title: Gene expression profile in the tethered Nrf1-MafG (T-N1G) heterodimer-expressing small Maf-deficient cells [RNA-seq]

first: Fumiki

last: Katsuoka

overall_design: polyA RNA-seq was performed using RNA from immortalized MafF-/-::MafG-/-::MafK-/- (F0G0K0) MEFs and T-N1G-expressing F0G0K0 MEFs, treated with Vehcle, 0.3 µM or 1.0 µM  Proteasome Inhibitor (MG132)  for 12hr.

first: Akihito

last: Ohtsuki

first: Nozomi

last: Hatanaka

first: Haruna

last: Okuyama

first: Masayuki

last: Yamamoto


title: Systematic analysis of effects of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs [ChIP-seq]

status: Public on Jan 28 2022

city: La Jolla

first: Christopher

middle: K

last: Glass

state: CA

address: 9500 Gilman Drive

department: CMM

country: USA

institute: University of California, San Diego

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 75 TFs in K562 cells as determined by ChIP-sequencing. We found a dominant pattern of a relaxed range of spacing between collaborative factors, including forty-six factors exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. Spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. A remarkable range of tolerance was further established for PU.1 and C/EBPβ, which exhibit relaxed spacing, by introducing synthetic spacing alterations ranging from 5-bp increase to >30-bp decrease using CRISPR/Cas9 mutagenesis. These findings provide implications for understanding mechanisms underlying enhancer selection and for interpretation of non-coding genetic variation.

overall_design: ChIP-seq for wild-type ER-HoxB8 cells

first: Thomas

middle: A

last: Prohaska

first: Marten

last: Hoeksema

first: Nathan

middle: J

last: Spann

first: Mashito

last: Sakai

first: Zeyang

last: Shen


title: Systematic analysis of effects of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification

status: Public on Jan 27 2022

city: Baltimore

first: Patrick

last: Cahan

state: MD

address: 733 North Broadway, MRB 653

department: ICE and Biomedical Engineering

institute: Johns Hopkins University School of Medicine

summary: Elucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. To overcome the limited scope and scalability of experimental methods in mapping this control, computational GRN reconstruction methods have been developed to infer relationships from transcriptomic data. We developed a new computational GRN reconstruction tool called Epoch, which takes advantage of single-cell RNA sequencing to address a number of shortcomings of existing tools including low precision, low sensitivity, and the limited ability to describe network topology changes over time. We used Epoch to identify the dynamic networks and key transcriptional regulators underpinning directed differentiation of mouse embryonic stem cells, and further demonstrate that modulating signaling pathways drives topological network changes that shape cell fate potential. By integrating signaling pathways with GRN structures, we traced how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively.

overall_design: We perform single-cell RNA-sequencing on five time points spanning day 0 through day 4 of mouse embryonic stem cell day 0 through day 4 using the MULTI-seq protocol for sample multiplexing.

first: Emily

middle: Y

last: Su

first: Abby

last: Spangler


title: Immune-related gene expression profiling of Epithelial Ovarian Cancer after Neoadjuvant chemotherapy

city: Madrid

first: Cristian

last: Suarez Cabrera

state: Madrid

address: Av. Complutense 40

country: Spain

institute: CIEMAT

summary: We explore the potential of immune-associated expression profiles in a series of Epithelial Ovarian Cancer (EOC) patients undergoing neoadjuvant chemotherapy (NACT), comparing primary lesions before patients underwent carboplatin/paclitaxel-based NACT (so-called ´biopsy samples´) and interval debulking surgery (IDS) samples from residual lesions after treatment with chemotherapy (so-called ´surgery samples´).

overall_design: nCounter immune profiling was used to carry out an immune-related gene expression analysis in 27 patients with EOC, comparing biopsy and surgery samples. Biopsy samples were collected from primary lesions before EOC patients underwent carboplatin/paclitaxel-based neoadjuvant chemotherapy, according to availability. Surgery samples were extracted from residual lesions after treatment with chemotherapy, according to availability. We analyzed only biopsy samples from 8 patients, only surgery samples from 2 patient and paired biopsy-surgery samples from 17 patients. We also compared responder and non-responder patients using the chemotherapy response score (CRS). This score stratifies patients with no/minimal tumor response (CRS1), partial response (CRS2) and total/near-total tumor response (CRS3) to NACT.

first: Iris

last: Lodewijk

first: Alejandra

last: Bernardini

last: Suárez-Cabrera

first: Elsa

last: Bernal

first: Rodrigo

last: Sánchez

first: Juan

middle: L

last: Garcia

first: Katerin

last: Rojas

first: Lucía

last: Morales

first: Sarah

last: Wang

first: Xinwei

last: Han

first: Marta

last: Dueñas

first: Jesús

middle: M

last: Paramio

first: Luis

last: Manso


title: mRNA-seq of Arabidopsis bz1728, nobiro6, nbr-t1 and taf12-t1 mutants

status: Public on Jan 26 2022

city: Yokohama

first: June-Sik

last: Kim

state: Kanagawa

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

country: Japan

institute: RIKEN CSRS

summary: The NGS-aided mRNA-seq analysis was conducted to survey transcriptome changes by each of following mutations; bz1728, bz1728nobiro6, nbr6-t1 or taf12-t1.

overall_design: Root and shoot tissues separately collected from the 12-d-old seedlings of wildtype (Col-0), bz1728, bzip1728nobiro6, nbr6-t1 or taf12-t1 were subjected to the NGS-aided transcriptome analysis with quadruple replicates.

first: Kazuo

last: Shinozaki

first: Taiji

last: Kawakatsu


title: Single-cell chimerism and transcriptional profiling identify long-term human skin-resident host T cells after stem cell transplantation

city: Freising

first: Gustavo

middle: P.

last: de Almeida

state: Bavaria

address: Liesel-Beckmann-Str. 1

department: Division of Animal Physiology and Immunology

country: Germany

institute: Technical University of Munich

summary: Tissue-resident memory T cells (TRM) have recently emerged as crucial cellular players for host defense in a wide variety of tissues and barrier sites. Insights into the maintenance and regulatory checkpoints of human TRM cells remain scarce, especially due to the difficulties associated with tracking T cells over time and spatially in humans. We therefore aimed to identify and characterize skin resident T cells in humans defined by their long-term in situ lodgement. Allogeneic hematopoietic stem cell transplantation paired with myeloablative chemotherapy unmasked long-term sequestration of host T cell subsets in the human skin despite complete donor T cell chimerism in the blood. Single-cell chimerism analysis paired with single-cell transcriptional profiling comprehensively characterized these bona fide long-term skin resident T cells and revealed differential tissue maintenance for distinct T cell subsets, specific TRM cell markers such as galectin-3, but also tissue exit potential with retention of the transcriptomic TRM cell identity. Analysis of 26 patients revealed profound interindividual variation in the tissue maintenance of host skin T cells and excluded a role for host T cells for the pathogenesis of chronic GvHD. Our data exemplify the power of exploiting a clinical situation as a proof-of-concept for the existence of bona fide human skin TRM cells and reveal long-term persistence of host immunity in the peripheral tissue but not in the circulation or bone marrow for a subset of patients.

overall_design:
Comparison of skin versus blood CD4 and CD8 T cell bulk transcriptomes and lymphocytes from blood versus skin of a healthy donor and a HSCT patient

**Samples GSM5776288-GSM5776291 do not have raw data due to patient privacy concerns**

middle: P

first: Sascha

last: Schäuble

first: Christina

last: Zielinski


title: In human CD16+ monocytes NK1R signaling upregulates inflammatory pathways and negatively regulates apoptosis

status: Public on Jan 30 2022

city: Philadelphia

first: Steven

middle: D

last: Douglas

state: PA

address: 3615 Civic Center Blvd

department: Pediatrics

institute: The Children's Hospital of Philadelphia

summary: The neurokinin-1 receptor (NK1R) pathway has immunomodulatory properties and is implicated in the pathophysiology of neurodegenerative, infectious and inflammatory diseases. Monocytes are important cells in inflammation and they consist of subsets with distinct phenotypes and biological functions. NK1R signaling is elevated in conditions that alter the balance of monocyte subsets, such as in HIV infection. In order to understand the pro-inflammatory mechanism of NK1R signaling in monocyte subsets, a transcriptome study was performed using RNA sequencing. Human primary peripheral monocytes were treated with the NK1R cognate ligand, substance P (SP), prior to being sorted into CD16- and CD16+ subsets. SP treatment led to the differential expression of 38 genes in CD16- monocytes and 12 genes in CD16+ monocytes. The canonical NF-κB pathway was activated in both subsets, as indicated by the upregulation of transcripts including IL1α, IL1β, IL6, CCL3 and CCL4. In the CD16+ monocytes, NK1R signaling led to priming of the NLRP3 inflammasome and upregulated transcripts including NLRP3 and CARD16. Alternative splicing analysis showed that NK1R signaling resulted in differential exon usage of CASP1 and CARD16 in CD16+ monocytes. SP treatment altered the properties of both CD16- and CD16+ monocytes, leading to an inflammatory response with activation of the canonical NF-κB pathway in both monocyte subsets, and priming of the NLRP3 inflammasome in the CD16+ subset.

