title: Single-nucleus total RNA-sequencing of C2C12 myoblasts via in situ polyadenylation

status: Public on Aug 01 2022

city: New York

first: David

middle: William

last: McKellar

address: 101 6th Ave

institute: New York Genome Center

summary: Current methods for single-nucleus RNA-sequencing (snRNAseq) are limited to assaying endogenously polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNAs enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based single-nucleus transcriptomics to the total transcriptome. We apply standard snRNAseq and total snRNAseq to C2C12 myoblasts. Our in situ polyadenylation strategy relies on a brief, low-cost add-on to a widely used protocol for single-cell/single-nucleus RNA-sequencing, and thus could be broadly and quickly adopted. Single-nucleus RNA-sequencing of the total transcriptome will enable new insights into  gene regulation and biology.

overall_design: Single-nucleus total RNA-sequencing was performed using a modified version of the Chromium protocol. C2C12 cells were grown to 90% confluence and collected with 0.25% TrypLE (Thermo Fisher Scientific). Nuclei were isolated similar to Petrany et al, Nature Communications, 2020. Cells were pelleted by centrifugation at 500 x g, at 4ºC, for 5 minutes, and resuspended in 6 ml of chilled homogenization buffer (0.25M sucrose, 1% bovine serum albumin, 1X phosphate-buffered saline, 0.2U/µl SUPERase•In RNase Inhibitor (Thermo Fisher Scientific #AM2694), nuclease-free H2O). 1ml of chilled 2.5% Triton-X100 diluted in 1X phosphate-buffered saline was then added. Cells were then incubated on ice for 5 minutes, then pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes. Nuclei were then resuspended in 1X phosphate-buffered saline and counted using trypan blue. Five million nuclei were suspended in 200ul of 1X phosphate-buffered saline, then 800ul of ice-cold methanol was added dropwise to fix. Nuclei were then stored at -20ºC overnight.     On the day of the experiment, nuclei were removed from -20ºC and incubated on ice for five minutes. Nuclei were then pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes and resuspended in 200 µl wash resuspension buffer (0.04% bovine serum albumin, 1mM DTT, 0.2U/µl Protector RNase Inhibitor (Roche #3335402001), 3X SSC buffer (Thermo Fisher Scientific #15557044 ), nuclease-free H2O). Nuclei were then pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes and washed in 200µl of 1X wash buffer (40µl 5X yPAP Reaction Buffer, 4µl 40U/µl Protector RNase Inhibitor, 156µl nuclease-free H2O), 1.6U/µl Protector RNase Inhibitor (Roche #3335402001), nuclease-free H2O). In situ polyadenylation was then performed by suspending nuclei in 50µl of yPAP enzyme mix (10µl 5X yPAP Reaction Buffer, 2µl of 600U/µl yPAP enzyme, 1µl 25mM ATP, 2µl 40U/µl Protector RNase Inhibitor, 35µl nuclease-free H2O) and incubating at 37ºC for 25 minutes without agitation. Nuclei were then washed with 500µl of nuclei suspension buffer (based on nuclei suspension buffer from Cao et al, Nature, 2019: 10mM Tris-HCl pH7.5, 10mM NaCl, 3mM MgCl2, 1% bovine serum albumin, 0.2U/µl Protector RNase Inhibitor, nuclease-free H2O) and pelleted by centrifugation at 1000 x g, at 4ºC, for 5 minutes. Nuclei were finally resuspended in 200µl nuclei suspension buffer, counted using a Countess 3 (Thermo Fisher Scientific) and the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher Scientific, #L3224), then diluted to the proper concentration. Nuclei for standard single-nucleus RNA-sequencing were processed similarly, but with no in situ polyadenylation step (counted immediately after wash buffer was added). 3,300 nuclei were then loaded onto the Chromium controller (10x Genomics) for a targeted capture of 2,000 nuclei. Libraries were generated using the standard Chromium v3 protocol. Final libraries were sequenced using the Illumina MiniSeq and Illumina NextSeq 500.

first: Iwijn

last: De Vlaminck

first: Benjamin

middle: D

last: Cosgrove


title: Time-resolved post-translational histone modifications during BCG vaccine induced trained immunity in human monocytes

status: Public on Aug 02 2022

city: Parkville

first: Boris

last: Novakovic

state: Victoria

address: Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road

country: Australia

institute: Murdoch Children's Research Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Trained immunity is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to certain microbial components, altering their responses to future exposures. This study profiled the distribution of two active histone modifications (H3K27ac and H3K4me1) of human monocytes responding to two different BCG vaccine strains.

overall_design: Time-course in vitro culture of human monocytes. Trained immunity was induced in culture through an initial exposure of primary human monocytes to BCG Denmark and BCG Bulgaria strains for 24 hours, followed by removal of stimulus and differentiation to macrophages for an additional 5 days. Cells were fixed for chromatin collection at baseline (day 0), 24 hours, and 6 days.

first: Richard

last: Saffery

first: Mihai

middle: G

last: Netea

first: Nigel

last: Curtis


title: Saliva and Lung Microbiome Associations With Electronic Cigarette Use and Smoking

status: Public on Jul 13 2022

city: Columbus

first: Peter

last: Shields

state: OH

address: 420 West 12th Ave

country: USA

institute: The Ohio State University

type: Expression profiling by high throughput sequencing

summary:
Background: The microbiome is increasingly being linked to cancer risk. Little is known about the lung and oral cavity microbiomes in healthy smokers (SM), and even less for electronic cigarette (EC) users, compared healthy never-smokers (NS).
Methods: In a cross-sectional pilot study of SM (N=8), EC users (N=10) and NS (N=10) saliva and bronchoscopy-collected bronchoalveolar lavage samples were collected. Bacteria species were identified through metatranscriptome profiling by RNA-sequencing to study associations with the lung and oral microbiome. Pairwise comparisons and linear modeling was assessed with false discovery rates <0.1.
Results: Total bacterial load was similar for the SM, EC users and NS, and there was no differences in the bacterial diversity across groups. In the lung, there were 44 bacterial species that were statistically significantly different for SM/NS, 80% of which were decreased in the SM. There were 12 bacterial species that were different for SM/EC users, all of which were decreased, 10 of which were also identified in the SM/NS comparison. The 2 bacterial species unique to SM/EC comparison were  Neisseria sp. KEM232 and Curvibacter sp. AEP1-3. From the top 5 decreased species in SM/EC, 3 were also identified in the SM/NS comparison (Neisseria elongata, Neisseria sicca, and Haemophilus parainfluenzae) and 2 of these were unique to the SM/EC comparison (Neisseria zoodegmatis and Ottowia sp. oral taxon 894). There were 8 species increased in SM compared to NS, none of which are known to be clinically significant.  In the oral microbiome, 152 bacteria species were differentially abundant for the SM/NS analysis, and only 17 for the EC/NS comparison, all which were also present in SM/NS comparisons. There were 21 bacteria that were differentially abundant in both the lung and oral cavity for SM and NS, 95% also were decreased in the SM.
Conclusion: Smoking and EC use do not appear to materially affect the lung microbiome, although differences are noted of unclear clinical significance. Most differentially abundant bacteria decreased, which may be due to a toxic effect of cigarette smoke, including a change in humidity or heating. Given the low number of overlapping oral and lung microbes, the oral microbiome does not appear to be a good surrogate for smoking-related effects in the lung.

