomycin favors survival of unrestricted, hypertrophic RasV12-glands. Our findings indicate the necessity for refined therapeutic approaches that fundamentally acknowledge detrimental effects that stimulated immune responses have on tissues coping with damage and stress.

overall_design: Bulk RNAseq data of dissected Drosophila larval salivary glands derived from 4 genotypes (i.e., w1118 / RasV12 / lglRNAi;RasV12 / lglRNAi;RasV12-PP) each as triplicates at 96 h AED.

first: Robert

last: Krautz

first: Dilan

last: Khalili


title: HPV16 alignment using RNA sequencing data in head and neck quamous cell carcinoma (HNSCC)

status: Public on Dec 28 2020

city: Southampton

first: JAYA

last: THOMAS

address: MP824 Somers Building, Southampton General Hospital, Tremona Road,

department: Cancer Science

institute: University of Southampton

summary: Detection of HPV16 RNA expression of separately for each gene using RNA sequencing data from head and neck squamous cell carcinoma.

sample_organism:
Homo sapiens
Human papillomavirus 16

platform_organism:
Homo sapiens
Human papillomavirus 16

overall_design: HPV16 gene alignment in HNSCC

first: Christian

last: Ottensmeier


title: A global transcription study of the regulatory effects of potassium, glutamate and c-di-AMP in Bacillus subtilis

city: Greifswald

first: Ulrike

last: Mäder

address: F.-L.-Jahn-Str. 15A

department: Functional Genomics

institute: University Medicine Greifswald

summary: Since c-di-AMP has been implicated in the interplay of potassium and glutamate homeostasis, we analysed, using strand-specific tiling arrays (tiling step of 22 nucleotides), global gene expression in the wild type strain B. subtilis 168 at low (0.1 mM) and high (5 mM) potassium concentrations and in the presence of ammonium and glutamate as the nitrogen source as well as in the c-di-AMP free strain (∆disA, ∆cdaA, ∆cdaS) GP2222 and its isogenic suppressor mutant GP2223 that is able to grow at 5 mM potassium. In addition to strongly reduced expression of genes involved in fermentation and respiration (regulated by the NADH-responsive transcription factor Rex), genes of the SigO regulon involved in acid stress response and several competence genes was severely reduced in the mutant strain GP2222. In the wild type strain, the ktrAB and kimA genes were most strongly repressed by potassium. These genes encoding high affinity potassium uptake systems are controlled by a c-di-AMP sensitive riboswitch.Taken together, the analysis confirmed that c-di-AMP is involved in multiple cellular functions including genetic competence and revealed a particular role of genes involved in controlling NADH homeostasis.

sample_organism: Bacillus subtilis subsp. subtilis str. 168

overall_design:
Cells were grown aerobically at 37 °C in a modified Spizizen's minimal medium (KH2PO4 was replaced by NaH2PO4, named as MSSM) with ammonium or glutamate as the nitrogen source and with 0.1 mM or 5 mM potassium. Cells were harvested in the mid exponential phase at an optical density at 600 nm of 0.4-0.6.
Hybridizations of strand-specific tiling arrays were performed with RNA from two biological replicates. cDNA samples were labeled with Cy3.

first: Hermann

last: Rath

first: Larissa

last: Krüger

first: Jan

last: Gundlach

first: Jörg

last: Stülke

first: Uwe

last: Völker


title: MicroRNA and mRNA integrated transcriptional analysis in a Cx30-/- mouse model of non-syndromic hearing loss and deafness [mRNA]

status: Public on Dec 29 2020

city: Catania

first: Sebastiano

last: Cavallaro

address: Via Paolo Gaifami, 18

country: Italy

institute: IRIB-CNR

summary: The GJB6 gene is located just 35 kb telomeric to GJB2 in the so-called nonsyndromic hearing loss and deafness locus 1 (DFNB1). Knock out mouse models confirmed that inner ear expression of their protein products, connexin 30 (Cx30) and connexin 26 (Cx26), is crucial for hearing acquisition and normal development of the organ of Corti, however the coordinated regulation mechanism of Cx26 and Cx30 expression in the cochlea remains unclear. To investigate the mechanism underlying the etiopathogenesis of DFNB1, we used a microRNA (miRNA) and mRNA integrated expression profiling analysis on Cx30 -/- mice, which represent a model for humans in which large deletions in the DFNB1 locus lead to the down-regulation of both connexins and profound deafness.

overall_design: Messenger RNA expression profiling of cochlear tissue from Cx30 wild-type mice at P5, and their age-matched homozygous knock-out siblings (Cx30-/-) were analysed by using an 8x60K SurePrint G3 Mouse GE v2 Microarray. Four biological replicates for each of the two genotypes were used.