overall_design:
mRNA transcript analysis of primary human CD16- and CD16+ monocyte subsets under no treatment conditions (3 replicates), SP treatment  (2 replicates) or treatment with vehicle control (2 replicates) using the Illumina HiSeq 4000 Sequencing System

mRNA transcript analysis of primary human CD16- and CD16+ monocyte subsets under SP treatment  (6 replicates) or treatment with vehicle control (6 replicates) using the Illumina NovaSeq 6000 Sequencing System

first: Vasiliki

last: Pappa

first: Sergei

last: Spitsin

first: Peter

last: Gaskill


title: RNA-sequencing of murine wild-type and Cdx-mutant E9.5 yolk sacs

status: Public on Feb 01 2022

city: Ottawa

first: Tanya

middle: Elizabeth

last: Foley

state: ON

address: 451 Smyth Road

department: Cellular and Molecular Medicine

country: Canada

institute: University of Ottawa

summary: E9.5 yolk sacs were collected from wild type (CD1) and Cdx-mutant (DKO) embryos and processed for RNA-sequencing to identify Cdx-dependent changes in gene expression

overall_design: mRNA profiles of E9.5 wild type (WT) and tamoxifen-treated Cdx1-/-Cdx2fl/fl Actin::CreERT (DKO) yolk sacs

middle: E

first: David

last: Lohnes


title: Developmental single-cell transcriptomics of hypothalamic POMC progenitors reveal the genetic trajectories of multiple neuropeptidergic phenotypes

city: Ann Arbor

first: Malcolm

last: Low

state: MI

address: 1000 wall street

department: Molecular & Integrative Physiology

institute: University of Michigan

summary: The purpose of this study is to characterize the ontogenetic transcriptional programs that specify the identity and functioning of POMC neurons during early postnatal stage and adulthood. We performed Translating Ribosome Affinity Purification followed by RNA sequencing technology (Trap-seq) using Pomc-CreERT;Rosa26-eGFPL10a transgenic mice where POMC neurons are labeled with eGFPL10a tag with tamoxifen administration. GFP-conjugated beads were used to pull-down POMC neurons from hypothalamic extracts at early postnatal days (P12) and adulthood(P60).  Both the pull-down mRNA samples and the resultant supernatant mRNA samples were subjected to RNA sequencing process. Gene enrichment analysis identified 1143 and 1047 genes are highly enriched (P<0.05) in Pomc- eGFPL10a at P12 and P60 Trap-seq materials respectively, from which 653 genes are overlapped expressed in both datasets. Gene ontology analysis showed that these common expressed genes are associated with the establishment and maintenance of neuron structure and basic neuronal functions. The ontology annotations on the uniquely expressed gene from P12 and P60 are dramatically different from each other. The functions of P12 uniquely enriched genes emphasizing on the basic cellular metabolism, cellular modeling and biochemical changes, whereas, the P60 enriched genes are related to the maturation of POMC neurons such neurotransmitter development and POMC specific function establishment including the response to nutrient, and the construction of feeding behavioral circuit.

overall_design: Trap-seq on hypothalami from Pomc-CreERT;Rosa26-eGFPL10a transgenic mice at P12 and P60. Three replicates for each pull-down and superanant samples.

first: Hui

last: Yu

first: Marcelo

last: Rubinstein


title: Store Independent Ca2+ Entry Regulates the DNA Damage Response Pathway in Breast Cancer Cells

first: Rajini

last: Rao

address: 725 N Wolfe St, WBSB 201

department: Physiology

institute: The Johns Hopkins University School of Medicine

summary:
The mainstay of treatment for hormone responsive breast tumors is chemotherapy, followed by targeted endocrine therapy. The vast majority (80%) of estrogen receptor positive tumors also express wild type p53 protein that is the main determinant of the DNA damage response. Tumors that are ER+ and p53WT respond poorly to chemotherapy, although the underlying mechanisms are not completely understood. We describe a novel link between store independent Ca2+ entry (SICE) and resistance to DNA damaging drugs, mediated by the secretory pathway Ca2+-ATPase, SPCA2. In luminal ER+/PR+ breast cancer subtypes, SPCA2 levels are high and correlate with poor survival prognosis. Independent of ion pump activity, SPCA2 elevates baseline Ca2+ levels through SICE and drives cell proliferation. Attenuation of SPCA2 led to increased mitochondrial ROS production, DNA damage and activation of the ATM/ATR-p53 axis leading to G0/G1 phase cell cycle arrest and apoptosis. Resistance to DNA damaging agents including doxorubicin, carboplatin, and ionizing radiation could be reversed by downregulation of SPCA2 expression using curcumin. In conclusion, elevated SPCA2 drives pro-survival and chemotherapy resistance in ER+ p53WT breast tumors by suppressing the DNA damage response. Attenuation of SPCA2 expression by curcumin may have therapeutic potential in treating receptor positive breast cancer.
The goal of this study is to investigate store Independent Ca2+ entry regulation of the DNA Damage Response pathway in breast cancer cells

overall_design: MCF-7 cells were treated with either control shRNA or shRNA constructs targeting ATP2C2 to determine the effect of SPCA2 knockdown on the MCF-7 transcriptome. RNA was extracted from two biological replicates per sample class and used as the input on an Affymetrix microarray to determine changes in gene expression; thus the comparison was two biological replicates each for experimental versus control.

first: Josè

last: Llongueras

first: Donna

last: Dang

first: Monish

middle: R

last: Makena

first: Allatah

middle: X

last: Mekile

first: C

middle: C

last: Talbot Jr


title: The developmental impacts of natural selection on human pelvic morphology [ATAC-seq]

city: Cambridge

first: Mariel

last: Young

state: MA

address: 11 Divinity Avenue

department: Human Evolutionary Biology

institute: Harvard University

summary: We investigate the genetic architecture of pelvis development at the regulatory and transcriptomic level.

overall_design: Examination of open chromatin regions (ATAC-seq) and differentially expressed genes (RNA-seq) in the developing pelvis.

first: Terence

last: Capellini

first: Daniel

last: Richard

first: Pushpanathan

last: Muthuirulan


title: Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration

city: New York

first: Jingli

last: Cao

state: NY

department: Cell & Developmental Biology

institute: Weill Cornell Medical College

summary: The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in the case of zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with 5 of 6 elements directing injury-induced epicardial expression but not ontogenetic expression in hearts of larval animals. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration.

overall_design: ATAC-sequencing and RNA-sequencing of isolated epicardial cells from uninjured zebrafish hearts and hearts after amputation injury

first: Yingi

first: Jianhong

last: Ou

first: Lingyun

last: Song

first: Alexias

last: Safi

first: Gregory

last: Crawford

first: Kenneth

last: Poss


title: Systematic analysis of effects of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs [ATAC-seq]

first: Christopher

last: Glass

overall_design: ATAC-seq for wild-type ER-HoxB8 cells

first: Thomas

last: Prohaska

first: Marten

last: Hoeksema

first: Nathan

last: Spann

first: Mashito

last: Sakai

first: Zeyang

last: Shen


title: Systematic analysis of effects of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs [RNA-seq]

overall_design: RNA-seq for wild-type ER-HoxB8 cells


title: Multimodal profiling of the transcriptional regulatory landscape of the developing mouse cortex identifies Neurog2 as a key epigenome remodeler

city: Neuherberg

first: Boyan

last: Bonev

state: Deutschland

address: Ingolstädter Landstr. 1

institute: Helmholtz Zentrum München

type: Methylation profiling by high throughput sequencing

summary: How multiple epigenetic layers and transcription factors (TFs) interact to facilitate brain development is largely unknown. Here, we combined single-cell RNA-seq and single-cell ATAC-seq with cell-type-specific measurements of enhancer activity, DNA methylation and three-dimensional genome architecture to systematically map the regulatory landscape of neural differentiation in the mouse neocortex in vivo. This allowed us to identify thousands of new enhancers, their predicted target genes and the temporal relationships between enhancer activation, epigenome remodeling and gene expression. We characterize specific neuronal transcription factors associated with extensive and frequently coordinated changes across multiple epigenetic modalities. In addition, we functionally demonstrate a new role for Neurog2 in directly mediating enhancer activity, DNA demethylation, increasing chromatin accessibility and facilitating chromatin looping in vivo. Our work provides a global view of the gene regulatory logic of lineage specification in the cerebral cortex.

sample_organism:
Escherichia coli
Mus musculus

platform_organism:
Escherichia coli
Mus musculus

overall_design: scRNA-seq and scATAC-seq of the somsatosensory area of the E14.5 mouse cortex. Additionally, massively parallel reporter assay (MPRA) and MethylHiC from isolated cell populations, namely neuronal stem cells (NSC), intermediate progenitors (IPC),  projection neurons (PN), obtained from the somatosensory area of the E14.5 mouse cortex. Functional validation of Neurog2 using Hi-C, RNA-seq and NOME-seq in GFP and Neurog2 in vivo overexpression via in utero electroporation.

first: Florian

last: Noack

first: Silvia

last: Vangelisti

first: Gerald

last: Raffl

first: Madalena

last: Carido

first: Jeisimhan

last: Diwakar

first: Faye

last: Chong


title: Mapping the m1A, m5C, m6A and m7G Methylation Atlas in Zebrafish Brains under Hypoxic Conditions by MeRIP-seq

city: Zhan Jiang

first: Xiaoyu

last: Li

state: Guangdong

address: 57 People's Avenue

country: China

institute: Affiliated Hospital of Guangdong Medical University

summary: The epigenetic modifications play important regulatory roles in tissue development, maintenance of physiological functions and pathological process. RNA methylations, including newly identified m1A, m5C, m6A and m7G, are important epigenetic modifications. However, how these modifications are distributed in the transcriptome of vertebrate brains and whether their abundance is altered under pathological conditions are still poorly understood. In this study, we chose the model animal of zebrafish to conduct a systematic study to investigate the mRNA methylation atlas in the brain. By performing unbiased analyses of the m1A, m5C, m6A and m7G methylation of mRNA, we found that within the whole brain transcriptome, with the increase of the gene expression levels, the overall level of each of these four modifications on the related genes was also progressively increased. Further bioinformatics analysis indicated that the zebrafish brain has an abundance of m1A modifications. In the hypoxia-treated zebrafish brains, the proportion of m1A is decreased, affecting the RNA splicing and zebrafish endogenous retroviruses. Our study presents the first comprehensive atlas of m1A, m5C, m6A and m7G in the epitranscriptome of the zebrafish brain and reveals the distribution of these modifications in mRNA under hypoxic conditions. These data provide an invaluable resource for further research on the involvement of m1A, m5C, m6A and m7G in the regulation of miRNA and repeat elements in vertebrates, and provide new thoughts to study the brain hypoxic injury on the aspect of epitranscriptome.

overall_design: For each normoxia control and hypoxia experimental group, 10 adult male zebrafish (3-4 month old) were chosen for the analyses. Three repeats of each experiment were performed and totally 30 fishes per group were collected. The brain tissues (around 0.02 g per brain) were then stored for further analysis in liquid nitrogen. For each analysis (m1A, m5C, m6A, m7G and RNA-Seq), the normoxia group and hypoxic group each consisted of 30 mixed brain tissues were analyzed parallelly by RNA-seq and MeRIP-Seq.


title: Expression data of osteosarcoma 143B and metastatic variants

city: Akita City

first: Sei

last: Kuriyama

department: Molecular Medicine & Biochemistry

institute: Akita University

summary:
To determine the key factor to select specifc organ to be metastasized, 143B+mCherryIRES-luciferase2 cells were injected into heart, and the metastasized cells were obtained from the kidney or lymph node.
We repeated the intracardiac injections, the selection using puromycin, and finally the cells metastasized into kidney within 3 weeks.