sample_organism:
human lung metagenome
human saliva metagenome

overall_design: Oral and lung microbial profiles of smokers, e-cig users and never-smokers

first: Kevin

middle: L

last: Ying

first: Ewy

middle: A

last: Mathé


title: Non-equilibrium pronucleus histones require gametal factor Lsm1 induced major satellite RNA decay

status: Public on Jul 05 2022

city: Shanghai

first: Jiang

last: Zhu

address: 1239 Siping Road

country: China

institute: Tongji University

summary: The development of a multicellular organism with various organs and tissues is a complex but highly concerted process, in which cells acquire different identities through exquisite spatiotemporal regulations called cell fate decision. Here, we Lsm1 targets non-coding major satellite RNAs in mouse zygote, which is required for the non-equilibrium epigenetic modification, especially H3K9me3 establishment during early embryo development.

overall_design: We performed RNAseq experiment on mouse zygotes with and without Lsm1 knockdown. We also performed RIP-seq on FLAG-Tagged LSM1, using NO-Tagged LSM1 as control.


title: Hierarchical enhancers 1 confer the temporal and partially compensatory transcriptional regulation of Nkx2-5 during heart development

status: Public on Jul 03 2022

city: Beijing

first: Yaxi

last: Liu

address: 5 Yiheyuan Road

institute: Peking University

summary: We comprehensively interrogate enhancers U1 and U2 in controlling Nkx2-5 transcription during heart development. Serial genomic deletions in mice reveal a functional hierarchy of U1 and U2 as evidenced by stage-dependent phenotypic variations. Specifically, U1 and U2 function redundantly to confer Nkx2-5 expression at early stages, but U2 instead of U1 supported its expression at later stages of heart chamber formation and maturation. Combined deletions markedly reduce Nkx2-5 dosage as early as E7.5, despite being largely reinstated two days later, displaying heart malformations with precocious differentiation of cardiac progenitors. Together, we propose a model that the temporal and partially compensatory regulatory function of two enhancers dictates a TF?s dosage and specificity during development.

overall_design: 1) H3K27ac ChIP-seq and ATAC-seq data on E16.5 ventricles from Control and U2KO mice. DKO mice were generated by deleting both U1 and U2 locus and U2KO mice were generated by deleting only U2 locus using CRISPR-Cas9 genome editing. 2) Single cells of outflow tract (OFT) or heart tube (HT) were derived for scRNA-seq from DKO and Control mice at E8.25. 3) H3K27ac ChIP-seq data on E9.5 hearts from Control and DKO mice.  4) NKX2-5 ChIP-seq data on E9.5 hearts from DKO mice. 5) Hi-C with WT and U1U2-DKO E8.5 mouse hearts.

first: Chen

middle: C

last: Li

first: Jiejie

last: Zhang

first: Qianhao

last: Wang

first: Haiqing

last: Xiong

first: Xin

first: Aibin

last: He

first: Shanshan

last: Ai


title: EVALUATION OF KRASG12C INHIBITOR RESPONSES IN NOVEL MURINE KRASG12C LUNG CANCER CELL LINE MODELS

status: Public on Jul 01 2022

city: Aurora

first: Lynn

last: Heasley

state: CO

department: Craniofacial Biology

institute: University of Colorado Anschutz Medical Campus

summary: The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months. To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the  KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods.  Also, a novel murine KRASG12C  line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model. The three lines exhibit similar in vitro sensitivities to KRASG12C inhibitors (MRTX-1257, AMG-510), but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to marked shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with MRTX-1257 and the SHP2 inhibitor, RMC-4550 and the MRTX-849/RMC-4550 combination yielded tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice.   Notably, this synergistic combination response was lost in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs. These new models of murine KRASG12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors.

overall_design: In vitro RNA was collected from cell lines and submitted to RNAseq

first: Daniel

last: Sisler


title: KAS-Analyzer: an integrated toolkit for exploring KAS-seq data

status: Public on Jul 27 2022

city: Chicago

first: Ruitu

last: Lyu

state: IL

address: 929 E 57th Street

department: Chemistry

institute: The University of Chicago

summary: Kethoxal-assisted ssDNA sequencing (KAS-seq) is gaining popularity as a robust and effective approach to study the dynamics of transcriptionally engaged RNA polymerases through profiling of genome-wide single-stranded DNA (ssDNA). Its latest variant, spKAS-seq, a strand-specific version of KAS-seq, has been developed to map genome-wide R-loop structures by detecting imbalances of ssDNA on two strands. However, user-friendly, open-source, and specific bioinformatic analyzer for KAS-seq data are still lacking. Here we present KAS-Analyzer as a flexible and integrated toolkit to facilitate the analysis and interpretation of KAS-seq data. KAS-Analyzer can perform standard analyses such as quality control, read alignment, and differential RNA polymerase activity analysis. In addition, KAS-Analyzer introduces many novel features, including, but not limited to: calculation of transcriptional indexes, identification of single-stranded transcribing enhancers, and high-resolution mapping of R-loops. We use benchmark datasets to demonstrate that KAS-Analyzer provides a powerful framework to study transient transcriptional regulatory programs. KAS-Analyzer is available at https://github.com/Ruitulyu/KAS-Analyzer.

overall_design: We perfomed KAS-seq and spKAS-seq in several popular human cell lines and UVB radiation treated NHEK cells.


title: Local IL-10 delivery modulates the immune response and enhances repair of volumetric muscle loss muscle injury

status: Public on Jun 26 2022

city: Fayetteville

first: Jeffrey

last: Wolchok

state: AR

address: 700 W. Research Blvd

department: Biomedical Engineering

institute: University of Arkansas

summary:
To explore the impact of targeting IL-10 signaling, tibialis anterior (TA) VML injuries were created and then treated in Sprague Dawley rats using an autologous minced muscle regenerative medicine repair strategy in combination with delayed exogenous IL-10 delivery.
We then performed gene expression profiling analysis using data obtained from RNA-seq from rat muscles (n=4 for IL-10 treatment, n=4 for PBS control , and n=4 for uninjured control) at day 14 post injury

overall_design: Comparative gene expression profiling analysis of RNA-seq data from rat Tibialis anterior muscle tissues between unjured, Pbs abd IL-10 treatment

first: Tai

last: Huynh


title: Analysis of gene expression changes upon ZBTB18 full length (FL), ZBTB18 full length-mutant (FL-mut), ZBTB18 short form Nte (SF-Nte) and ZBTB18 short form Nte (SF-mut) in SNB19 glioblastoma cell line

status: Public on Jun 20 2022

city: Freiburg

first: Maria Stella

last: Carro

address: Breisacherstraße 64

country: Germany

institute: Freiburg University Medical Center

summary: ZBTB18 is a transcriptional repressor and tumor suppressor in glioblastoma (GBM). Specifically, it acts as a repressor of mesenchymal genes. ZBTB18 interacts with C-terminal binding proteins 1 and 2 through a VLDL motif. In GBM, ZBTB18 is cleaved the the calpain 2 intracellular proteases which generates truncated Nte and Cte short forms. Here we have investigated gene expression changes which occur upon the expression of ZBTB18 full lenght or ZBTB18 short form Nte (SF-Nte), as well as upon expression of the mutated counterparts which no longer interacts with CTBP1/2.

overall_design: mRNA profiles of SNB19 cells transduced with the following lentiviral vectors: pCHMWS (empty vector, EV), pCHMWS-ZBTB18 FL,  pCHMWS-ZBTB18 FL-mut,  pCHMWS-ZBTB18 SF-Nte,  pCHMWS-ZBTB18 SF-mut.

first: Maria

middle: S

first: Dieter

middle: H

last: Heiland

first: Rana

last: Schulzki


title: Genes critical for development and differentiation of dopaminergic neurons are downregulated in Parkinson’s disease

status: Public on Jun 17 2022

city: Bangalore

first: Vijayalakshmi

last: Ravindranath

address: C.V. Raman Avenue

country: India

institute: Centre for Brain Research

summary: We performed transcriptome analysis using RNA sequencing on substantia nigra pars compacta (SNpc) from mice after acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment and Parkinson’s disease (PD) patients.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Comparative gene expression profiling analysis of RNA-seq data for substantia nigra pars compacta from MPTP mice treated for 14 days and 24 hours (treated vs. control) and for human PD (PD patients vs. Control).