first: Giulia

last: Gentile

first: Antonio

last: Spampinato

first: Maria

last: Guarnaccia


title: MicroRNA and mRNA integrated transcriptional analysis in a Cx30-/- mouse model of non-syndromic hearing loss and deafness [miRNA]

overall_design: MiRNA profiling of cochlear tissue from Cx30 wild-type mice at P5, and their age-matched homozygous knock-out siblings (Cx30-/-) were analysed by using G3 Mouse miRNA Microarray, Release 21, 8×60K (Agilent Technologies). Four biological replicates for each of the two genotypes were used. After the evaluation of quality control parameters for each scanned microarray images, the raw data of three replicates for wild-type and four replicates for knock-out experimental conditions were further analyzed.


title: scRNA-seq of P2 mouse cochlea

city: Palo Alto

first: Marie

last: Kubota

address: 240 Pasteur Drive

institute: Stanford University

summary:
Purpose and Methods: Cochlear sensory hair cells (HCs) are essential for our sense of hearing. They are embedded in the organ of Corti that lacks regenerative capacity, which is a major cause of hearing loss. However, some neonatal non-sensory cells in the organ of Corti display a limited regenerative ability. We used fluorescence-activated cell sorting (FACS) to isolate different cochlear cell types from postnatal day 2 (P2) mice to assess the individual cell groups’ potential to grow organoids and to generate new HCs. Single-cell RNAseq validated the composition of the cell types in the sorted cell groups.
Results and Conclusions: The greater epithelial ridge (GER), a transient tissue that only exists in the neonatal inner ear, harbored the most potent organoid-forming cells. GER-derived organoids expanded into large colonies when cultured adherently and gave rise to new hair cell marker-expressing cells in a sensory epithelia-resembling organization. The organoid forming ability of GER cells was synergistically enhanced when they were cultured at increasing density. The synergistic effect relied on direct cell-to-cell contact rather than released soluble factors.

overall_design: We used Sox2-GFP, Lfng-GFP, Fgfr3-CreERT2, and Ai14-tdTomato two-day-old transgenic mice to isolate five distinctive groups of cochlear cells. Single-cell RNAseq was performed on these cells using Illumina NextSeq 500 high output flow cell configuration with a 150 bp paired-end sequencing.

first: Mirko

last: Scheibinger

first: Taha

last: Jan

first: Stefan

last: Heller


title: Gene expression data of 2D duodenum organoids with or without continuous flow

status: Public on Dec 23 2020

city: Tokyo

first: Shinya

last: Sugimoto

address: 35 Shinanomachi, Shinjuku-ku

institute: Keio University School of Medicine

summary: We developed a monolayer culture system for small intestinal organoids, and then placed the culture plate on a rotator to generate a constant flow in the overlay medium. This mechanical stimulation facilitated the formation of villus-like structures from the monolayer of small intestinal organoids.

overall_design:
We analyzed gene expression of 2D normal duodenum organoids with or without medium rotation. Total RNA was extracted from organoids with or without a four-day medium rotation, and after 0, 1, 2 or 4 days of medium rotation, and sequenced with HiSeq X Ten (Illumina).
*** RNA sequencing controlled-access data have been deposited to the Japanese Genotype-phenotype Archive under the accession number JGAS000256. ***

first: Kohta

last: Toshimitsu

first: Toshiro

last: Sato


title: MicroRNA and mRNA integrated transcriptional analysis in a Cx30-/- mouse model of non-syndromic hearing loss and deafness

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

overall_design: Refer to individual Series


title: Identification of differentially expressed Oncorhynchus mykiss microRNA in plasma, mucus, and water following acute stress using high throughput sequencing.