overall_design: RNA was extracted from normal cell culture lysates to compare the stable expressions of 143B, 143Bkid(renal metastasis), and 143B-lymph(lymph node specific)


title: Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites

status: Public on Jan 21 2022

first: Gregory

last: Donahue

address: 3400 Civic Center Blvd, Bldg 421

institute: The University of Pennsylvania


title: Calcium signaling is impaired in PTEN-deficient T cell acute lymphoblastic leukemia

status: Public on Jan 25 2022

city: Marseille cedex 09

first: Saran

last: Pankaew

address: Parc Scientifique et Technologique de Luminy

country: France

institute: Center of Immunology Marseille Luminy

summary: PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ+ T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Ptendel blasts were unable to release calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol.

overall_design: A comparative single cell RNA-seq study of PTEN-proficient versus PTEN-deficient murine T lymphocytes.

last: Pankeaw

first: Delphine

last: Potier

first: Clémence

last: Grosjean

first: Mathis

last: Nozais

first: Julie

last: Quessada

first: Marie

last: Loosveld

first: Élisabeth

last: Remy

first: Dominique

last: Payet-Bornet


title: Gene expression profile of modified MSCs

city: Cape Town

first: Victoria

last: Damerell

state: Western Cape

address: Anzio Road

department: Human Biology

country: South Africa

institute: University of Cape Town

summary: Analysis of gene expression changes in MSCs in response to TBX3 overexpression

overall_design: Expression microarrays (Affymetrix Clariom S Pico Array Human) are performed to investigate the effect of TBX3 overexpression in MSCs.

first: Melvin

last: Ambele

first: Shanel

last: Salisbury

first: Alexis

last: Neumann-Mufweba

first: Chrisna

last: Durandt

first: Michael

middle: S

last: Pepper

first: Sharon

last: Prince


title: Testing conditions for ribosome profiling in Drosophila melanogaster

city: Leeds

last: Aspden

address: Woodhouse

department: FBS

country: United Kingdom

institute: University of Leeds

summary: Comparison of experimental conditions for ribosome profiling.

overall_design: Ribo-seq of Drosophila melanogaster S2 cells from 4 different conditions: untreated or treated with cycloheximide, each undergoing two different RNase I footprinting conditions.

first: Michaela

last: Agapiou

first: Isabel

last: Birds


title: SETDB1/NSD dependent H3K9me3/H3K36me3 dual heterochromatin maintains gene expression profiles by bookmarking poised enhancers

city: Montpellier

first: Amandine

last: BARRAL

state: Hérault

address: 141 rue de la cardonille

institute: IGH - CNRS

summary: Gene silencing by heterochromatin plays crucial roles in cell identity and development. However, the function of heterochromatin components is not fully understood. Here, we characterize the localization, the biogenesis and the function of an atypical heterochromatin, which is simultaneously enriched in the typical heterochromatin mark H3K9me3 as well as in H3K36me3, histone mark usually associated with gene expression. This dual heterochromatin forms on 3,015 regions in the genome of mouse embryonic stem cells and relies on the histone methyltransferases SET Domain Bifurcated 1 (SETDB1) and Nuclear Set Domain containing proteins (NSD) to generate H3K9me3 and H3K36me3, respectively. Upon SETDB1 removal, dual domains loose both trimethyl marks, gain epigenomic signatures of active enhancers, and come into long range contact with upregulated genes, suggesting that it is a major pathway by which gene expression is controlled by heterochromatin. In differentiated tissues, a large subset of these dual domains is destabilized and become enriched in active enhancer marks, providing a mechanistic insight into the involvement of heterochromatin in development and the maintenance of cell identity.

overall_design: Characterization of H3K9me3/H3K36me3 dual heterochromatin in mouse embryonic stem cells using ChIP-seq, CIDOP-seq, RNA-seq, ATAC-seq and HiC methods

last: Barral

first: Gabrielle

last: Pozo

first: Lucas

last: Ducrot

first: Giorgio

last: Papadopoulos

first: Sandrine

last: Sauzet

first: Andrew

last: Oldfield

first: Giacomo

last: Cavalli

first: Jérôme

last: Déjardin


title: The 2´-O-methylation landscape of ribosomal RNA in Dictyostelium discoideum

status: Public on Jan 24 2022

city: Copenhagen

first: Nicolai

last: Krogh

address: Blegdamsvej 3B

department: Department of Cellular and Molecular Medicine

country: Denmark

institute: University of Copenhagen

summary: A sequencing-based profiling method (RiboMeth-seq) for ribose methylations was applied to study methylation patterns in Dictyostelium discoideum

overall_design: Cell cultures were analysed in triplicates

first: Jan

last: Diesend

first: Ulf

last: Birkedal

first: Jonas

last: Kjellin

first: Jingwen

last: Zhang

first: Kim

last: Jablonski

first: Fredrik

last: Söderbom

first: Henrik

last: Nielsen

first: Christian

last: Hammann


title: Molecular pathogenesis of several neurodevelopmental disorders converges on MeCP2-TCF20 complex interactions

city: Houston

first: Jian

last: Zhou

address: 1250 Moursund St

institute: BCM

summary: Neurodevelopmental disorders (NDDs) are genetically heterogeneous, yet often share certain features such as intellectual disability or motor incoordination. MeCP2 dysfunction is associated with MECP2 duplication syndrome and Rett syndrome (RTT), the phenotypes of which overlap with other NDDs, yet the precise molecular pathogenesis remains unclear. Using proximity-dependent biotinylation (BioID), we identified a novel TCF20 complex that interacts with MeCP2 through its subunit PHF14. TCF20 regulates the expression of key neuronal genes, many of which are co-regulated by MeCP2. Reducing Tcf20 partially rescued the behavioral deficits caused by MECP2 overexpression, underscoring a functional relationship between MeCP2 and TCF20 in NDD pathogenesis. We identified a patient with a missense mutation in PHF14 that abolishes the MeCP2-PHF14-TCF20 interaction who exhibits neurological features seen in RTT. These data demonstrate the critical role of MeCP2-TCF20 complex interaction for brain function.

overall_design: RNA-seq was performed on whole-cell RNA from prefrontal cortex tissue from 3 Tcf20+/- and 3 WT mice, 3 Phf14+/- and 3 WT mice, 5 MeCP2 KO and 5 WT mice.

first: Yingyao

last: Shao

first: Hari Krishna

last: Yalamanchili

first: Zhandong

last: Liu

first: Huda

last: Zoghbi


title: Attenuation of SARS-CoV-2 Infection by Losartan in Human Kidney Organoids

status: Public on Jan 23 2022

city: Calgary

first: Jeff

last: Biernaskie

state: Alberta

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

institute: University of Calgary

summary: COVID-19 associated acute kidney injury (COVID-AKI) is a common complication of SARS-CoV-2 infection in hospitalized patients. It is unclear how susceptible human kidneys are to direct SARS-CoV-2 infection and whether pharmacologic manipulation of the renin-angiotensin II signaling (RAS) pathway modulates this susceptibility. Using induced pluripotent stem cell derived kidney organoids, SARS-CoV-1, SARS-CoV-2 and MERS-CoV tropism, defined by the paired expression of a host receptor (ACE2, NRP1 or DPP4) and protease (TMPRSS2, TMPRSS4, FURIN, CTSB or CTSL), was identified primarily amongst proximal tubule cells. Losartan, an angiotensin II receptor blocker being tested in COVID-19 patients, inhibited angiotensin II mediated internalization of ACE2, upregulated interferon stimulated genes (IFITM1 and BST2) known to restrict viral entry, and attenuated the infection of proximal tubule cells by SARS-CoV-2. Our work highlights the susceptibility of proximal tubule cells to SARS-CoV-2 and reveals a putative protective role for RAS inhibitors during SARS-CoV-2 infection.

overall_design:
Cell Lines:
Human iPSC lines were purchased from Gibco (human episomal iPSC line, Cat. #A18944-derived from neonate female umbilical cord blood) or were derived from cryopreserved human dermal fibroblast (Cell Applications, Inc. Cat. #106-05n, lot #1481) using CytoTune™-iPS 2.0 Sendai reprogramming kit (Life Technologies Cat. #A16517) at the Harvard Stem Cell Institute iPSC Core Facility, as a gift from Dr. Martin Pollak at Harvard Medical School. iPSCs were confirmed to be of normal karyotype and maintained at 37 °C with 5% CO2 with daily medium changes of mTeSR1 medium in 6 well plates coated with Matrigel® (Corning, Cat. #354277). iPSC colonies were passaged using GCDR (Stem Cell Technologies, Cat. #05275) and transferred to T25 flasks coated with Matrigel® prior to differentiation. iPSCs were routinely tested and confirmed negative for mycoplasma. Vero E6 cells (ATCC, Cat. #C1008) were maintained in a humidified 37°C incubator with 5% CO2 and cultured in DMEM (Thermo Fisher) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (Thermo Fisher, Cat. #10378-016; herein referred to as 1% PSQ) and 10% heat-inactivated FBS (Thermo Fisher; herein referred to as complete DMEM).

Generation of kidney organoids:
Kidney organoids were differentiated using the Takasato protocol with minor modifications51 that included APEL2 medium (Stem Cell Technologies, Cat. #05275) supplemented with 5% PFHM-II (Thermo Fisher, Cat. #12040077) (APEL2 + PFHM) in replacement of APEL. Induced PSCs were grown on a feeder-free system using Matrigel matrix coated plates. Cells were treated with 8 µM CHIR99021 (R&D Systems, Cat. #4423) for 4 days followed by recombinant human FGF-9 (200 ng/mL) and heparin (1 μg/mL) for an additional 3 days. At day 7, cells were dissociated into single cell suspension using trypsin-EDTA (0.05%) for 2 min and 250,000 or 500,000 cells were centrifuged to make a pellet at 400 x g for 2 min. The pellet was transferred onto a six-well transwell plate (Corning, Cat. # 07-200-170) with four or nine pellets per well, respectively. Pellets were incubated with a pulse of 5 µM CHIR99021 in APEL2 + PFHM medium for 1 hr at 37 °C. After 1 hr, the medium was changed to APEL2 + PFHM supplemented with FGF-9 (200 ng/mL, R&D Systems, Cat. #273-F9-025) and heparin (1 μg/mL, Millipore Sigma, Cat. #4784) for an additional 5 days, and then were maintained in APEL2 + PFHM medium for 14 days with medium change every other day.