title: UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes [RNA-seq]

city: Jerusalem

first: Inbar

last: Plaschkes

address: The Hebrew University of Jerusalem - Ein Kerem

department: The Faculty of Medicine - Ein Kerem

country: Israel

institute: HUJI

summary: Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of trypanosomatids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and play an essential role in chromosome ends protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its, previously described, DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression that plays a role in the control of antigenic variation in T. brucei.

overall_design: Differential expression of UMSBP2 knock down Trypanosoma brucei brucei compared to wild type ( 3 replicates for each of the 2 conditions)

first: Awakash

last: Soni

first: Olga

last: Klebanov-Akopyan

first: Esteban

last: Erben

first: Amnon

last: Harel

first: Katreena

last: Yamin

first: Itay

last: Onn

first: Joseph

last: Shlomai


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type Xenopus tropicalis Tail and Notochord During Metamorphosis

status: Public on Jun 14 2022

city: Bethesda

first: Ryan

last: Dale

state: MD

address: Rm 10D39, 10 Center Drive

department: National Institute of Child Health and Human Development

institute: National Institutes of Health

summary: Our experiment revealed the notochord specific metamorphosis-induced genes to clarify the molecular mechanisms of tail regression.

overall_design: Compared tail before (St60) and during (St63) metamorphosis. Compared whole tail and notochord-removed-tail.

first: Keisuke

last: Nakajima

first: Yun-Bo

last: Shi

first: Hongen

last: Zhang


title: Effect of depletion of GMDS-AS1 on gene expression of HCT116 colorectel cancer cells

status: Public on Jun 10 2022

first: Deji

last: Ye

address: 320 Yueyang Road

institute: Shanghai Institute of Nutrition and Health, Chlinese Academy of Sciences

summary:
To detect the biological function of LncRNA GMDS-AS1 in colorectal cancer tumorigenesis, we constructed lentivirus-infected stable GMDS-AS1-knockdown (KD) HCT116 cell lines using shRNA (GMDS-AS1 shRNA2) and a nontarget control shRNA
Next we performed gene expression profiling analysis using data obtained from RNA-seq of control and GMDS-AS1 KD cells, three biological replicates were applied.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for control and GMDS-AS1 KD HCT116 cells.

first: Hanshao

last: Liu


title: Negative regulation of endothelial to haematopoietic transition by Nupr1

first: Di

address: No. 45 West Street, Beijing

institute: Xuanwu Hospital, Capital Medical University

summary: we uncovered that nuclear protein 1 (Nupr1), a transcription regulatory gene preferentially expressed in HECs, was a negative regulator of EHT. Nupr1 deficiency resulted in plentiful emergence of HSC-competent cells in the AGM region, including HECs and HSPCs. Further single-cell transcriptome combined serial functional assay revealed that loss of Nupr1 prominently accelerate EHT process, also promote the specification of HECs and strengthen their subsequent hematopoietic differentiation potential towards HSPC. Interestingly, we found that numerous inflammatory signals were specifically upregulated in Nupr1-/- EHT relevant populations, as elevated inflammatory response signals have been proved to promote EHT and HSPCs generation.

overall_design: Cd31+, Cd44+ cells form VEC-Cre and Nupr1fl/fl mice were isolated by Fluorescence-activated cell sorting (FACS)  and analyzed using scRNAseq.

first: Haizhen

last: Wang

first: Jie

last: Zhou


title: Rif1 functions in complex with PRC1.6 to regulate mouse embryonic stem cells fate potential

status: Public on Jun 09 2022

city: Leiden

first: Jiale

last: Chen

address: Einsteinweg 55

country: Netherlands

institute: Leiden University

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

overall_design: Refer to individual Series


title: Vascular integrity is essential for maintaining tissue homeostasis

first: Junhao

last: Hu

address: Build 13, No.100 Kaike Road, Pudong District

institute: Interdisciplinary Research Center of Biology and Chemistry

summary:
Purpose: analyze the transcriptomic differences in PBS and Riociguat treated whole lung lysate
Methods: C57BL6 mice were given PBS or Riociguat treatment (i.g.). 24h later, homogenize lung and extract mRNA
Results: Riociguat has merely effect on normal mice lung

overall_design: C57BL6 mice were given PBS or Riociguat treatment (i.g.). 24h later, homogenize lung and extract mRNA.  mRNA profiles of whole lung lysate were generated by sequencing.

first: Hao

last: He


title: ZBTB12-HERVH-LncRNA axis is a molecular barrier for dedifferentiation of human stem cells

status: Public on May 31 2022

city: Santa Barbara

first: Dasol

last: Han

state: California

address: University of California Santa Barbara

institute: University of California Santa Barbara

summary: Development is generally viewed as one-way traffic of cell state transition from primitive to developmentally advanced states. However, molecular mechanisms that ensure the unidirectional transition remain largely unknown. NanoCAGE sequencing identified an evolutionarily conserved zinc finger repressor, ZBTB12, as a molecular barrier for dedifferentiation of human pluripotent stem cells (hPSCs). Single cell RNA sequencing revealed that ZBTB12 is essential for three germ layer differentiation by blocking hPSC dedifferentiation. Mechanistically, ZBTB12 cooperates with NANOG to fine-tune the expression of human endogenous retroviruses (HERVs). Active HERVH loci drive the expression of overlapping long non-coding RNAs (lncRNAs) whose downregulation by ZBTB12 is necessary for dedifferentiation blockage and pluripotency exit. Overall, we have identified a ZBTB12-HERVH-lncRNA axis as molecular machinery that safeguards the unidirectional transition of stem cell fates.

overall_design: Single cell transcriptomic profiling from hESCs expressing control or ZBTB12 shRNA before and after spontaneous differentiation (8 days), using 10X genomics Chromium Next GEM system


title: Loss of pex5 sensitizes zebrafish to fasting due to deregulated mitochondria, mTOR, and autophagy

status: Public on Jun 05 2022

city: Gwangju

first: Yunju

last: Jo

address: 123, Cheomdangwagi-ro

country: South Korea

institute: Gwangju Institute of Science and Technology

summary: Animal models have been utilized to understand the pathogenesis of Zellweger spectrum disorders (ZSD), but the link between the clinical manifestations and molecular pathways has not yet been clearly established. We generated a peroxin 5 homozygous mutant zebrafish (pex5-/-) to gain insight into the molecular pathogenesis of peroxisome dysfunction. pex5-/- displays hallmarks of ZSD in humans and die within one month after birth. Fasting rapidly depletes the liver lipids and glycogen in pex5-/- and expedites their mortality. Mechanistically, abnormal mitochondrial activity and mechanistic target of rapamycin (mTOR) signaling act together to induce metabolic alterations to deplete the hepatic nutrients and accumulate the damaged mitochondria. Accordingly, the use of chemical interventions blocking either the mitochondrial activities or the mechanistic target of rapamycin complex 1 or a combination of both improves the metabolic imbalance shown in the fasted pex5-/- livers and extends the survival of the animals. Furthermore, the use of an autophagy activator also effectively ameliorated the early mortality of the fasted pex5-/-. These results suggest that fasting may be detrimental to patients with peroxisome dysfunction and that modulating the mitochondria, mTOR, or autophagy activities may provide a therapeutic option to alleviate the symptoms of peroxisomal diseases associated with metabolic dysfunction.