city: Waterloo

first: Heather

last: Ikert

state: Ontario

address: 200 University Ave W

department: Biology

country: Canada

institute: University of Waterloo

summary: Circulating plasma microRNAs (miRNAs) are well established as biomarkers of several diseases in humans and have recently been used as indicators of environmental exposures in fish. However, the role of plasma miRNAs in regulating acute stress responses in fish is largely unknown. Tissue and plasma miRNAs have recently been associated with excreted miRNAs in humans however external miRNAs have never been measured in fish. The objective of this study was to characterize the plasma miRNA profile in response to acute stress in rainbow trout (Oncorhynchus mykiss), as well as miRNA profiles in novel external samples, (fish epidermal mucus and the surrounding water). RNA was extracted and sequenced from plasma, mucus, and water collected from rainbow trout and their surrounding environment prior to and one-hour following a three-minute air exposure, a known inducer of an acute stress response in fish. Following small RNA-Seq and pathway analysis, we identified differentially expressed plasma miRNAs that targeted biosynthetic, degradation, and metabolic pathways. We successfully isolated miRNA from trout mucus and the surrounding water and detected differences in miRNA expression one-hour post air stress. The altered miRNA profiles in mucus and water were unique to the altered plasma miRNA profile, indicating that the plasma miRNA response was not associated with or immediately reflected in external samples. This research expands our understanding of the role of plasma miRNA in the acute stress response of fish and is the first study to report on the successful isolation and profiling of miRNA from fish mucus and water samples. Measurements of miRNA from plasma, mucus, and water can be further studied and have the potential to be applied in environmental monitoring as non-lethal indicators of acute stress in fish.

overall_design: Six blood plasma samples, six epidermal mucus samples, and six water samples were analyzed. Each of these had three experimental replicates for the control treatment and three experimental replicates for the stressed treatment (18 samples total).


title: Influence of L-lactate and low glucose concentrations on the metabolism and the toxin formation of Clostridioides difficile

city: Braunschweig

first: Rebekka

last: Biedendieck

address: Rebenring 56

department: Institute of Microbiology

institute: Technische Universtität Braunschweig

summary: Metabolomic and transcriptomic analysis of changes in the exponential and stationary phase of Clostridioides difficile after cultivation in casamino acids medium (reference) and supplemented with L-lactate or glucose and the connection to toxin production.

overall_design: Clostridioides difficile 630∆erm (DSM28645) was grown in a casamino acids medium (reference)  and supplemented with 2 g/L glucose (Glc) or 0.5 g/L L-lactate (Lac). Samples were taken at exponential growth (1/2 ODmax) and at the beginning of the stationary phase (ODmax).

first: Julia

middle: D

last: Hofmann

first: Annika-Marisa

last: Michel

first: Dietmar

last: Schomburg

first: Dieter

last: Jahn

first: Meina

last: Neumann-Schaal


title: The molecular basis of socially-mediated phenotypic plasticity in a eusocial paper wasp

city: London

first: Benjamin

middle: Aaron

last: Taylor

state: London

address: Gower Street

department: Genetics, Evolution & Environment

institute: University College London

summary: We report analyses the individual-level brain gene expression profiles of Polistes dominula paper wasps following the loss of a queen, a perturbation that induces some individuals to undergo a significant phenotypic shift and become queens while others maintain an apparently unchanged worker phenotype. We use a machine learning approach to map individual transcriptomes on a continuum between worker and queen states and then match these scores to detailed phenotypic data. We find a strong response of caste-associated gene expression to queen loss, with individuals’ expression profiles becoming intermediate between queen and worker states.

overall_design: Brain mRNA profiles for 96 individuals: 26 queens; 12 workers from control nests; 24 individuals from nests three days after queen removal; 34 individuals from nests twelve days after queen removal.

first: Alessandro

last: Cini

first: Christopher

last: Wyatt

first: Max

last: Reuter

first: Seirian

last: Sumner


title: IL-22-dependent dysbiosis and mononuclear phagocyte depletion contribute to steroid-resistant gut graft-versus-host disease in mice II

status: Public on Dec 27 2020

city: Duarte

first: Defu

last: Zeng

address: 1500 East Duarte Road

department: Immunology & Theragnostics

institute: The Beckman Reseach Institute

summary: CD4+ and CD8+ T cells can reciprocally differentiate into Th/Tc1, Th/Tc17 and Th/Tc22. Although alloreactive Th/Tc1 cells play a critical role in initiating pathogenesis of gut acute graft-versus-host disease (Gut-aGVHD), the pathogenic T cells in steroid-resistant Gut-aGVHD (SR-Gut-aGVHD) remains unclear. Here, we show that in murine models of SR-Gut-aGVHD, the pathogenesis is associated with reduction of IFN-g+ Th/Tc1 and IL-17A+IL-22- Th/Tc17 cells but expansion of IL-17-IL-22+ Th/Tc22, particularly Tc22 cells. The IL-22 from Th/Tc22 cells causes dysbiosis.