Treatment with RAAS modulators:
Kidney organoids on day 28 or hTEC at passage 5 were pretreated with or without losartan (100 µM, Tocris, Cat. #3798) for 30 min prior to Ang II (1 µM, Millipore Sigma, Cat. #A9525) treatment for 24 hr. DMSO was used as a vehicle control.

scRNA-Seq Library Construction:
Single (n=1) technical replicates of each treatment were kidney organoid group derived from the 1481 iPSC line were prepared for sequencing. Organoids were dissociated to single cells using TrypLE Select (Thermo Fisher Scientific) at 37°C with mechanical trituration. Single cell suspensions were visually inspected under a microscope, counted using an automated hand-held cell counter (Millipore Scepter™ 2.0 Cell Counter), and resuspended at a concentration of 15,000 single-cells in 50 microliters of FACS buffer (0.1% BSA–PBS). All four samples were processed according to 10X Genomics ChromiumTM Next GEM Single Cell 3' Reagent Kits v3.1 as per manufacturer’s protocol. Briefly, single cells were partitioned together with barcoded Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent D1000 ScreenTape System. Libraries were pooled and first sequenced on NovaSeq 100 (plus 25 additional) Cycle SP dual lane flow cell to approximate the number of recovered cells in each sample. We recovered 4,235 cells in Control, 5,702 cells in losartan-alone, 5,800 cells in angiotensin II alone, and 5,855 cells in angiotensin II plus losartan treated groups with an estimated doublet rate of ≈4%. Based on these, we determined proportions for Illumina NovaSeq 100 (plus 25 additional) Cycle S2 dual lane flow cell with a targeted sequencing depth of ~30,000 reads per cell. Illumina sequencer's BCL files were demultiplexed using cellranger mkfastq (a wrapper around Illumina's bcl2fastq). Downstream barcode processing, alignment to pre-built GRCh38, and single-cell 3′ gene counting were performed using CellRanger software version 3.0.1 (10X Genomics). All four libraries were aggregated using cellranger aggr (with mapped normalization). The resulting filtered feature-barcode matrix was imported into Seurat v3 for quality control, dimensionality reduction, cell clustering, and differential expression analysis using default or recommended parameters.

first: Waleed

last: Rahmani

first: Hyunjae

last: Chung

first: Sarthak

last: Sinha

first: Maxwell

last: Bui-Marinos

first: Rohit

last: Arora

first: Arzina

last: Jaffer

first: Jennifer

last: Corcoran

first: Justin

last: Chun


title: Estradiol-dependent gene expression profile in the amygdala of ovariectomized SHRs

city: Tokyo

first: Sabine

last: Gouraud

address: 3-10-2  Osawa, Mitaka

institute: International Christian University

summary: Pre-menopausal women exhibit a lower arterial pressure (AP), a lower sympathetic outflow and a greater baroreceptor reflex than age-matched men. However, AP and subsequent cardiovascular diseases dramatically increases in post-menopausal women. Estrogen level is believed to play a role in the regulation of the basal blood pressure, emotion and hormonal response in the different periods of life of a woman but the molecular mechanisms are still not well understood. Amygdala is a major nucleus of the limbic system involved in the control of cardiovascular, behavior and hormonal response to stress or fear. Since variations in estrogen level are associated with cardiovascular, behavior, and hormonal response changes, we hypothesized that it could also be associated with different neuronal functions in amygdala, characterized by a differential gene expression profile. Young Female SHRs were ovariectomized (OV) and half of them received an estradiol treatment (OV+E2) for one month. We investigated whether the amygdala of ovariectomized rats exhibit gene expression differences after estradiol treatment by using microarray technique.

overall_design: Two groups of female adult SHRs (4-weeks-old) were formed : ovariectomized rats (OV, n=4) and ovariectomized rats treated with estradiol (OV+E2, n=5) (Alzet minipump, 2 ug/day/rat) for one month. Rats were sacrificed by decapitation, brains were extracted, amygdala (right and left part) was dissected under microscope. Total RNAs were extracted from the amygadala and 200ng total RNAs of each rats were processed in the microarray experiment.

first: Linh

middle: T

last: Pham

first: Ko

last: Yamanaka

first: Makiko

last: Onishi

first: Yasunori

last: Miyamoto

first: Hidefumi

last: Waki


title: Diverse MicroRNAs-mRNA networks regulate the priming phase of mouse liver regeneration and of direct hyperplasia

city: Monserrato

first: Rajesh

last: Pal

state: Cagliari

address: Cittadella Universitaria, SP. 8 Monserrato

department: Department of Biomedical Sciences

country: Italy

institute: University of Cagliari

summary: Adult hepatocytes are quiescent cells, that can be induced to proliferate in response to a reduction in liver mass (liver regeneration) or by agents endowed with mitogenic potency (primary hyperplasia). The latter condition is characterized by a more rapid entry of hepatocytes into the cell cycle but the mechanisms responsible for the accelerated entry into the S phase are unknown. Illumina microarray was used to profile mRNA expression in CD-1 mice livers 1, 3 and 6 hours after 2/3 partial hepatectomy (PH) or a single dose of TCPOBOP, a ligand of the constitutive androstane receptor. Ingenuity pathway and DAVID analyses were performed to identify deregulated pathways

overall_design:
Illumina microarray were used to profile mRNA expression in CD-1 mice livers 1, 3 and 6 hours after 2/3 partial hepatectomy (PH) or a single dose of TCPOBOP, a ligand of the constitutive androstane receptor.
21 samples in total (TCPOBOP + PH). 3 replicates at each time point: CO, 1,3, and 6 hour

first: Marta

last: Kowalik

first: Marina

last: Serra

first: Cristina

last: Migliore

first: Silvia

last: Giordano

first: Amedeo

last: Columbano

first: Andrea

last: Perra


title: Real-time quantitative PCR analysis of murine kidney epithelial cell line (MCT) cells

city: Bern

last: Karolin

address: Freiburgstrasse 15

country: Switzerland

institute: University of Bern

summary: We could show that nephrotoxic genes show increased expression once MCT cells are treated with Cyclosporine A and p38 kinase inhibitor (SB203580) but not with a NFAT inhibitor (11R VIVIT).

overall_design: Nephrotoxic genes (Microarray: PAMM-094Z); same amount of total RNA was used for cDNA, and thereafter for qRTPCR.

first: Geneviève

last: Escher

first: Stefan

last: Rudloff

first: Daniel

last: Sidler


title: Computational identification of clonal cells in single-cell CRISPR screens

city: Dallas

first: Yihan

last: Wang

state: Texas

address: 5323 Harry Hines Blvd.

institute: UT Southwestern

summary: Single-cell CRISPR screens are powerful tools to understand genome function by linking genetic perturbations to transcriptome-wide phenotypes. However, since few cells can be affordably sequenced in these screens, biased sampling of cells could affect data interpretation. One potential source of biased sampling is clonal cell expansion. Here, we present a computational pipeline for clonal cell identification in single cell screens using multiplexed sgRNA barcodes. We find that the cells in each clone share transcriptional similarities and we infer the segmental copy number changes in clonal cells. These analyses suggest that clones are genetically distinct. Finally, we show that the transcriptional similarities of clonally expanded cells contribute to false positives in single-cell CRISPR screens. As a result, experimental conditions that reduce clonal expansion or computational filtering of clonal cells will improve the reliability of single-cell CRISPR screens.

overall_design: We performed single-cell CRISPR screen on 2045 enhancers in MDA-MB-231 cells. We used 10X genomics 3’ V3 kit and Cell Hashing staining to prepare the single-cell transcriptome libraries. The sgRNA is expressed by using the CROP-seq design, and a sgRNA enrichment library was also prepared for each single-cell library. In total, we performed six 10X runs, with a total number of 50000 singlets. For the processed data, we uploaded the gene expression matrix (HDF5 format) from 10X cellranger pipeline in together with the sgRNA information and Cell Hashing information of each cell.

first: Gary

last: Hon


title: Hyaluronidase-1 impairs endothelial barrier in a subtype of age-related macular degeneration

status: Public on Jan 20 2022

city: Singapore

first: Christine

last: Cheung

state: Singapore

address: 59 Nanyang Drive

country: Singapore

institute: Lee Kong Chian School of Medicine

summary: Polypoidal choroidal vasculopathy (PCV) is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Here, we cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls. When BOECs were exposed to heterogeneous flow, single-cell analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of endothelial glycocalyx. Hyaluronidase-1 was correspondingly detected in PCV patient vitreous humor and plasma samples. Notably, knockdown of hyaluronidase-1 in PCV BOECs modulated their abnormal cell migration and barrier permeability due to glycocalyx impairment. Therefore, hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

overall_design: scRNA-seq of patient/donor-derived BOECs subjected to orbital flow

first: Kan

last: Wu


title: Epigenetic state determines inflammatory sensing in neuroblastoma

city: Philadelphia

first: Priyankara

last: Wickramasinghe

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

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

overall_design: Refer to individual Series


title: Multi-omic analysis reveals divergent molecular events in scarring and regenerative wound healing

city: Stanford

first: Michael

last: Januszyk

state: CA

address: 257 Campus Dr

department: Surgery

institute: Stanford University

summary: Regeneration is the “holy grail” of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward either scarring or regenerative fates. We profile scarring versus YAP inhibition-induced wound regeneration at the transcriptional (single-cell RNA-sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell surface barcoding, we integrate these data to reveal fibrotic and regenerative “molecular trajectories” of healing. We show that disrupting YAP mechanical signaling yields regenerative repair orchestrated by fibroblasts with activated Trps1 and Wnt signaling. Finally, by performing in vivo gene knockdown and overexpression in wounds, we identify Trps1 as a key regulatory gene that is necessary and partially sufficient for wound regeneration. Our findings serve as a multimodal map of wound regeneration and could have therapeutic implications for pathologic fibroses.

overall_design: Mouse dorsal excisional wound tissue transcriptomes were profiled using single cell RNA-sequencing. A total of 90 samples derived from 5 timepoints and 2 treatment groups were sequenced.

first: Shamik

last: Mascharak

first: Heather

last: Talbott


title: Next Generation Sequencing to Identify Targets of the C. albicans Dfi1 Pathway

city: Boston

first: Carol

last: Kumamoto

state: Massachusetts

address: 150 Harrison Ave., Tufts University, Department of Molecular Biology and Microbiology, Jaharis 429

department: Molecular Biology and Microbiology

institute: Tufts University

summary:
Purpose: To identify downstream genetics targets of the Dfi1 pathway during Dfi1 activating conditions.
Methods: WT and dfi1 null cells were grown in minimal media, then treated with either 4uM of the calcium ionophore A23187 or an equal volume of 100% ethanol vehicle. After 30 min of treatment, cells were collected for RNA extraction and analysis. RNA was extracted using the Qiagen RNAeasy kit, then sent to the Tufts University Core Facility for library prep and sequencing.
Results: Analysis of this data resulted in the identification of a number of possible targets of the Dfi1 pathway, as reported in the manuscript "Bypass of Dfi1 regulation of Candida albicans invasive filamentation by iron limitation".