overall_design: Total RNAs of whole liver tissues from fasted WT and pex5 homozygous mutant zebrafish at 6 dpf were used to identify differential gene expression between the two genotype under fasting condition

first: Dongryeol

last: Ryu

first: Seong-Kyu

last: Choe


title: Transcriptome analysis of tomato seedlings infected by Pepino Mosaic Virus

status: Public on May 24 2022

city: Athens

first: Stamatis

last: Rigas

state: Attiki

address: Iera Odos 75

department: Biotechnology

country: Greece

institute: Agricultural University of Athens

summary: Pepino Mosaic Virus (PepMV) consists a major pathogenic threat in the greenhouses worldwide and has a devasting impact on tomato global production. PepMV belongs to the Potexvirus genus of the Flexiviridae family. PepMV has a viral RNA genome of approximately 6400 nucleotides long that contains five open reading frames (ORFs) and 4 major genotypes have been characterized. TomCr3, an indigenous virulent PepMV strain derived from the Chilean (CH2) genotype, was isolated in Crete, Greece and was used for the mechanical infection of Belladonna F1 hybrid tomato seedlings. Here, we present the results of a deep RNA-sequencing (RNA-seq) analysis performed to characterize the dynamic expression profile of tomato genes upon PepMV infection, including tomato transcription factors.

overall_design: The TomCr3 strain of PepMV was isolated from naturally infected tomato plants in 2012 grown in a greenhouse in Ierapetra area from the Crete island in Southern Greece (35.0119° N, 25.7407° E). The virus was preserved in the laboratory through serial infections of tomato plants (Solanum lycopersicum L., hybrid Belladonna). Mechanical inoculations of tomato plants were performed by applying a suspension culture of PepMV prepared by grinding 0.5g of TomCr3 infected leaves in 5mL of Infection Buffer (0.1M Na2HPO4, 0,2% w/v Na2SO3) onto young leaves at the fourth leaf stage. Mock infected tomato leaves were used as a control. Exactly 10 days post inoculation (dpi), approximately 0.1 g of tissue was cut from the upper two or three leaves of each plant. The material was immediately frozen in liquid nitrogen and stored at -80°C until processing.

first: Dikran

last: Tsitsekian


title: Profiling the peripheral immune response to TNF-alpha stimulation in patients with untreated juvenile idiopathic arthritis using scRNAseq

status: Public on Jun 06 2022

city: Atlanta

first: Greg

last: Gibson

state: GA

address: North Ave NW

institute: Georgia Institute of Technology

summary: We report the application of next-generation sequencing tehcnologies to profile the effects of tumor necrosis factor alpha (TNFα) treatment on the transcriptomes of cells derived from juvenile idopathic arthritis (JIA) patients and healthy control subjects. Whole blood from de-identified subects with JIA was collected following children's provided assent and parental signed consent. Controls included healthy adults under the age of 25 years without any medical conditions or family history of autoimmune disease. All study procedures were approved by the Emory IRB. Blood samples from each subject was then separated into two aliquots, with one supplemented with TNFα. After 24hr ex-vivo stimulation, PBMCs were isolated and subject to cDNA library sequencing on a NextSeq 550.

overall_design: Peripheral blood single-cell mRNA profiles for 6 JIA patients and 2 healthy controls, with each subject having a corresponding unstimulated and a 24 TNFα ex-vivo stimulated sample.

first: Kathleen

middle: J

last: Imbach

first: Astrid

last: Kosters

first: Nicole

last: Treadway

first: Viashali

last: Prahalad

first: Dalia

last: Arafat

first: Meixue

last: Duan

first: Talia

last: Gergely

first: Shanmuganathan

last: Chandrakasan

first: Eliver

last: Ghosn

first: Sampath


title: Spatial profiling of chromatin accessibility in mouse and human tissues

status: Public on May 20 2022

city: New Haven

first: Yanxiang

last: Deng

state: CT

address: 55 Prospect Street

institute: Yale University

summary: We present spatially resolved chromatin accessibility profiling of tissue sections via next-generation sequencing by combining in situ ATAC-seq chemistry and microfluidic deterministic barcoding. Mouse and human tissues were profiled by spatial-ATAC-seq.

overall_design: Spatially resolved chromatin accessibility sequencing in mouse and human tissues.

first: Rong

last: Fan


title: Deletion and Complementation of Alr3614 in Anabaena PCC7120

status: Public on May 12 2022

first: Marcus

last: Ziemann

state: Baden württemberg

address: Schänzlestraße 1

institute: Albert-Ludwigs-Universität Freiburg

summary: To examine Alr3614 influence on the expression of Anabaena sp. PCC 7120, we have used customized microarrays to identify genes potentially regulated by this short DNA-binding protein. We compared the expression of an Alr3614-deletion strain with an Alr3614-complementation strain.

overall_design: The Anabaena sp. PCC 7120 cultures incubated under optimal conditions before cell harvest and two different experiments were performed for each strain.

first: Viktoria

last: Reimann

first: Wolfgang

last: Hess

first: Jens

last: Georg


title: The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries [scRNA-seq]

status: Public on May 01 2022

city: Boston

first: CBDM

last: Lab

state: MA

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: Specific microbial signals induce the differentiation of a distinct pool of RORγ+ regulatory T cells (Tregs) crucial for intestinal homeostasis. We discovered highly analogous populations of microbiota-dependent Tregs that promote tissue regeneration at extra-gut sites, notably acutely injured skeletal muscle and fatty liver. Tissue damage elicited the emigration of RORγ+ Tregs from the gut to compromised tissues, wherein they regulated the dynamics and tenor of early inflammation and helped balance the proliferation versus differentiation of local stem cells. Reining in IL-17A-producing T cells was a major mechanism underlying these rheostatic functions. Our findings highlight the importance of gut-trained Treg emissaries in controlling the response to sterile injury of non-mucosal tissues.

overall_design: We investigated the transcriptional heterogeneity of skeletal muscle regulatory T cells (Tregs) 3 days after cardiotoxin-induced injury via single-cell RNA and Tcr sequencing (scRNA-seq/ scTcr-seq). For standard genome-wide scRNA-seq analyses, Tregs from hindlimb muscles of Foxp3-Gfp, Foxp3-cre x Maffl/fl (MafΔTreg)  and Foxp3-cre x Mafwt/wt (Maf-wt) were sorted by flow cytometry and encapsulated using the Chromium Single Cell 3′ v2 platform. For coupled scRNA-seq and scTcr-seq, Tregs or CD4+ T cells from muscles and colon of six individual Foxp3Gfp mice were sorted and encapsulated using the Chromium Single Cell 5′ v3 and V(D)J platform (10x Genomics).

first: Bola

last: Hanna

first: Diane

last: Mathis

first: Christophe

last: Benoist


title: Design principles and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

status: Public on May 03 2022

city: Guelph

first: Rebecca

last: Shapiro

state: ON

address: 50 Stone Road East

department: Molecular and Cellular Biology

country: Canada

institute: University of Guelph

summary: Here, we sought to assess target-specificity and detect potential off-target effects of a novel CRISPR activation (CRISPRa) system in Candida albicans. We used the CRISPRa system to target ALS1 for overexpression, and we also created a CRISPRa strain that does not target any gene for overexpression for use as a control. Differential gene expression analysis between the CRISPRa-ALS1 strain and the non-targeting strain allowed us to determine if ALS1 was the only gene overexpressed by our CRISPRa-ALS1 strain, or if the system non-specifically targeted other genes as well.

overall_design: Differential gene expression profiling analysis of RNA-seq data for a CRISPRa-ALS1 overexpression strain and a non-targeting CRISPRa control strain.