overall_design: Examination of gut microbiome difference in the mouse model of acute gut GVHD

first: Qingxiao

last: Song


title: IL-22-dependent dysbiosis and mononuclear phagocyte depletion contribute to steroid-resistant gut graft-versus-host disease in mice


title: IL-22-dependent dysbiosis and mononuclear phagocyte depletion contribute to steroid-resistant gut graft-versus-host disease in mice I

summary: CD4+ and CD8+ T cells can reciprocally differentiate into Th/Tc1, Th/Tc17 and Th/Tc22. Although alloreactive Th/Tc1 cells play a critical role in initiating pathogenesis of gut acute graft-versus-host disease (Gut-aGVHD), the pathogenic T cells in steroid-resistant Gut-aGVHD (SR-Gut-aGVHD) remains unclear. Here, we show that in murine models of SR-Gut-aGVHD, the pathogenesis is associated with reduction of IFN-g+ Th/Tc1 and IL-17A+IL-22- Th/Tc17 but expansion of IL-17-IL-22+ Th/Tc22, particularly Tc22 cells. IL-22 from Th/Tc22 cells causes dysbiosis. Using a Gut-aGVHD model induced by alloreactive IFN-g-/- CD8+ T cells, we show that the Gut-aGVHD pathogenesis requires both dysbiosis and depletion of CX3CR1hi mononuclear phagocytes (MNP) that regulate intestinal bacterial translocation. Absence of IFN-g leads to preferential expansion of Tc22 that induce dysbiosis by augmenting RegIIIg production, and depletion of CX3CR1hi MNP via its PD-1 interaction with tissue PD-L1. Interestingly, SR-Gut-aGVHD is also associated with depletion of CX3CR1hi MNP that reduces expansion of Tc22 under steroid treatment. Our studies indicate that expansion of Th/Tc22, dysbiosis, and depletion of CX3CR1hi MNP cells play critical roles in SR-Gut-aGVHD pathogenesis. These results provide new avenue towards studies in patients and call for caution in clinical testing of IL-22 agonists or IFN-g antagonist in patients.

overall_design: Experiments of the relationship between the SR-Gut-aGVHD pathogenesis and the regulation of IFN-γ+ Th/Tc1, IL-17A+IL-22- Th/Tc17 and IL-17-IL22+Th/Tc22 cells.


title: Phenotypic variation within and across transcriptomic cell types in mouse motor cortex

status: Public on Dec 24 2020

city: Solna

first: Leonard

last: Hartmanis

address: Solnavägen 9

department: Cell & Molecular Biology

institute: Karolinska Institutet

summary: Cortical neurons exhibit astounding diversity in gene expression as well as in morphological and electrophysiological properties. Most existing neural taxonomies are based on either transcriptomic or morpho-electric criteria, as it has been technically challenging to study both aspects of neuronal diversity in the same set of cells. Here we used Patch-seq to combine patch-clamp recording, biocytin staining, and single-cell RNA sequencing of over 1300 neurons in adult mouse motor cortex, providing a comprehensive morpho-electric annotation of almost all transcriptomically defined neural cell types. We found that, although broad families of transcriptomic types (Vip, Pvalb, Sst, etc.) had distinct and essentially non-overlapping morpho-electric phenotypes, individual transcriptomic types within the same family were not well-separated in the morpho-electric space. Instead, there was a continuum of variability in morphology and electrophysiology, with neighbouring transcriptomic cell types showing similar morpho-electric features, often without clear boundaries between them. Our results suggest that neural types in the neocortex do not always form discrete entities. Instead, neurons follow a hierarchy consisting of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families.

overall_design: Neurons from all layers of primary mouse motor cortex were profiled transcriptomically, electrophysiologically and morphologically using Patch-seq, using various Cre-driver lines to ensure a diverse sampling of known cell types. Neurons in acute slices were patch-clamped and stimulated with brief current impulses to record their electrophysiological activity, filled with biocytin for subsequent morphological recovery and reconstruction, and their RNA was extracted and sequenced.

first: Rickard

last: Sandberg


title: MITF reprograms the extracellular matrix and focal adhesion in melanoma

status: Public on Dec 22 2020

city: reykjavik

first: Ramile

last: Dilshat

address: Sturlugata 8

department: Biochemistry and Molecular biology

country: Iceland

institute: University of Iceland

summary: To assess the effects of permanent loss of MITF in melanoma cells, we used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technique to generate MITF knockout (KO) cell lines in the human hypo-tetraploid SkMel28 melanoma cell line (containing four copies of MITF). We targeted exon 6 (containing the DNA binding domain) of MITF the resulting isogenic cell line is hereafter referred to as ΔMITF-X6. We also performed siRNA mediated transient knock down of MITF along with control siCTRL in 501Mel cells. Furthermore, we used a stable doxycycline inducible over-expression cell line containing FLAG tagged MITF and a control cells with empty vector FLAG in A375P. From our RNA-sequencing studies we found that MITF plays a critical role as a repressor of extracellular matrix gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.