overall_design: mRNA profiles of WT and dfi1 null cells treated with the calicium ionophore A23187 or vehicle control, in triplicate.

first: Ashlee

last: Junier

first: Anne

last: Weeks


title: Long-Term Hepatitis B Virus Infection Induces Cytopathic Effects in Primary Human Hepatocytes, and Can be Partially Reversed by Antiviral Therapy

status: Public on Jan 22 2022

city: SHANGHAI

first: Wenjing

last: Zai

address: Dongan Road

institute: Fudan University

summary: Transcriptomic analysis was then applied to identify genes or pathways that could be rescued by RNAi.

overall_design: A combination of two siRNA (siHBV, 100 nM) to degrade all kinds of HBV RNAs (targeting both HBs and HBx sequences) was transfected by commercial reagents at 7 days post HBV infection, and the cells were collected for RNA-seq 6 days later.

first: Kongying

last: Hu

first: Jianyu

last: Ye

first: Jiahui

last: Ding

first: Chao

last: Huang

first: Yaming

last: Li

first: Zhong

last: Fang

first: Min

first: Cong

first: Jieliang

last: Chen

first: Zhenghong

last: Yuan


title: Transcriptional profiling of conditional knockout mice lacking Izumo1r in FoxP3+ Tregulatory cells

status: Public on Jan 19 2022

first: CBDM

last: Lab

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: We reported transcriptional characterization of splenic, ear skin and back skin of Izumo1R flox X Foxp3Cre mice and littermate controls. We report few changes observed in splenic Tregs but changes seen in the whole skin transcriptome and gamma delta compartment starting at approximately 8 weeks of age. We report upregulation of inflammatory genes overlapping with genes upregulated in the skin following topical administration of Imiquimod.

overall_design: For the IMQ treated samples, dorsal and ventral sides of the ear were treated with 125ug of 5% Imiquimod cream until indicated date. All other data are from untreated mice at indicated age. Foxp3/GFP+ Treg cells were sorted and processed for RNA-Seq. TCRB+ CD4+ Foxp3GFP- cells were sorted from the skin as Tcon. TCRb-CD19-GammaDeltaTCR+ cells from the skin were sorted as skin gamma delta T cells. All sorted samples were pre-gated on DAPI- single cells.

first: Payam

last: Zarin

first: Christophe

last: Benoist

first: Diane

last: Mathis


title: Genome-wide analysis of gene expression regulated by FSCN1 in cervical cancer HeLa cells

city: Wuhan

first: Dong

state: Hubei

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

department: Center for Genome Analysis

institute: ABLife, Inc.

summary: FSCN1 has been reported to be dysregulated in cervical cancers. However, the genome-wide regulated targets of FSCN1 is still unclear in cervical cancers. Here, the gene expression profile of HeLa cells transfected with FSCN1 shRNA (shFSCN1) were compared with cells transfected with empty vector (shCtrl). The results showed that shFSCN1 extensively affects the transcriptional level of 5, 043 genes in HeLa cells. There were more up-regulated genes (3, 870) than down-regulated ones (1, 173) after FSCN1 was knocked down in HeLa cells. GO analysis showed that the up-regulated genes were associated with transcription regulation and DNA binding. The down-regulated genes were enriched in some cancer associated pathway including angiogenesis and cell adhesion. In particular, FSCN1 positilvely regulated ANGPTL4 in HeLa cells. Compared to normal tissue, both FSCN1 and ANGPTL4 showed a higher expression in cervical tumor tissue. Moreover, ANGPTL4 was also positilvely correlated with expression of FSCN1 in cervical tumor tissue of TCGA. In conclusion, our study provide important cues for further study on the regulatory mechanism of FSCN1 in cervical cancer.

overall_design: Transcrptional profile of  Hela cells with/without FSCN1 Knockdown were generated

first: Yanliang

last: Liu

first: Li

last: Hong


title: Epigenetic state determines inflammatory sensing in neuroblastoma [ATAC-seq]

first: Priyankara

last: Wickramasinghe

summary: Immunotherapy has revolutionized cancer treatment, but many cancers are not impacted by currently available immunotherapeutic strategies. Here, we investigated inflammatory signaling pathways in neuroblastoma, a classically “cold” pediatric cancer. By testing the functional response of a panel of 20 diverse neuroblastoma cell lines to three different inflammatory stimuli, we found that all cell lines have intact interferon signaling and all but one lack functional cytosolic DNA sensing via cGAS-STING. However, dsRNA sensing via TLR3 was heterogeneous, as was signaling through other dsRNA sensors and TLRs more broadly. Seven cell lines showed robust response to dsRNA, six of which are in the mesenchymal epigenetic state, while all unresponsive cell lines are in the adrenergic state. Genetically switching adrenergic cell lines towards the mesenchymal state fully restored responsiveness. In responsive cells, dsRNA sensing results in the secretion of pro-inflammatory cytokines, enrichment of inflammatory transcriptomic signatures, and increased tumor killing by T cells in vitro. Using single cell RNA sequencing data, we show that human neuroblastoma cells with stronger mesenchymal signatures have a higher basal inflammatory state, demonstrating intra tumoral heterogeneity in inflammatory signaling that has significant implications for immunotherapeutic strategies in this aggressive childhood cancer.

overall_design: RNA-seq performed for various Neuroblastoma cell lines

first: Adam

last: Wolpaw

first: Chi

middle: V

last: Dang


title: Epigenetic state determines inflammatory sensing in neuroblastoma [RNA-seq]


title: Expression profiling of Ciona intestinalis initial tailbud stage embryos expressing mutant forms of the transcription factor Ci-XBP1 in the notochord.

city: New York

first: Anna

last: Di Gregorio

address: 345 E 24th Street, room 804A

department: Basic Science and Craniofacial Biology

institute: NYU College of Dentistry

summary:
XBP transcription factors are well-known regulators of the the unfolded protein response and are required for plasma cell differentiation. However, recent studies have shown that the XBP1 gene is also expressed in the developing notochord of Ciona and various vertebrates, and to date its role in the formation of this organ is largely uncharacterized.  We identified putative targets of Ci-XBP1 through a microarray screen, using RNAs extracted from embryos expressing mutant forms of this transcriptional regulator in the notochord.
We expressed in the Ciona notochord a dominant-negative version of Ci-XBP1 (XBP DBD::GFP) by cloning its DNA-binding domain (DBD) downstream of the Ci-Brachyury (Ci-Bra) promoter. We created a constitutive activator form of Ci-XBP by fusing its DBD to the VP16 transactivation domain (Bra>XBP DBD::VP16::GFP). These constructs were introduced into 1-cell stage embryos and grown to the initial tailbud (iTb) stage to identify Ci-XBP notochord target genes.

overall_design: Genes expressed in embryos carrying the XBP DBD::GFP and XBP DBD::VP16::GFP were compared genes expressed in wild-type controls (Bra>GFP) at the initial tailbud using whole-genome custom Affymetrix microarrays.


title: Transcriptome analysis of young and old zebrafish ventricles

city: Jena

first: Philipp

last: Koch

address: Beutenbergstraße 11

department: Core Facility Life Science Computing

institute: Leibniz Institute on Aging - Fritz Lipmann Institute

summary: The gradual decline of tissue functionality is the main reason why humans suffer from age-related diseases. The prevalence for cardiovascular diseases increases with increasing age. In order to prevent age-related cardiac diseases, it is of importance to understand the respective age-associated risk factors. We have therefore compared the ventricular transcriptome of old and young hearts of the model organism zebrafish. We identified the immune system as activated in the old and found muscle organization to deteriorate upon aging. We show an accumulation of immune cells, mostly macrophages, in the old zebrafish ventricle.

overall_design: 6 samples: ventricle of 3 young and 3 old individuals

first: Marco

last: Groth

first: Hanna

last: Reuter

first: Christoph

last: Englert


title: Structure and Function of an Effector Domain in Antiviral Factors and Tumor Suppressors SAMD9 and SAMD9L

city: San Antonio

first: Yan

last: Xiang

state: TX

address: 7703 FLOYD CURL DR

department: Microbiology, Immunology and Molecular Genetics

institute: UT Health San Antonio

summary: SAMD9 and SAMD9L (SAMD9/9L) are antiviral factors and myeloid tumor suppressors. They are critical for innate immune defense against several viruses, particularly the poxviruses. SAMD9/9L mutations with a gain-of-function (GoF) in inhibiting cell growth cause multisystem developmental disorders including many pediatric myelodysplastic syndromes. Predicted to be multi-domain proteins with an architecture like that of the NOD-like receptors, SAMD9/9L molecular functions and domain structures are largely unknown. Here, we identified a SAMD9/9L effector domain that functions by binding to double-stranded nucleic acids (dsNA) and determined the crystal structure of the domain in complex with DNA. Aided with precise mutations that differentially perturb dsNA binding, we demonstrated that the antiviral and antiproliferative functions of the wild-type and GoF SAMD9/9L variants rely on dsNA binding by the effector domain. Furthermore, we showed that GoF variants inhibit global protein synthesis, reduce translation elongation, and induce proteotoxic stress response, which all require dsNA binding by the effector domain. The identification of the structure and function of a SAMD9/9L effector domain provides a therapeutic target for SAMD9/9L associated human diseases.

overall_design: RNA-seq of BT20 cells transduced with SAMD9-R1293W with or without inducer for SAMD9-R1293W expression


title: MUC1-C INTEGRATES CHRONIC TYPE II INTERFERON SIGNALING WITH CHROMATIN REMODELING PATHWAYS IN IMMUNOSUPPRESSION OF PROSTATE CANCER

city: Buffalo

first: Qiang

last: Hu

address: Elm & Carlton Streets

institute: Roswell Park Cancer Institute

summary: The cancer stem cell (CSC) state is intimately associated with suppression of the immune tumor microenvironment (TME). The oncogenic MUC1-C protein drives dedifferentiation of castrate resistant prostate cancer (CRPC) CSCs in association with induction of the BAF, NuRD and PBAF chromatin remodeling complexes. The present work demonstrates that MUC1-C is necessary for expression of IFNGR1 and activation of the type II interferon-gamma (IFN- pathway in CRPC cells. We show that the MUC1-CARID1A/BAF pathway induces IFNGR1 transcription and that MUC1-CNuRD signaling suppresses FBXW7 in stabilizing the IFNGR1 protein. MUC1-C and NuRD were also necessary for expression of the downstream STAT1 and IRF1 transcription factors. Additionally and in contrast, our results demonstrate that MUC1-C and the PBRM1/PBAF pathway are necessary for IRF1-induced expression of (i) IDO1, WARS and PTGES, which metabolically suppress the immune TME, and (ii) the ISG15 and SERPINB9 inhibitors of T cell function. Of translational relevance, we show that MUC1 associates with expression of IFNGR1, STAT1 and IRF1, as well as the downstream IDO1, WARS, PTGES, ISG15 and SERPINB9 immunosuppressive effectors in CRPC tumors. Consistent with these results, MUC1 associates with immune cell-depleted “cold” CRPC TMEs. These findings demonstrate that MUC1-C integrates chronic activation of the type II IFN-G pathway with induction of chromatin remodeling complexes in linking CSC dedifferentiation and immune evasion.