first: Nicholas

last: Gervais

first: Victoria

last: Acquaviva

first: Alyssa

last: La Bella

first: Madison

last: Best

first: Lauren

last: Wensing

first: Jehoshua

last: Sharma

first: Deeva

last: Uthayakumar

first: Alejandro

last: Chavez

first: Felipe

last: Santiago-Tirado

first: Ana

last: Flores Mireles


title: The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries [bulk RNA-seq]

overall_design: To analyze the transcriptional differences between RORγ+ Tregs from the colon and regenerating muscle, Foxp3-Thy1.1x RorcGFP/wt male mice were injected with cardiotoxin, and 3 days later RORγ+ Tregs (CD4+TCRβ+Foxp3Thy1.1+RORγGFP+) were sorted from both tissues, and their transcriptional profiles were determined using RNA-seq. To assess the impact of RORγ+ Tregs on muscle regeneration, we analyzed the transcriptomes of whole muscle samples from Foxp3-cre x Maffl/fl (MafΔTreg) mice versus Foxp3-cre x Mafwt/wt (Maf-wt) littermate controls 7 days after cardiotoxin-induced muscle injury. For sorted muscle satellite cells, samples were analyzed from rIL-17A-treated mice against PBS-treated controls at 0, 1 and 3 days after cardiotoxin-induced muscle injury. To study the impact of RORγ+ Tregs on non-alcoholic steatohepatitis (NASH) development, we analyzed the transcriptomes of whole liver samples from MafΔTreg mice versus Maf-wt littermate controls 7 days after feeding them a choline-deficient, amino-acid-defined, high-fat diet (CDAHFD).


title: Gene expression signatures in human oral squamous cell carcinoma (OSCC) cell lines

status: Public on Apr 30 2022

city: Nagoya

first: Akinobu

last: Ota

state: Aichi

address: 1-1 Yazakokarimata

department: Food and Nutritional Environment

country: Japan

institute: Kinjo Gakuin University

summary: To investigate the effect of PBK inhibitor OTS514 on gene expression, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to suppress the cell proliferation of human OSCC cell lines.  The OSCC cells were treated with OTS514 (20 nmol/L) for 24 h in vitro.

overall_design: OSCC cell lines (HSC-2, HSC-3, SAS, and OSC-19) were treated with OTS514 (20 nmol/L) for 24 h in vitro.

first: Mikako

last: Kato

first: Sivasundaram

last: Karnan


title: The developmental transcriptome and epigenome of Owenia fusiformis reveal the development and evolution of mitraria larvae

status: Public on May 04 2022

city: London

first: Yan

last: Liang

state: London

address: mile end road

department: SBBS

country: United Kingdom

institute: Queen Mary Univerisity of London

summary: Owenia fusiformis has a unique mitraria larvae form which is different to other marine spiralians. we utilize RNA-seq and ATAC-seq to profile the gene expression and chromatin accessibility to understand their development and evolution.

overall_design: Collected the  fresh samples from the zygotes to the juvenile stages, profiling the gene expression; Collected the fresh samples from blastula to competent larvae, profiling the chromatin accessibility.

first: Chema

last: Martin


title: The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries

first: CBDM

last: Lab


title: IL-11 is a therapeutic target in idiopathic pulmonary fibrosis

status: Public on May 06 2022

city: Singapore

first: Eleonora

last: Adami

address: 8 College Road

department: CVMD

country: Singapore

institute: Duke-NUS Medical School

summary: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease where invasive pulmonary myofibroblasts secrete collagen and destroy lung integrity. Here we show that IL-11 is upregulated in the lung of IPF patients, associated with disease severity and is secreted from IPF fibroblasts. In vitro, IL-11 stimulates lung fibroblasts to become invasive, ACTA2+ve, collagen secreting myofibroblasts, in an ERK-dependent fashion. In mice, fibroblast-specific transgenic expression or administration of Il-11 drives lung fibroblast-to-myofibroblast transformation and causes lung fibrosis. Il11ra1 deleted mice, whose lung fibroblasts are unresponsive to pro-fibrotic stimulation, are protected from fibrosis in the bleomycin mouse model of pulmonary fibrosis. We generated an IL-11 neutralising antibody that blocks lung fibroblast activation downstream of multiple stimuli and reverses myofibroblast activation. In therapeutic studies, anti-IL-11 treatment both prevented and reversed lung fibrosis, which was accompanied by diminished Erk activation. These data prioritise IL-11 as a drug target for lung fibrosis and IPF.

overall_design: For human  in vitro samples, 3 commercial and 2 patient-derived normal lung fibroblasts were stimulated with either TGFB or IL11 and compared with baseline accounting for the sample effect (matched design) and the source effect. Mouse in vitro samples were also treated similarly and were analyzed using a matched design. Mouse in vivo samples have 4 replicates per treatment group.

first: Benjamin

last: Ng

first: Jinrui

last: Dong

first: Giuseppe

last: D’Agostino

first: Sivakumar

last: Viswanathan

first: Anissa

last: Widjaja

first: Wei-Wen

last: Lim

first: Nicole

last: Ko

first: Jessie

last: Tan

first: Sonia

middle: P

last: Chothani

last: Huang

first: Chen

last: Xie

first: Chee

last: Pua

first: Ann-Marie

last: Chacko

first: Nuno

last: Guimarães-Camboa

first: Sylvia

middle: M

last: Evans

first: Adam

last: Byrne

first: Toby

last: Maher

first: Jiurong

first: Dianhua

last: Jiang

first: Paul

middle: W

last: Noble

first: Sebastian

last: Schafer

first: Stuart

last: Cook


title: Transcriptomic Profiling of Peripheral Edge of Lesions to  Elucidate the Pathogenesis of Psoriasis Vulgaris

status: Public on Apr 28 2022

city: Klong Luang

first: Onjira

last: Jumlongpim

state: Pathumthanii

address: Chulabhorn international college of medicine, 99 Moo 18

department: Dermatology

country: Thailand

institute: Chulabhorn International College of Medicine, Thammasat University

summary:
To identify the gene expression in the peripheral edge of the lesional (PE) skin of psoriasis vulgaris.

Methods: Full-thickness skin biopsies of PE skin and uninvolved skin were taken from psoriasis patients. Transcriptomic profiling was constructed by high-throughput next-generation sequencing (NGS) technology.

Result: A total of 1,202 DEGs were identified. Of these, 653 (54%) were upregulated and 549 (46%) were downregulated. These DEGs might play a pivotal role in psoriasis pathogenesis.

This study accelerates psoriasis-associated gene discovery in the whole picture of PE skin.

overall_design:
PE skin and uninvolved (UN) skin biopsies were taken patients with moderate to severe psoriasis vulgaris.

RNA-seq was performed using Next-Generation sequencing.