overall_design: Four replicates RNA-seq data for empty vector control cell line and MITF-X6 (MITF-KO) cells. Triplicate RNA-seq data for siCTRL and siMITF in 501Mel cells. Triplicates RNA-seq data for MITF overexpression and empty vector overexpression cell lines in A375P


title: A phase 1 study of guadecitabine plus cisplatin in platinum refractory germ cell tumors [Methylation]

status: Public on Dec 25 2020

city: Urbana

first: Michael

last: Spinella

state: IL

address: 2001 S Lincoln Ave

department: Comparative Biosciences

country: USA

institute: University of Illinois at Urbana-Champaign

type: Methylation profiling by genome tiling array

summary: Germ cell tumors (GCTs) can be cured with cisplatin-based therapy, although a clinically significant number of patents are refractory and die of progressive disease.  Based on preclinical studies indicating that refractory testicular GCTs are hypersensitive to hypomethylating agents (HMA), we conducted a phase I trial [NCT02429466] of the next-generation HMA guadecitabine (SGI-110) in combination with cisplatin in patients with recurrent, cisplatin-resistant GCTs.  The primary endpoint was safety and toxicity including DLT and MTD.   A total of 14 patients were enrolled.  The majority of patients were heavily pretreated.  MTD was determined to be 30 mg/m2 guadecitabine followed by cisplatin 100 mg/m2. DLTs were neutropenia and thrombocytopenia. Three patients had partial responses by RECIST criteria, two of these patients qualified as complete responses by serum tumor marker criteria with survival of 16 and 26 months.  Three patients also had stable disease for an overall response rate of 23% and clinical benefit rate of 46%. Genome-wide analysis pre- and post-guadecitabine indicated dramatic and widespread global DNA demethylation in cell-free plasma samples including in the promoters of genes of cancer related pathways.  In summary, the combination of guadecitabine followed by cisplatin was tolerable and demonstrated biological activity in patients with platinum refractory GCTs.

overall_design: Tumor Biopsy was obtained from patient  at baseline C1D1 (pretreatment; prior to first 30 mg/m2 quadecitabine dose) and then on C1D8 (posttreatment; after 5 consecutive doses of guadecitabine and just prior to cisplatin). Bisulphite converted DNA from the 2 samples were hybridised to the Infinium MethylationEPIC beadchip array

first: Zeeshan

last: Fazal


title: Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide

city: Shanghai

first: Fei

last: Li

address: 320 Yue-Yang Road Shanghai

institute: Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences


title: Defining the tuberculosis lung landscape during disease and latency using single cell technologies

first: Maxim

middle: N.

last: Artyomov

state: MO

address: 660 S. Euclid Avenue, Campus Box 8118

department: Immunology&Pathology

institute: Washington University in St.Louis

summary: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world’s population. The majority of infected persons are asymptomatic, but latent TB infection (LTBI) can progress to active clinical disease in 5-10% of infected individuals. The immune mechanisms that govern progression from latent to active pulmonary TB (PTB) remain poorly defined. An in-depth understanding of immune factors correlating with TB disease, as well as protection during TB, is necessary for developing new immunotherapies to promote immune control of Mtb. Experimentally Mtb-infected non-human primates (NHP) mirror the disease progression and pathology observed in humans and can recapitulate both PTB and LTBI. In the present study, we have characterized the lung immune landscape in NHPs with LTBI and PTB using high-throughput technologies including single-cell RNA sequencing (scRNA-seq) and Time of flight cytometry (CyTOF). We show that the three defining features of PTB in macaque lungs are the influx of plasmacytoid DCs (pDCs), an Interferon (IFN)-exhibiting alveolar macrophage population and predominant activated T cell responses. These features contribute to uncontrolled inflammation and disease without mediating Mtb control. In contrast, a CD27+ Natural killer (NK) cell subset accumulated in the lungs of LTBI macaques and in circulation in individuals with LTBI, thus providing novel insights into the protective lung landscape that functions during TB latency. A comprehensive understanding of the lung immune landscape as described here will improve our overall understanding of TB disease immunopathogenesis and provide novel targets for design of new therapies and vaccines for TB control.

overall_design: To delineate the lung landscape at a high resolution at the single cell level, we performed 10X scRNA-seq on dematricized single cells that underwent CD45+ enrichment from the lungs of uninfected (control, n=3), PTB (n=5) and LTBI (n=3) macaques.