overall_design: DU-145 cells stably expressing a tet-MUCshRNA vector were treated with vehicle or DOX for 7 days were stimulated with 10 ng/ml IFN-g for 24 hours.. Total RNA from cells cultured in triplicates was isolated and used for library preparation.

first: Hagiwara

last: Masayuki

first: Kufe

last: Donald

first: Long

last: Mark

first: Hu

last: Qiang


title: MRP5 and MRP9 Play a Concerted Role in Male Reproduction and Mitochondrial Function

city: College Park

first: Iqbal

last: Hamza

state: MD

address: 8127 Regents Drive, 1109 Animal Sciences

institute: University of Maryland

summary: Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated.  In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 mutants are unviable due to their inability to export heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway and MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 in mammals is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 deficiency. Here, we show that MRP5 and MRP9 double KO mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only in the absence of MRP5. Both ABCC transporters localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes and RNA-seq show significant alterations in genes related to mitochondria function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

overall_design: RNAseq analysis was perform on RNA extracted from the testes of three Wildtype and MRP5/MRP9 DKO age matched mice

first: Ian

last: Chambers

first: Praveen

last: Kumar

first: Jens

last: Lichtenberg

first: John

last: Phillips

first: Maureen

last: Kane

first: David

last: Bodine


title: FMRP regulates mRNAs encoding distinct functions in the cell body and dendrites of CA1 pyramidal neurons [TRAP-seq, RNA-seq]

first: Caryn

last: Hale

state: New York

address: 1230 York Avenue

institute: Rockefeller University

summary: We report cell-type and compartment-specific TRAP-seq, RNAseq, FMRP-CLIP, and PAPERCLIP from CA1 hippocampal neurons.  We use TRAP-seq and PAPERCLIP data to precisely define the dendritic transcriptome, including mRNAs that harbor APA and AS events that are differentially localized.  Further, we use FMRP-CLIP in order to define mRNAs that undergo compartment-specific regulation by FMRP.

overall_design: Cell-type and compartment-specific TRAP-seq, RNAseq, FMRP-CLIP, and PAPERCLIP from mouse microdissected CA1.

first: Robert

middle: B

last: Darnell

first: Thomas

last: Carroll


title: Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA base-editing activity, functionality and specificity

city: La Jolla

first: Dario

last: Meluzzi

address: 9500 Gilman Drive #0648

department: School of Medicine

institute: UC San Diego

summary: We use RNA-seq to study the transcriptome wide specificity profiles of A-to-I RNA editing by split-ADAR2 constructs.

overall_design:
All RNA-seq experiments were carried out in HEK293FT cells which were  grown in DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher) in an incubator at 37 °C and 5% CO2 atmosphere. Plasmid transfection for the split-ADAR2 samples was carried out according to standard protocols. RNA was extracted using the RNeasy mini kit. RNA-seq libraries were prepared from 250ng RNA using the NEBNext Ultra RNA Library Prep Kit for Illumina.
Basecalls were performed using Illumina RTA3, and fastq files were then generated using Illumina bcl2fastq2 v2.20. Both tasks were carried out by the UCSD IGM Genomics Center. RNA-seq read pairs with 100 bases per read mate were aligned to the GRCh38 reference genome using STAR aligner version 2.7.1a or 2.7.3a (Dobin A et al 2013). The genome index was built using primary assembly annotations from GENCODE release 32. Default parameters were used to run STAR, except for the following relevant settings: --clip3pAdapterSeq AGATCGGAAGAGCACACGTCTGAACTCCAGTCA, AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT, --outSAMtype BAM Unsorted, --outBAMcompression 0, --quantMode GeneCounts, --outReadsUnmapped Fastx, --genomeLoad LoadAndRemove, --alignSJDBoverhangMin 1, --peOverlapNbasesMin=10, --peOverlapMMp=0.05, --outSAMmultNmax 1, --alignEndsType EndToEnd, --outFilterMismatchNmax -1, --outFilterMismatchNoverReadLmax 0.2, --outFilterMultimapNmax 1. The reads of the resulting uniquely aligned pairs were sorted by genomic coordinate using samtools sort (Li H. et al 2009). Duplicated read pairs were marked using samtools markdup and were removed from subsequent RNA-editing analysis. The uniquely aligned reads for each sample were down-sampled using samtools view with option -s. Down-sampling fractions were calculated by dividing the smallest number of uniquely aligned reads among all samples by the number of uniquely aligned reads available for the sample being down-sampled. Down-sampling was not performed on the reads of the control samples. The down-sampled reads where then fed to samtools mpileup, whose output was parsed to extract the counts of each base found at each A-site and T-site in the GRCh38 reference genome. Insertions and deletions were ignored. Reference sites with read coverage of less than 10 were discarded. From the remaining reference A- and T-sites a final list was selected by choosing sites common to all samples and with at least one G or C at a reference A- or T-site, respectively, in at least one sample. For each pair of compared control and treatment samples, and for each reference A- or T- site selected as described above, a Fisher exact test was carried out to calculate the p-value for the change in proportion of G or C bases, respectively, at that site. The p-values for all selected reference sites were adjusted for multiple testing using the Benjamini-Hochberg method. The measured base proportions for each site (row) and sample (column) are reported in processed data file pm_adar_rned_008-mcplns_seq_rna_ed_bfscr_0_res-bpr-0.tsv.gz. The adjusted p-values for the comparison (column) of base proportion between each sample and the control sample at each site (row) are reported in processed data file pm_adar_rned_008-mcplns_seq_rna_ed_bfscr_0_res-pad-0.tsv.gz.

first: Dhruva

last: Katrekar

first: Nathan

last: Palmer

first: Prashant

last: Mali


title: Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites (RNA-Seq)

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

institute: The University of Pennsylvania

summary: Histone acetylation, a post-translational modification associated with transcriptional activation, is governed by nuclear acetyl-CoA pools that can vary depending on the metabolic state of the cell. The metabolic enzyme acetyl-CoA synthetase 2 (ACSS2) is proposed to regulate nuclear acetyl-CoA levels, using local acetate to produce acetyl-CoA that is utilized for histone acetylation. We hypothesize that during gene activation, a local transfer of intact acetate occurs between histones to upregulate transcription via sequential action of epigenetic and metabolic enzymes. Here we present converging lines of evidence in support of this acetate transfer to serve rapid gene induction. Using stable isotope labeling, we detect local transfer of intact acetate between histone acetylation sites both in vitro using purified mammalian enzymes and in vivo using quiescence exit in Saccharomyces cerevisiae as a change-of-state model. We delineate the enzymatic components required for this transfer mechanism, finding that ACSS2, histone deacetylase and histone acetyltransferase enzymes are necessary for efficient acetyl-group transfer in vitro. We show that Acs2, the yeast orthologue of ACSS2, is recruited to the genome during quiescence exit, and observe dynamic changes of histone acetylation in the vicinity of Acs2 peaks in vivo. Strikingly, we find that Acs2 is preferentially associated with the most upregulated growth genes, suggesting that acetyl-group transfer might play an important role in increased gene expression. Overall, our data reveal direct transfer of acetate between histone lysine residues to facilitate rapid transcriptional induction, an exchange that may be critical during metabolic alterations and changes in nutrient availability.

overall_design: RNA-seq in a time-course in S. cerevisiae during quiescence exit. Four time points are measured: glucose-deprived quiescent cells (Q), 30 min post-quiescence exit (E30), 240 min post-quiescence exit (E240), and logarithmic growth (LOG). At each time point, three biological replicates were measured.

first: Mariel

last: Mendoza

first: Gabor

last: Egervari

first: Simone

last: Sidoli

first: Greg

first: Desi

last: Alexander

first: Payel

last: Sen

first: Benjamin

middle: A

last: Garcia

first: Shelley

last: Berger


title: Effect of arcA overexpression on murine liver gene expression profiles

status: Public on Jan 18 2022

city: St. Louis

first: Brian

middle: Jesse

last: DeBosch

state: MO

address: 660 S. Euclid Ave, Box 8208

department: Pediatrics and Cell Biology & Physiology

institute: Washington University School of Medicine

summary: We demonstrate the effects of arcA overexpression on murine liver gene expression by high-throughput RNA sequencing

overall_design: Examination of transcriptome in murine liver 5 weeks after infection with adeno-associated virus 8 (AAV8) encoding eGFP or arcA.

first: Yiming

last: Zhang


title: Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites (ChIP-Seq)

department: Cell & Developmental Biology

overall_design: ChIP-seq in a time-course in S. cerevisiae during quiescence exit, measuring four histone acetylation post-translational modifications (H3K9ac, H3K18ac, H3K23ac, and H3K27ac) as well as the enzyme ACSS2; ACSS2 is also measured using TAP-tag in a second strain of yeast. Four time points are measured: glucose-deprived quiescent cells (Q), 30 min post-quiescence exit (E30), 240 min post-quiescence exit (E240), and logarithmic growth (LOG). The replicate structure is as follows: H3K9ac, H3K18ac, H3K23ac, H3K27ac and non-tagged ACSS2 have two replicates each in each time point, with a corresponding two replicates of input in each time point to adjust for sonication efficiency bias; while TAP-tagged ACSS2 has two replicates in Q and E30 and one sample in E240 and LOG, with a corresponding input from this strain with identical replicate structure.


title: Effect of ADI-PEG 20 treatment on murine liver chromatin landscape at single cell resolution.

summary: We demonstrate the effects of 4-week ADI-PEG 20 on murine liver chromatin accessibility by single-cell ATAC-sequencing

overall_design: Examination of single-cell chromatin accessibility changes in 16-week Western diet-fed murine livers after 4 weeks of ADI-PEG 20 treatment.