Alterations of mRNA in PE skin compared to those in UN skin were identified as differentially expressed genes (DEGs).

first: Suphagan

last: Boonpethkaew

first: Jitlada

last: Meephansan

first: Pattarin

last: Tangtanatakul

first: Wipasiri

last: Soonthornchai

first: Jongkonnee

last: Wongpiyabovorn

first: Ratchanee

last: Vipanurat

first: Komine

last: Komine


title: Single cell RNA sequencing of paired myelodysplastic syndrome and secondary acute myeloid leukemia bone marrow aspirate samples

city: Nashville

middle: Brent

last: Ferrell

state: TN

address: 777 Preston Research Bldg., 2220 Pierce Ave.

department: Medicine

institute: Vanderbilt University Medical Center

summary: This experiment is designed to compare single cell gene expression in paired samples from two individuals who had MDS that progressed to sAML.

overall_design: Bone marrow mononuclear cells were used for scRNAseq analysis with 10x Chromium controller. Patient 17 samples were run on the same 10x chip in different lanes and sequenced together. Patient 3 samples were hashtagged and combined in one lane of a 10x chip and sequenced together.

first: Tiffany

last: Guess

first: P

middle: B


title: RNA-seq experiments on heart regeneration in WT, foxm1 and dusp6 mutants.

status: Public on Jun 22 2022

city: Pittsburgh

first: Michael

last: Tsang

state: PA

address: 3501 Fifth Ave. BST3-5062

department: Cell Biology

institute: University of Pittsburgh

summary: The study compares gene expression profile at several stages post amputation of the adult zebrafish ventricular heart between zebrafish mutants and WT siblings. The first experiment was to identify genes that are activated in response to cardiac injury at 3 and 7 days post amputation (dpa). Dusp6 mutant hearts were reported to show an enhanced regenerative response. For this experiment, bulk RNA seq was obtained from WT and Dusp6 mutant hearts and genes increased at 3 and 7 dpa were identified. The forkhead transcription factor, foxm1, showed increased expression in cardiomyocytes and follow up studies show that it is required to regulate cardiomyocyte proliferation. This was further explored with RNA-seq experiments comparing WT and foxm1 mutant hearts at 3dpa. We identified genes normally expressed in proliferating cells to be decreased in the foxm1 mutants.

overall_design: Ventricular resection was performed on WT, dusp6 and foxm1 mutant hearts. Uninjured adult hearts were used as control to compare to hearts at 3 and 7 days post amputation (dpa).

first: Zuppo

last: Daniel

first: Missinato

last: Maria

first: Tsang

last: Michael


title: Mesoderm-derived PDGFRA+ cells regulate emergence of hematopoietic stem cells in the dorsal aorta

status: Public on Apr 26 2022

city: UNSW Sydney

first: Yizhou

last: Huang

state: NSW

address: High Street

institute: University of New South Wales

summary:
During embryonic development, the first hematopoietic stem cells (HSCs) arise from a transient population of endothelial cells lining the ventral surface of the dorsal aorta, via a process of endothelial to hematopoietic transition (EHT) at embryonic day (E) 10.5. This region contains resident PDGFRA+ stromal cells (PSCs), but their identity and role in HSC generation in the AGM are not well understood. Using a library of compound transgenic mice, we identified a population of PDGFRA+/Nestin-GFP (N-GFP)-/PDGFRB-/CD31- cells with PSC activity in the E10.5 and E11.5 mouse AGM. Freshly isolated PSCs were adept at forming blood vessels with CD31+ luminal endothelium enveloped by PDGFRB+ pericytes, when transplanted subcutaneously into mice. Conditional ablation of PDGFRA+ or Nestin+ cells led to either complete or partial loss of PSCs respectively, with severe loss of endothelial and pericyte-like cells, and concomitant loss of blood formation in the AGM. Lineage tracing studies using tamoxifen induction in PDGFRACreERT2/R26eYFP embryos showed that stromal, sub-endothelial, endothelial and long-term repopulating hematopoietic stem cells (LT-HSCs) cells in the E11.5 AGM were progeny of PDGFRA cells. Using transgenic reporter mice, we showed that MesP1 (mesoderm) derived PDGFRA+ cells dominated the sub-endothelial and deeper ventral stroma in the AGM at E10.5 and E11.5 but were replaced by Wnt1 (neural crest) derived cells at E13.5. Re-aggregation of E11.5 Mesp1 derived PSC cells with E13.5 aortic or adult cardiac non-hemogenic endothelial cells resulted in the generation of endothelial cell derived LT-HSCs. RNA-sequencing analysis of non-hemogenic E13.5 endothelial cells showed up-regulation of EHT genes, WNT, BMP and Notch cell signalling pathways when re-aggregated with E11.5 Mesp1 derived PSCs. LT-HSC generation from these re-aggregates was suppressed by dose-dependent inhibition of PDGF-AA/PDGFRA signalling. Taken together, we report that PSC populations in the AGM are temporally dynamic, and that MesP1-derived PSCs regulate hemogenic potential of the endothelium and that this cooperativity is dependent on PDGF-AA/PDGFRA signalling.
This submission represents the RNAseq component of the study.

overall_design: E11.5- and E13.5 AGM PSCs were isolated from Mesp1DsRed and Wnt1DsRed embryos and cultured as previously described. Dissected AGMs were transferred into collagenase Type II (263 U/ml; Worthington Biosciences, Lakewood, NJ) and placed on a shaker at 37˚C for 15 min. The supernatant was passed through a 40 µm filter into a fresh tube and inactivated with 100% FCS. Cells were washed twice in 2% FCS in PBS and plated in alpha-MEM (Invitrogen, Carlsbad, CA) with 20% FCS, and penicillin/ streptomycin/ glutamine (P/S/G, Invitrogen)2 and cultured in the incubator at 37˚C, 5% CO2 for 72 h. At the end of 72 hrs, cells were washed in PBS to remove non-adherent cells and cultures were continued in fresh medium. Cells were passaged on reaching 80% confluence. After passaging, cells were placed back in tissue culture flasks with alpha-MEM + 20% FCS + P/S/G for bulk passaging. Cells were routinely cryopreserved in 10% DMSO and 90% culture medium.   Re-aggregates were made by reconstituting FAC sorted cells from one AGM equivalent or 50,000 adult cardiac endothelial cells with 200,000 AGM PSCs. Dissociated cells were resuspended in 10μl of IMDM+ (IMDM Invitrogen) containing 20% fetal calf serum, 4mM L-glutamine, 50units/ml penicillin/streptomycin, 0.1mM mercaptoethanol, 100ng/ml IL, 100ng/ml SCF and 100ng/ml Flt3L (Peprotech). Re-aggregates were made by centrifugation in a yellow tip occluded by parafilm at 300 x g for 5 min and cultured on top of a 0.65μm Durapore filter (Millipore, Cat. No. DVPP02500) at the gas-liquid interface as described. Tissues were maintained in 5% CO2 at 370C in a humidified incubator.

first: Vashe

last: Chandrankanthan

first: Dominik

last: Beck


title: Copy number variations may contribute to congenital heart defect risk greatly by disrupting long noncoding RNAs

status: Public on Apr 24 2022

city: Shanghai

first: Yibo

last: Lu

address: No.1678, Dongfang Road, Shanghai

department: Shanghai Children's Medical Center

institute: Shanghai Jiao Tong University

summary: We identified lncRNAs locating in CNV locus which were coexpressed with multiple congenital heart defect associated genes. To validate our findings, we performed overexpression and knockdown experiments to characterize the trascriptomic profiles regulated by HSALNG0104472.

overall_design: Paired RNA-seq of lncRNA HSALNG0104472 knockdown cardiomyocytes (AC16), HSALNG0104472 overexpression cardiomyocytes, control cardiomyocytes for overexpression of HSALNG0104472 and control cardiomyocytes for knockdown of HSALNG0104472. Three biological replicates were obtained for each sample.

first: Qing

last: Fang

first: Bo

last: Wang


title: Spatial Total RNA-Sequencing of regenerating mouse hindlimb muscle and Type 1-Lang reovirus-infected mouse heart

status: Public on Apr 20 2022

city: New York

first: David

last: McKellar

state: NY

address: 101 6th Ave

institute: New York Genome Center

summary: Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying endogenously polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNAs enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We apply this Spatial Total RNA-Sequencing (STRS) approach to spatially map noncoding RNAs, newly transcribed RNAs, and non-host RNAs at the tissue-scale in the study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify noncoding transcripts which exhibit zonal variation in skeletal muscle regeneration, and highlight host transcriptional responses highly associated with local viral RNA abundance. Our in situ polyadenylation strategy relies on a brief, low-cost add-on to a widely used protocol for spatial RNA-sequencing, and thus could be broadly and quickly adopted. Spatial RNA-sequencing of the total transcriptome will enable new insights into spatial gene regulation and biology.