first: Ekaterina

last: Esaulova

first: Shibali

last: Das

first: Dhiraj

last: Singh

first: Jose

last: Choreño-Parra

first: Amanda

last: Swain

first: Laura

last: Arthur

first: Javier

last: Rangel-Moreno

first: Mushtaq

last: Ahmed

first: Allison

last: Bucsan

first: Chivonne

last: Moodley

first: Smriti

last: Mehra

first: Ethel

last: García-Latorre

first: Joaquin

last: Zuniga

first: Jeffrey

last: Atkinson

first: Deepak

last: Kaushal

middle: N

first: Shabaana

last: Khader


title: Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide [ATAC-Seq]

first: Fei

last: Li

summary: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat due to the lack of effective drugs or vaccines against SARS-CoV-2. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a second second-generation antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry into host cells, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and SARS-CoV-2-infected Ad-ACE2-transduced Tmprss2 knockout (Tmprss2-KO) and wild-type (WT) mice. TMPRSS2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cells, however, such antiviral efficacy of enzalutamide was lacking in human lung cells and organoids. Although Tmprss2 knockout effectively blocked SARS-CoV-2 infection in ACE2-transduced mice, Accordingly, enzalutamide showed no antiviral activity due to the AR-independent TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19 through reducing TMPRSS2 expression in lung cells.

overall_design: ATAC-seq was performed in AR-positive lung cancer cells A549, H2126 and H1437 and prostate cancer cells LNCaP


title: FGF2 effects on the transcriptome of the human dermal fibroblasts

first: Ronghu

last: Ke

address: No.12 Road WU Nu Mu Qi

institute: Fudan University

summary:
The current study was to determine the effects of Fibroblast Growth Factor 2 (FGF2) on the transcriptome of adult human dermal fibroblasts. Transcriptional profiles of adult human dermal fibroblasts grown in culture medium (with FGF2 or not) were compared. Comparison of the transcriptomes will allow to identify significantly differentially expressed genes exposure to FGF2, which in turn will allow for identification of the pathways affected by these factors in the human adult fibroblasts.
Methods: Human dermal fibroblasts were  FGF2 (10ng/ML) treatment for 48h hours. Then, the RNA was extracted for library construction
Results: FGF2-responsive genes were significantly involved in ECM-receptor interaction, PI3K-Akt signaling and Hippo pathway.

overall_design: RNA profiles of FGF2-treated human dermal fibroblasts were generated by sequencing.

first: Baojin

last: Wu

first: Hao

last: He

first: Junhao

last: Hu


title: Identification of transcripts simultaneously bound by Mmi1 and Mei2

status: Public on Dec 21 2020

city: JOUY-EN-JOSAS

first: Alicia

last: Nevers

address: Domaine de Vilvert

department: Micalis

institute: Institut national de la recherche agronomique (Inrae)

summary: We determined the repertoire of transcripts simultaneously bound by Mmi1 and Mei2 through sequential immunoprecipitations followed by high-throughput sequencing of associated RNAs (seqRIP-seq)

overall_design: Total (WCE) and Immunoprecipitated (IP) RNAs from the Pnmt41-TAP-Mmi1 Pnmt1-3xFLAG-Mei2 and Pnmt41-GFP-Mmi1 Pnmt1-3xFLAG-Mei2 (negative control) strains were analyzed by high-throughput sequencing from biological duplicates.

first: Vedrana

last: Andric

first: Ditipriya

last: Hazra

first: Sylvie

last: Auxilien

first: Alexandra

last: Menant

first: Marc

last: Graille

first: Benoit

last: Palancade

first: Mathieu

last: Rougemaille


title: Expression profiling of the wild type, mot2∆, mamRNA∆ and mot2∆ mamRNA∆ strains

summary: Transcriptome analyses of wild type, mot2∆, mamRNA∆ and mot2∆ mamRNA∆ strains. We determined the role of mamRNA in the accumulation of Mmi1-targeted transcripts observed in mot2∆ cells.