title: Phase 1 Trial With Challenge to Assess the Safety and Biomarkers of Protection in Malaria-naive Adults of Immunization Via Mosquito Bite With Radiation-Attenuated Plasmodium Falciparum Sporozoites (IMRAS) - Day 0-28

city: Seattle

first: Raphael

last: Gottardo

state: WA

address: 1100 Fairview Ave N

department: VIDD

institute: Fred Hutch

summary: This study is to assess the safety, tolerability, and biomarkers of protection in healthy malaria-naive adults, who will receive bites from Anopheles stephensi mosquitoes either infected with Plasmodium falciparum Sporozoites (PfRAS) (true-immunization) or noninfected (mock-immunization).

overall_design: Comparison of RNAseq results from two cohorts each with two treatments, true-immunization and mock-immunization, across multiple timepoints from baseline to post-1st vaccination (Day 0, 1, 3, 7, 14, 28) to assess biomarkers of clinically-defined protection. This Series includes the data from Cohort 1.

first: Judith

last: Epstein

first: Ying

last: Du

first: Fergal

last: Duffy

first: Jason

last: Carnes

first: Kenneth

last: Stuart


title: Transcriptome-wide map of m6A RNAs  in A375 NC cells and shMETTL3 cells

status: Public on Jan 17 2022

city: Nanjing

first: Zhanghui

last: Yue

state: Jiangsu

address: 12 Jiangwangmiao Street, Xuanwu Area

institute: Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences & Peking Union Medical College

summary: The newly discovered dynamic N6-methyladenosine (m6A) modification plays a critical role in gene expression from a post-transcriptional level. we profiled the transcriptome-wide m6A modification in mRNAs and lncRNAs in non-targeting control A375 cells(NC) and METTL3 knockdown A375 cells(shMETTL3). Methylated RNA immunoprecipitation sequencing results revealed that the RNA m6A modification is conserved. METTL3 knockdown altered the expression of RNAs m6A modified sites in A375 cells. Finally, we show that the transcriptome-wide m6A alterations occurring in mRNAs and lncRNAs following METTL3 knockdown suggest this process plays important regulatory roles during A375 growth. This study provides a framework for applying the m6A modification regulated by METTL3 to melanoma research.

overall_design: Examination of m6A modification change and gene expression alternation in A375 NC and shMETTL3 cells

first: Jianfang

last: Sun

first: Yan

last: Wang


title: Total RNA-seq of L. mexicana promastigotes and axenic amastigotes following UV-cross-linking and orthogonal organic phase separation

status: Public on Jan 14 2022

city: Durham

first: Wenbin

last: Wei

address: Stockton Road

department: Biosciences

institute: Durham University

summary: We compared the relative abundance of RNAs in the UV- cross-linked (CL) and non-cross-linked (NC) samples using total RNA-seq of both promastigotes and axenic amastigotes of L. mexicana parasites. Irradiation of the Leishmania parasites with UV-dose of 525 mJ/cm2 followed by sequential AGPC phase partitioning and proteinase-treatment of the final interface provided the CL samples for the RNA-seq analyses. The abundance of RNA species in CL and NC samples were different; ncRNAs and protein coding RNAs respectively predominate the NC samples and CL samples in both promastigotes and amastigotes. Similarly, the CL samples of the two life cycle stages showed a higher Pearson correlation (median correlation 0.86) as compared to the correlation between the CL and NC samples in promastigotes (median correlation 0.69) and amastigotes (median correlation 0.84). Crucially, in addition to the similar distribution of RNA species in the CL samples of both life cycle stages, the abundance of the RNAs at the interface after CL was unaffected by the size of RNAs, suggesting that the orthogonal organic phase separation method recovered all CL Leishmania RNAs above 60bp without any systematic bias.

overall_design: Profiling of relative abundance of RNA species of L. mexicana promastigotes and axenic amastigotes following UV cross-linking and orthogonal organic phase saparation. Two biological replicates of the NC and CL samples of both life cycle stages of the Leishmania parasites were analysed.

first: Kalesh

last: Karunakaran


title: Single cell immune profiling reveals a developmentally distinct CD4+ GM-CSF+ T cell lineage that induces GI tract GVHD

city: Milwaukee

first: Clint

last: Piper

state: WI

address: 8701 W Watertown Plank Rd

institute: Medical College of Wisconsin

summary: Gastrointestinal (GI) tract involvement is a major determinant for subsequent morbidity and mortality arising during graft versus host disease (GVHD). CD4+ T cells that produce GM-CSF have emerged as central mediators of inflammation in this tissue site as GM-CSF serves as a critical cytokine link between the adaptive and innate arms of the immune system. However, cellular heterogeneity within the CD4+ GM-CSF+ T cell population due to the concurrent production of other inflammatory cytokines has raised questions as to whether these cells have a common ontology or if there exists a unique CD4+ GM-CSF+ subset that differs from other defined T helper (TH) subtypes. Using single cell RNA sequencing analysis, we identified two CD4+ GM-CSF+ T cell populations that arose during GVHD and were distinguishable by the presence or absence of IFN-γ co-expression. CD4+ GM-CSF+ IFN-γ- T cells which emerged preferentially in the colon had a distinct transcriptional profile, employed unique gene regulatory networks, and possessed a non-overlapping TCR repertoire when compared to CD4+ GM-CSF+ IFN-γ+ T cells as well as all other transcriptionally defined CD4+ T cell populations in the colon. Functionally, this CD4+ GM-CSF+ T cell population contributed to pathological damage in the GI tract which was critically dependent upon signaling through the IL-7 receptor but was independent of type 1 interferon signaling. Thus, these studies help to unravel heterogeneity within CD4+ GM-CSF+ T cells that arise during GVHD and define a developmentally distinct colitogenic TH GM-CSF+ subset that mediates immunopathology.

overall_design: Single cell RNA + TCR sequencing of donor T cells in the colon 21 days following hematopoietic stem cell transplantation

first: William

last: Drobyski

first: Moujtaba

middle: Y

last: Kasmani

first: Weiguo

last: Cui


title: RNAseq for Identification of Doxycycline transcriptomic impact in N2 & CB4856 C. elegans worms

city: Lausanne

first: Gaby

last: El Alam

state: Vaud

address: Station 15

department: SV LISP

institute: École polytechnique fédérale de Lausanne

summary: Purpose: Transcriptomic exploration for the identification of genes affected upon Doxycycline treatment in each of the two different worm strains studied

overall_design: Design: Wild-type C. elegans (N2 Bristol and CB4856 Hawaii) were received from Andersen’s group. Bacterial strain E. coli OP50 and E. coli HT115 were obtained from the Caenorhabditis Genetic Center (CGC). Worms of each strain were cultured on plates seeded with E. coli OP50, then eggs were obtained by alkaline hypochlorite treatment of gravid adults. A synchronized L1 population was obtained by incubating the egg suspension in M9 butter overnight at room temperature. Approximately 2000 L1 worms were transferred onto plates with or without 15 μg/mL Dox seeded with E. coli HT115. L4 worms were harvested after 50 h by three times of washing with M9 buffer. Tubes containing worm pellets were immediately submerged in liquid nitrogen for snap freezing and stored at -80ºC until use.  Three biological replicates were collected for each condition.

first: Arwen

last: Gao

first: Kevin

last: Hof


title: Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques

status: Public on Jan 15 2022

city: Hamilton

first: Craig

last: Martens

state: MT

address: 903 S. 4th St

department: DIR

institute: NIAID

summary: Older age is a key predictor of severe COVID-19. To gain insight into this relationship, especially with respect to immune responses, we utilized the rhesus macaque model of SARS-CoV-2 infection. Two cohorts of eight older (16-23 years) and eight younger (3-5 years) rhesus macaques were inoculated with SARS-CoV-2. Four animals per group were euthanized at 7- and 21-days post inoculation (dpi). Our time-resolved evaluation included viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis. Differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between age cohorts. Transcriptional signatures of inflammation-associated genes in cells isolated from bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both younger and older animals. These findings suggest that age does not substantially skew major facets of acute disease in this model. However, age-specific divergence of immune responses emerged during the post-acute phase of infection (7-21 dpi). Older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, in contrast to persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling in SARS-CoV-2-infected rhesus macaques suggests that age may delay or impair the induction of anti-viral cellular immune responses and delay efficient return to immune homeostasis following acute infection.

overall_design: Sixteen Rhesus macaques (8 older and 8 younger) were inoculated with SARS-CoV-2. BALF samples were taken from the animals at pre-infection (-7 days post inoculation) and at 3 and 7 days post inoculation for assessment by single cell RNA-Sequencing on the 10X genomics platform. Additionally, 4 of the older and 4 of the younger animals were necropsied at 7 days post inoculation for assessment of lung tissue by single cell RNA sequencing.

first: Emily

last: Speranza

first: Jyothi

middle: N

last: Purushotham

first: Julia

last: Port

first: Benjamin

last: Schwarz

first: Meaghan

last: Flagg

first: Brandi

last: Williamson

first: Friederike

last: Feldmann

first: Manmeet

last: Singh

first: Lizzette

last: Pérez-Pérez

first: Gail

middle: L

last: Sturdevant

first: Lydia

last: Roberts

first: Aaron

last: Carmody

first: Jonathan

last: Schulz

first: Neeltje

last: van Doremalen

first: Atsushi

last: Okumura

first: Jamie

last: Lovaglio

first: Patrick

middle: W

last: Hanley

first: Carl

last: Shaia

first: Ronald

last: Germain

first: Sonja

last: Best

first: Vincent

middle: J

last: Munster

first: Catharine

last: Bosio

first: Emmie

last: de Wit


title: Genes regulated by JAK1/2 inhibitor ruxolitinib and myxoid liposarcoma-specific fusion onccogene FUS-DDIT3 in HT1080 cells.

city: Göteborg

first: Emma

last: Jonasson

address: Medicinaregatan 1F, floor 5

department: Sahlgrenska Center for Cancer Research

country: Sweden

institute: University of Gothenburg

summary: Sarcomas and leukemias that are characterized by FET (FUS, EWSR1, TAF15) fusion oncogenes consist of more than 20 entities. Myxoid liposarcoma, one of the most common FET sarcoma types, is defined by the FUS-DDIT3 or the less common EWSR1-DDIT3 fusion oncogene. Previously, JAK-STAT signaling have been connected to cancer stem cell properties and chemotherapy resistance in myxoid liposarcoma, but the role of FUS-DDIT3 is not known. Therefore, we treated HT1080 fibrosarcoma cells expression FUS-DDIT3 with JAK1/2 inhibitor ruxolitinib and performed RNA sequencing of treated and control cells. Additionally, we analyzed native HT1080 cells to be able to compare the induced gene expression changes due to FUS-DDIT3 expression with those induced by JAK-STAT pathway inhibition.