overall_design: Spatial total RNA-sequencing was performed using a modified version of the Visium protocol. After dissection, tissues were embedded in OCT compound and frozen fresh in liquid nitrogen. 10um thick tissue sections were mounted onto the Visium Spatial Gene Expression v1 slides. For heart samples, one tissue section was placed into each 6x6mm capture area. For skeletal muscle samples, two tibialis anterior sections were placed into each capture area. After sectioning, tissue sections were fixed in methanol for 20 minutes at -20oC. Next, H&E staining was performed according to the Visium protocol, and tissue sections were imaged. After imaging, the slide was placed into the Visium Slide Cassette. In situ polyadenylation was then performed using yeast Poly(A) Polymerase (Thermo Scientific, cat #74225Z25KU). First, samples were equilibrated by adding 100ul of 1X wash buffer (20ul 5X yPAP Reaction Buffer, 2ul 40U/ul Protector RNase Inhibitor, 78ul nuclease-free H2O) to each capture area and incubating at room temperature for 30 seconds. The buffer was then removed. Next, 75ul of yPAP enzyme mix (15ul 5X yPAP Reaction Buffer, 3ul of 600U/ul yPAP enzyme, 1.5ul 25mM ATP, 3ul 40U/ul Protector RNase Inhibitor, 52.5ul nuclease-free H2O) was added to each reaction chamber. The reaction chambers were then sealed and the slide cassette was incubated at 37oC for 25 minutes. The enzyme mix was then removed. Prior to running STRS, optimal tissue permeabilization time for both heart and skeletal muscle samples was determined to be 15 minutes using the Visium TIssue Optimization Kit from 10x Genomics. Following in situ polyadenylation, the standard Visium library preparation was followed to generate cDNA and final sequencing libraries. The libraries were then pooled and sequenced according to guidelines in the Visium Spatial Gene Expression protocol using either a NextSeq 500 or NextSeq 2000.

first: Madhav

last: Mantri

first: Iwijn

last: De Vlaminck

first: Benjamin

last: Cosgrove


title: Decoding the PITX2 controlled genetic network in atrial fibrillation

city: Cincinnati

first: Jeffrey

last: Steimle

email: jeffrey.steimle@cchmc.org, jeffrey.steimle@bcm.edu, jsteimle@uchicago.edu, jsteimle@alumni.nd.edu

state: OH

address: 240 Albert Sabin Way

department: Molecular Cardiovascular Biology

institute: Cincinnati Children's Medical Hospital Center

summary: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia and a major risk factor for stroke, often arises through ectopic electrical impulses derived from the pulmonary veins (PV). Sequence variants in enhancers controlling expression of the transcription factor PITX2, which is expressed in the cardiomyocytes (CMs) of the PV and left atrium (LA), have been implicated in AF predisposition. Single nuclei multiomic profiling of RNA and analysis of chromatin accessibility combined with spectral clustering uncovered distinct PV- and LA-enriched CM cell states. Pitx2 mutant PV and LA CMs exhibited gene expression changes consistent with cardiac dysfunction through cell-type-distinct, PITX2-directed, cis-regulatory grammars controlling target gene expression. The perturbed network targets in each CM were enriched in distinct human AF-predisposition genes, suggesting combinatorial risk for AF-genesis. Our data further reveals that PV and LA Pitx2 mutant CMs signal to endothelial and endocardial cells through BMP10 signaling with pathogenic potential. This work provides a multiomic framework for interrogating the basis of AF-predisposition in the PV of humans.

overall_design: The Tg(Ckmm-cre)5Khn/0 (MCK-cre) mice were crossed to the germline Pitx2tm1Jfm (Pitx2–) allele to generate MCK-cre; Pitx2+/– mice, which are crossed with homozygous Pitx2tm1.1Sac (Pitx2flox/flox) to generate the Pitx2 control (Pitx2flox/+) and Pitx2 mutant (MCK-cre; Pitx2flox/–) mice. Mice were raised to adulthood (6-8 months) and genotypes confirmed by PCR prior to experimentation. For single nuclei RNA and ATAC sequencing (snRNA-seq and snATAC-seq), the left atrium (LA) and the pulmonary vein (PV) were dissected from the adult mouse heart. The LA dissection includes both the chamber and the appendage, while the PV includes the proximal connection to the atrium, the primary PV, and the branches prior to entry into the lungs.  Nuclei were harvested from the two tissue sources in pools of 8 Pitx2 control and 8 Pitx2 mutant mice using a previously published method with modifications (Mo, A. et al. Epigenomic Signatures of Neuronal Diversity in the Mammalian Brain. Neuron 86, 1369–1384 (2015).). In brief, the tissues were minced with sharp scissors in 250μl of HB++ (HB: 0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH, pH 7.8; ++: 0.15mM spermine, 0.5mM spermidine, 1mM DTT) and centrifuged at 500g for 5 minutes to remove excess red blood cells and fat and replaced with 250μl of fresh HB++. Sample was transferred to a 2mL Dounce homogenizer and the tube washed with an additional 250μl of HB++ to collect any remaining sample and transferred. Tissue was homogenized 30 times with loose and tight pestle before adding 32μl of 5% NP40 (in HB) and Dounced with tight pestle an additional 75 times. Sample was strained through a 40μM strainer and washed with 9.5mL of NWB (PBS with 1% BSA and 0.2U RNAse inhibitor) and spun down at 500g at 4°C for 5 minutes. The supernatant removed and nuclei resuspended in 500μl of NWB and 900μl of SCB (90% Nuclei PURE 2M Sucrose Cushion Solution and 10% Nuclei PURE Sucrose Cushion Solution; Sigma NUC-201: S9308 and S9058). The 1400μl of nuclei suspension is layered on top of 500μl SCB in a 2mL LoBind Eppendorf tube and centrifuged at 13,000g at 4°C for 45 minutes. The supernatant removed leaving ~50μl of nuclei pellet, which is resuspended in 1mL of NWB. Pellet nuclei at 500g at 4°C for 5 minutes, remove supernatant leaving ~50μl of nuclei pellet. Nuclei were quantified using the inCyto C-Chip DHC-F01.  The snRNA-seq libraries were generated using the 10x Chromium Single Cell 3’ v3 reagent kit and in parallel the snATAC-seq libraries were generated using the 10x Chromium Single Cell ATAC v1 with a target of 10,000 nuclei per library according to the manufacturer’s instructions. The libraries were sequenced on an Illumina NovaSeq6000 with the Genome and RNA Profiling Core at Baylor College of Medicine and an Illumina NextSeq 500. Raw sequencing data was handled by the 10x Genomics Cell Ranger software (cellranger-3.0.1 and cellranger-atac-1.2.0) and mapped to the mouse mm10 genome. For snRNA-seq, the gene counts were quantified using cellranger count to the mouse transcriptome including pre-mRNA (v3.0.0) and passed to Seurat for downstream analysis. For snATAC-seq, samples were individually aligned using cellranger-atac count and aggregated using cellranger-atac aggr (--nosecondary --normalize=none) using the peak output from each individual sample. Following initial clustering (see below for downstream analysis) and per cluster and per sample peak calling using MACS2 callpeak (v2.1.1.20160309) with parameters -f BAMPE -g mm -B -q 0.1 samples were re-aggregated using cellranger-atac aggr using a union set of peaks and passed to Seurat and Signac for downstream analysis.