overall_design: Total RNAs from wild type (duplicate), mot2∆ (duplicate), mamRNA∆ (duplicate) and mot2∆mamRNA∆ (duplicate) strains were analyzed by high-throughput sequencing


title: RNA-seq analysis of human T CD4+ cells after their interaction with autologous antigen-specific vitamin D3-generated tolerogenic dendritic cells

city: Badalona

first: Eva

last: Martínez-Cáceres

state: Barcelona

address: Campus Can Ruti, Carretera de Can Ruti s/n

department: Immunology

country: Spain

institute: Germans Trias i Pujol University Hospital and Research Institute

summary: The use of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases. Among the different alternatives available, the use of vitamin D3 for the generation of tolDC (vitD3‑tolDC) constitutes one of the most robust approaches due to their immune regulatory properties, which are currently being tested in clinical trials. However, the mechanisms that vitD3‑tolDC are triggering for the induction of tolerance remain elusive. For this reason, we have performed a full transcriptomic characterization of T cells upon their interaction with autologous, antigen-specific vitD3-tolDC. Our results evidenced a strong down-modulation of genes involved in cell cycle and cell response to immune-related stimuli in T CD4+ cells after their interaction with vitD3-tolDC. Consequently, our results show the induction of a strong antigen-specific hyporresponsiveness, which manifests the regulatory properties of these cells and therefore their therapeutic potential in the clinic.

overall_design: Analysis of the antigen-specific effect of vitamin D3-generated tolerogenic dendritic cells over autologous T CD4+ cells compared to mature immunogenic dendritic cells

first: Juan

last: Navarro-Barriuso


title: Acute BAF perturbation causes immediate changes in chromatin accessibility

city: Vienna

first: Stefan

last: Kubicek

address: Lazarettgasse 14

institute: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

summary: Loss-of-function mutations in genes coding for subunits of the large, multifarious BRG1/BRM associated factor (BAF) chromatin remodeling complexes are frequently causative for cancer or developmental diseases. Cells lacking the most frequently mutated subunits, like the ATPase SMARCA4, typically exhibit drastic chromatin accessibility changes, especially of important regulatory regions. However, so far it remains unknown how these changes are established over time, and whether they are causative for intra-complex synthetic lethalities abrogating the formation or activity of BAF complexes. Here, we utilize the dTAG system to induce acute degradation of BAF subunits in wildtype and BAF mutant backgrounds and analyze the resulting chromatin accessibility changes with high kinetic resolution. We observe that chromatin alterations are established faster than the duration of one cell cycle and that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in a near-complete loss of chromatin accessibility at BAF-controlled sites, especially at super-enhancers, providing a mechanism for intra-complex synthetic lethalities.

overall_design:
ATAC-seq, RNA-seq, PRO-seq experiments were performed in duplicates, ChIP-seq once. All experiments contain respective control samples.
ChIP antibody details: H3K27ac (Abcam, catalog# ab4729, lot# 3211741-1), ARID1A (Abcam, catalog# ab182560, lot# GR269670-9), IgG (Cell Signaling, catalog# CS 2729S, lot# 9), BRD4 (Abcam, catalog# ab128874, lot# GR3251918-7).

first: Sandra

last: Schick

first: Sarah

last: Grosche

first: Katharina

last: Kohl

first: Danica

last: Drpic

first: Martin

last: Jaeger

first: Nara

last: Marella

first: Michael

last: Schuster

first: André

middle: F

last: Rendeiro

first: Anna

last: Koren

first: Mark

last: Petronczki

first: Christoph

last: Bock

middle: C

last: Mueller

first: Georg

last: Winter


title: The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

status: Public on Dec 19 2020

city: Seattle

first: Robert

middle: K.

last: Bradley

state: Washington

address: 1100 Fairview Ave N

department: Public Health Sciences/Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary: Pancreatic adenosquamous carcinoma (PASC) is an aggressive cancer whose mutational origins are poorly understood. An early study reported high-frequency somatic mutations affecting UPF1, a core nonsense-mediated mRNA decay (NMD) factor, in PASC, but subsequent studies did not observe these lesions. The corresponding controversy about whether UPF1 mutations are important contributors to PASC has been exacerbated by a paucity of functional studies. Here, we modeled two UPF1 mutations to find no significant effects on pancreatic cancer growth, acquisition of adenosquamous features, UPF1 splicing, UPF1 protein levels, or NMD efficiency. We subsequently discovered that ~40% of UPF1 mutations reportedly present in PASCs are identical to standing genetic variants in the human population, suggesting that they may be non-pathogenic inherited variants rather than pathogenic mutations. Our data suggest that UPF1 is not a common functional driver of PASC and motivate further attempts to identify unique genetic features defining these malignancies.

overall_design: RNA-seq of HEK 293T cell lines carrying mutations in UPF1.

first: Jacob

middle: T

last: Polaski

first: Ram

last: Kannan

middle: K


title: DNA methylome signatures of prenatal exposure to synthetic glucocorticoids in hippocampus and peripheral whole blood of female guinea pigs in early life

status: Public on Dec 18 2020

city: Toronto

first: Stephen

last: Matthews

address: 1 King's College Circle

institute: University of Toronto

type: Methylation profiling by high throughput sequencing

summary: We examined the hypothesis that prenatal synthetic glucocorticoids (sGC) administration alters DNA methylation signatures in Guinea pig offspring hippocampus and whole blood. Guinea pigs were treated with sGC or saline in late gestation. Genome-wide modifications of DNA methylation were analyzed using reduced representation bisulfite sequencing in juvenile female offspring.