overall_design: RNA sequencing was performed on HT1080 fibrosarcoma cells expressing FUS-DDIT3-EGFP, treated with JAK 1/2 inhibitor ruxolitinib and untreated controls (n=3-4). For comparison, native HT1080 cells (n = 4) were included.

first: Anders

last: Ståhlberg

first: Soheila

last: Dolatabadi

first: Lisa

last: Andersson


title: RNA-seq of control and USP7 siRNA transfected ES and 2-cell like cells

city: Boston

first: Fei

last: Ji

address: 185 Cambridge St

institute: Massachusetts General Hospital

summary: 2-cell-like cells (2CLCs) that resemble 2-cell stage embryos can arise with a very low frequency in ES cell cultures. Here, we found a novel factor, Usp7 which promotes 2CLC transition from ESCs and its effect is dependent of DNA replication. Endogenous retroviruses and 2-cell stage specific genes are significantly upregulated in siUSP7-transfected 2CLCs.

overall_design: RNA-seq of siControl and siUSP7 transfected cells. GFP+ are 2CLCs.

first: T

last: Nakatani

first: F

first: R

last: Sadreyev

first: M

last: Torres-Padilla


title: Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae [II]

city: Berkeley

first: Molly

last: Brothers

address: UC Berkeley 440 Barker Hall

department: Molecular and Cell Biology

institute: University of California, Berkeley

summary: The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. For the following datasets, we created a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII. We mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein. We also used a temperature-sensitive allele of SIR3 (sir3-8) fused to M.ECOGII to induce m6A methylation over time. Time courses involved a switch from restrictive temperature (37°C) to permissive temperature (25°C).

overall_design: 29 samples total. Replicates of each experiment are included (indicated by multiple samples of the same genotype and condition).

first: Jasper

last: Rine


title: An adjuvant strategy enabled by modulation of the physical properties of fungal ligands expands viral glycoprotein immunogenicity

status: Public on Jan 13 2022

city: Los Angeles

first: Roberto

last: Spreafico

address: UCLA

department: Institute for Quantitative and Computational Biosciences

institute: University of Califonia Los Angeles

summary: Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganism, but if and how the physical properties of PRR ligands influence development of the immune response remains unknown. Through the study of fungal mannans we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

overall_design: Examination of skin and lymph node immune responses to mannans or beta-glucans by RNA-seq

first: Francesco

last: Borriello

first: Ivan

last: Zanoni


title: Real-time quantitative PCR analysis of human dental pulp stem cells

city: São Paulo

first: Fabio

middle: Dupart

last: Nascimento

state: SP

address: Rua Doutor Gabriel Piza, 636 apto 23

department: Biochemistry

country: Brazil

institute: Federal University of Sao Paulo

summary: Excised pulp tissue was digested in a solution containing 3 mg/ml collagenase type I (Merck KGaA, Darmstadt, Germany) and 4mg/ml dispase (Merck KGaA, Darmstadt, Germany) for 1 h at 37°C. After digestion, 5 volumes of α-MEM medium containing 10% FCS were added. This solution was centrifuged at 120 × g for 10min, at room temperature. The precipitated material was resuspended in α-MEM medium and filtered through filters with pores of 70 μM. This procedure resulted in a single-cell suspension for in vitro culture. Isolated DPSCs were randomly assigned to the following experimental groups: Group 1–LPS stimulus and laser irradiation; Group 2–LPS stimulus and no laser irradiation; Group 3–Control group, no LPS or laser treatment.

overall_design: The isolated dental pulp stem cells were stimulated to an inflammatory state, using a lipopolysaccharide (LPS) model, and then subjected or not to laser photobiomodulation. Each experiment was statistically evaluated according to the sample distribution. A total of 85 genes related to inflammation and osteogenesis were evaluated regarding their expression by RT-PCR.

first: I

last: Tersariol

middle: O

last: Carrilho


title: Brain antigens stimulate proliferation of T lymphocytes with a pathogenic phenotype in multiple sclerosis patients

city: Houston

first: John

middle: William

last: Lindsey

address: 6431 Fannin St, Ste 7.044 MSB, Neurology Dept

institute: University of Texas Health Science Center At Houston

summary: A method to stimulate T lymphocytes with a broad range of brain antigens would facilitate identification of the autoantigens for multiple sclerosis and enable definition of the pathogenic mechanisms important for multiple sclerosis.  In previous work we found the obvious approach of culturing leukocytes with homogenized brain tissue does not work because the brain homogenate suppresses antigen-specific lymphocyte proliferation.  We now report a method that substantially reduces the suppressive activity.  We used this non-suppressive brain homogenate to stimulate leukocytes from multiple sclerosis patients and controls.  We also stimulated with common viruses for comparison.  We measured proliferation, selected the responding CD3+ cells with flow cytometry, and sequenced their transcriptomes for mRNA and T cell receptor sequences.  The mRNA expression suggested the brain-responding cells from MS patients are potentially pathogenic.  The T cell receptor repertoire of the brain-responding cells was clonal with minimal overlap with virus antigens.

overall_design: We studied 5 multiple sclerosis patients and 3 controls for a total of 8 subjects.  For each subject, we sequenced RNA from T cells responding to stimulation with brain antigens, T cells responding to stimulation with influenza, and peripheral blood cells immediately ex vivo.

first: J


title: Genome-wide analysis of replication timing in ESC and 2CLC

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

overall_design: Refer to individual Series


title: T cell characteristics associated with toxicity to immune checkpoint blockade in patients with melanoma [bulk RNA-seq]

city: Stanford

first: Aaron

middle: M

last: Newman

state: California

address: 265 Campus Drive

department: Institute for Stem Cell Biology and Regenerative Medicine, Department of Biomedical Data Science

institute: Stanford University

summary: Severe immune-related adverse events (irAEs) occur in up to 60% of melanoma patients treated with immune checkpoint inhibitors (ICIs). However, it remains unknown whether a common baseline immunological state precedes irAE development. Leveraging CyTOF, single-cell RNA sequencing, bulk RNA sequencing, and T cell receptor sequencing to analyze pretreatment blood from metastatic melanoma patients treated with ICIs, we investigated cellular factors associated with severe irAE development regardless of organ system involvement. Our results demonstrate circulating T cell characteristics associated with ICI-induced toxicity, with implications for improved diagnostics and clinical management.

overall_design:
To investigate cellular determinants of severe irAE development, we performed bulk RNA sequencing (bulk RNA-seq) of peripheral blood mononuclear cell (PBMC) samples obtained at baseline from 61 patients with metastatic melanoma and five healthy controls. The 61 patient samples consisted of two independently profiled bulk cohorts (bulk cohort 1, n = 26; bulk cohort 2, n = 27) and eight patient samples used for technical experiments.

>>>Submitter states that raw data will be submitted to dbGaP due to patient privacy concerns<<<

first: Alexander

last: Lozano

first: Aadel

last: Chaudhuri

first: Aishwarya

last: Nene


title: RB1 loss promotes a noncanonical activator function of LSD1 and sensitizes castration-resistant prostate cancer to LSD1 inhibition [ChIP-seq]

first: Changmeng

last: Cai

state: MA

address: 100 William T Morrissey Blvd

institute: University of Massachusetts Boston

summary: Genomic loss of RB1 is a common alteration in castration-resistant prostate cancer (CRPC) and is associated with poor patient outcomes. RB1-loss is also a driver event that promotes the neuroendocrine transdifferentiation of prostate cancer (PCa). The loss of Rb protein disrupts the Rb-E2F repressor complex and thus hyperactivates E2F transcription activators. While the impact of RB1-loss on PCa progression and linage plasticity has been previously studied, the underline mechanisms remain unclear. Using an integrated cistromic and transcriptomic analysis, we have characterized Rb activities in multiple CRPC models by identifying Rb directly regulated genes and revealed that Rb has distinct binding sites and targets in TP53-mutated CRPC. Significantly, we show that RB1-loss promotes the noncanonical activator function of LSD1/KDM1A, which stabilizes chromatin binding of E2F1, and hence sensitizes CRPC tumor to the LSD1 inhibitor treatment. These results provide new molecular insights of Rb activity in PCa progression and suggest LSD1 as a potential therapeutic target in CRPC with RB1-loss.

overall_design: RB ChIP-Seq were performed in both C4-2 and VCaP cells without any treatment. V5 (E2F1) ChIP-seq were performed in both C4-2-tetE2F1 WT and K185R mutant cell lines by treating with/out 50μM GSK2879552 for 24 hours under doxycycline pretreatment or not. All ChIP-seq samples contain replications.

first: Wanting

last: Han

first: Mingyu

last: Liu

first: Dong


title: Lidocaine and bupivacaine downregulate MYB protein and LncRNA DANCR through interaction with miR-187-5p in MCF7 cells

city: Taipei

first: Liang‑Chuan

last: Lai

state: Not Listed

address: No.1 Jen Ai road section 1

department: Graduate Institute of Physiology

country: Taiwan

institute: National Taiwan University

type: Non-coding RNA profiling by high throughput sequencing

summary: In order to identify microRNAs (miRNAs) that were involved in the effects of anesthetics on breast cancer cells, Illumina next generation miRNA sequencing was performed to search for any differentially expressed miRNAs in responding to lidocaine and bupivacaine treatments in MCF7 breast cancer cells. The filtering criteria for identifying differentially expressed miRNAs in the lidocaine and bupivacaine groups were fold change ≥ 2.0X and P-value ≤ 0.05. In total, there were 1,228 significantly upregulated miRNAs in lidocaine group and 133 miRNAs in bupivacaine group. On the contrary, there were 25 significantly downregulated miRNAs in lidocaine group and 36 miRNAs in bupivacaine group.

overall_design: MCF7 cells were incubated at 37°C in 5% CO2 and grown to 80-90% confluence before treatment. MCF7 were treated respectively with 4 mM lidocaine, 1 mM bupivacaine, or left untreated for 6 hours.

first: Chiao-yi

last: Lin


title: T cell characteristics associated with toxicity to immune checkpoint blockade in patients with melanoma [BCR and TCR scRNA-Seq]

overall_design:
To investigate the association of TCR and BCR diversity with severe irAE development, we performed single-cell V(D)J sequencing (scVDJ-seq) on peripheral blood mononuclear cells (PBMCs) obtained pre-treatment from patients with metastatic melanoma.
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Authors state "raw data are not available due to patient privacy concerns".

middle: X