overall_design: sGC to saline control comparisons for the hippocampus (HPC: n=6/condition, total of 12) and for whole blood (BL: n=6/condition, total of 12) within the same female offspring

first: A

last: Sasaki

first: S

middle: G


title: SPT6 Promotes Epidermal Differentiation and Blockade of an Intestinal Phenotype through Control of Transcriptional Elongation

city: San Diego

first: George

last: Sen

state: CA

address: 9500 Gilman Drive

institute: UCSD

summary: In adult tissue, stem and progenitor cells must tightly regulate the balance between proliferation and differentiation to sustain homeostasis. It is assumed that the rate-limiting step during the induction of somatic tissue differentiation is the recruitment of RNA polymerase II (Pol II) to promoters. Here we show that ~30% of induced differentiation genes already contain stalled Pol II at the promoters in epidermal stem and progenitor cells which is then released into productive transcription elongation upon differentiation. Central to this process is the histone chaperone, SPT6 which is necessary for the elongation of these differentiation genes. Upon SPT6 depletion there is a loss of human skin differentiation and stratification. Unexpectedly, loss of SPT6 also caused the spontaneous transdifferentiation of epidermal cells into an intestinal phenotype due to the stalled transcription of the master regulator of epidermal fate P63. Our findings suggest that control of transcription elongation through SPT6 plays a prominent role in adult somatic tissue differentiation and the inhibition of alternative cell fate choices.

overall_design: RNA-seq of SPT6 knockdown in regenerated epidermis using organotypic culture. ChIP-seq with SPT6 and RNA Pol II antibodies in differentiated primary keratinocytes, ATAC-seq in differentiated primary keratinocytes.

first: Jingting

last: Li


title: CRISPRi knockdown of essential genes in Pseudomonas aeruginosa

city: Muenster

first: Anne-Sophie

last: Stolle

address: Von-Esmarch-Str. 56

institute: Institut für Infektiologie

summary: To understand molecular pathways responding to loss of essential proteins in the inner- or outer-membrane, we knocked down essential genes in the inner- and outer membrane using a CRISPRi approach in Pseudomonas aeruginosa. CRISPRi is an inducible system with which even essential genes can be targeted by a specifig sg-RNA. In this study, we investigated the transcriptomic changes upon loss of BamA, LptD and FtsH. Loss of BamA and LptD, both outer membrane proteins, resulted in strong upregulation of genes involved in LipidA modification and upregulation of several two component systems. Depletion of FtsH resulted in strong upregulation in genes responsible for amino acid biosynthesis and regulatory genes. Surprisngly, knockdown of bamA and lptD resulted in (mild) upregulation of H1-T6SS genes.

overall_design: RNAseq experiment on three independent biological replicates was performed on sg-bamA, sg-lptD, sg-ftsH and sg-CTRL expressing P.aeruginosa dCas9 strains. RNA was isolated in the exponential growth phase.

first: John

last: Mekalanos


title: Ribosome-bound Get4/5 facilitate the capture of tail anchored proteins by Sgt2 in yeast

city: Göttingen

first: Katherine

last: Bohnsack

address: Humboldtallee 23

institute: University Medical Centre Göttingen

summary: The guided entry of tail-anchored proteins (GET) pathway assists in the proper delivery of tail-anchored (TA) proteins to the ER. Here we uncover how the yeast GET pathway components Get4/5 mediate capture of TA proteins by Sgt2, which interacts with TA sequences and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of TA proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting of proteins containing internal TM domains. Exposure of nascent, internal TM domains at the tunnel exit induces high-affinity ribosome-binding of SRP, which in turn prevents ribosome-binding of Get4/5. In this way, the position of TM domains within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

overall_design: Wild-type BY4741 yeast cells or cells expressing genomically C-terminally His-Tev-ProtA-tagged Get4 were analysed by CRAC (Bohnsack et al., 2012).

first: Ying

last: Zhang

first: Evelina

last: de Laurentiis

first: Mascha

last: Wahlig

first: Namit

last: Ranjan

first: Simon

last: Gruseck

first: Philipp

last: Hackert

first: Tina

last: Wölfle

first: Marina

last: Rodnina

first: Blanche

last: Schwappach

first: Sabine

last: Rospert