first: Francisco

last: Grisanti Canozo

first: Minjun

last: Park

first: Zachary

last: Kadow

first: M

last: Samee

first: James

middle: F

last: Martin


title: Small RNA sequencing of regenerating mouse hindlimb muscle and Type 1-Lang reovirus-infected mouse heart

type: Non-coding RNA profiling by high throughput sequencing

summary: Small RNAs, including microRNAs, play many roles in the regulation of gene expression. We performed small RNA-sequencing on two groups of samples to catalog which microRNAs are expressed during muscle regeneration in response to chemical injury and in heart tissue in response to viral-induced myocarditis.

overall_design: For skeletal muscle samples, following the injury time course, tibialis anterior muscles were dissected and snap frozen with liquid nitrogen. The Norgen Total RNA Purification Kit (Cat. 17200) was used to extract RNA from 10 mg of tissue for each sample. For heart samples, following the infection time course, hearts were dissected, embedded in OCT, and frozen in liquid nitrogen. RNA was extracted with Trizol (Invitrogen, Cat. 15596026) and glycogen precipitation for a small fraction of each of the heart samples. RNA quality was assessed via High Sensitivity RNA ScreenTape Analysis (Agilent, Cat. 5067-5579) and all samples had RNA integrity numbers greater than or equal to 7. Small RNA sequencing was performed at the Genome Sequencing Facility of Greehey Children’s Cancer Research Institute at the University of Texas Health Science Center at San Antonio. Libraries were prepared using the TriLink CleanTag Small RNA Ligation kit (TriLink Biotechnologies, San Diego, CA). Libraries were sequenced per lane with single-end 50× using a HiSeq2500 (Illumina, San Diego, CA).


title: Specific targeting of plasmids with Argonaute enables genome editing

status: Public on Apr 17 2022

city: Moscow

first: Anastasiia

last: Okhtienko

address: Akademika Kurchatova square 2

country: Russia

institute: Institute of Molecular Genetics, Russian Academy of Sciences

summary: Prokaryotic Argonaute nuclease induces DNA interference between plasmid and chromosomal DNA to promote genome recombineering

overall_design:
Argonaute-associated smDNA from E.coli transformed by plasmids of different constructions was analysed to elucidate the requirements for genomic DNA cleavage by CbAgo during DNA interference and integration of plasmid-encoded sequences into the homologous chromosomal locus

Second part of data on DNA interference research. Additional libraries from 2 clones were prepared to show the integration of plasmid-encoded genes into the homologous chromosomal locus promoted by bacterial Argonaut protein


title: Transcriptional profiles of melanoma metastases from patients responsive or resistant to BRAF/MEK inhibitor

status: Public on Apr 12 2022

city: Milan

first: Loris

last: De Cecco

address: via Venezian 1

country: Italy

institute: IRCSS Istituto Nazionale Tumori

summary: Transcriptional profiles of formalin-fixed-paraffin-embedded melanoma metastases including pre-treatment (PRE) and post-treatment (POST) specimens from 50 patients treated with BRAF inhibitors or with BRAF and MEK inhibitors

overall_design: 91 samples from 50 patients showing a long-term response (LR) or a short-term response (SR) to treatment are included


title: Hepatitis B virus core protein is not required for cccDNA transcriptional regulation

status: Public on Apr 01 2022

city: Wuhan

first: Youquan

last: Zhong

state: hubei

address: NO.115, Donghu road, Wuchang district, Wuhan city, Hubei province, China.

department: TaiKang Medical School,

institute: Wuhan University

summary:
Background & Aims: Hepatitis B virus (HBV) infection is a major health burden worldwide and currently there is no cure. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle for antiviral treatment. HBV core protein (HBc) has merged as a promising antiviral target, as it plays important roles in critical steps of viral life cycle. However, whether HBc could regulate HBV cccDNA transcription remains to be illustrated.
Methods: Synthesized HBV cccDNA and HBVcircle with or without HBc deficiency were transfected into hepatocytes. A recently reported Adeno-Associated Virus (AAV) mediated HBV cccDNA mouse model was employed. Two capsid assembly modulators (CAMs) were used. HBV replication markers were evaluated. Chromatin immunoprecipitation (ChIP) or ChIP sequencing assays were conducted with different transcription factors, histones and RNA polymerase 2.
Results: In HBV cccDNA and HBVcircle transfection assays, lack of HBc showed no effect on transcription of HBV RNA as well as HBV surface antigen production. Reconstitution of HBc did not change cccDNA derived HBV markers. Similar results were obtained in vivo, from mouse cccDNA model. ChIP data revealed similar transcription regulation of HBc deficient cccDNA chromatin with wide type cccDNA. Furthermore, CAMs treatment could not alter cccDNA transcription.
Conclusions: Our results indicate that HBc neither affects histone modifications and transcription factors binding of cccDNA, nor influences cccDNA transcription. Although CAMs could reduce HBc binding to cccDNA, it does not suppress cccDNA transcriptional activity. Thus, therapeutic targeting capsid or HBc is not sufficient to reduce cccDNA transcription.

sample_organism:
Homo sapiens
Hepatitis B virus

platform_organism:
Homo sapiens
Hepatitis B virus

overall_design: eight samples were analyzed

first: Yuchen

last: Xia

first: Wen

first: Chuanjian

last: Wu


title: N-acetyltransferase 10 drives cisplatin chemoresistance by enhancing ac4C-associated DNA repair in bladder cancer

status: Public on Apr 07 2022

city: Guangzhou

first: Ruihui

last: Xie

state: Guangdong

address: 107 Yan Jiang West Road

institute: Sun Yat-sen Memorial Hospital, SYSU

summary: To explore the molecular mechanism underlying NAT10-mediated cisplatin resistance in bladdder cancer, a genome-wide RNA-sequencing was conducted to compare gene expression profiles of T24 and UM-UC-3 cells with NAT10 knockdown and control. And acRIP-seq was performed to identified the downstream targets of NAT10.

overall_design: Detection of acetylated cytide in RNA with ac4C-immunoprecipitation (acRIP), along with RNA-seq after bladder cancer cell line T24 and UM-UC-3 were transfected with the NAT10 siRNA or control for 48h.

first: Jian

last: Huang

first: Xu

last: Chen

first: Tianxin

last: Lin


title: Investigation of Gene Regulation Induced by Merkel Cell Polyomavirus (MCV) Large T Antigen

city: Hershey

first: Hyun Jin

last: Kwun

address: 500 University Drive

department: Microbiology and Immunology

institute: Penn State Milton S. Hershey Medical Center

summary: We report the ability of MCV LT to regulate gene expression in human fibroblast cells.

overall_design: 12 samples were analyzed with three replicates per sample. Empty Vector_GFP was used as a control. Treatment groups included BJhTERT cells expressing MCV LT, MCV LT MUR deletion mutant (dMUR), and MCV LT with p21 knockdown.

first: Hyun

first: Alexander

last: Pham


title: Next generation sequencing facilitates quantitative analysis of control and NICD overexpression salivary gland imagnal ring tumor transcriptomes

city: New Orleans

first: Xian-Feng

last: Wang

state: LA

address: 1700 Tulane Avenue

institute: Tulane University

summary: We established a tumor model in which Notch overexpression can induce tumor formation in a transition-zone (TZ) region in the Drosophila salivary gland imagnal ring (ImR). TZ cells are polyploid cells and do not devide during normal development, however, polyploid TZ cells reenter to mitosis and form tumor after Notch induction. Growth and progression of the TZ tumor are fueled by a combination of polyploid mitosis, endoreplication, and depolyploidization. To determine the molecular mechanisms underlying tumor formation in the NICD-induced TZ tumors, we did RNA-seq and compared the transcriptomes between tumor and control. We found the mitosis of the salivary gland ImR tumor was due to the Drosophila Cdc25 homolog String (Stg), a M-phase inducer phosphatase, was significantly upregulated in the tumor sample. Besides, we found DNA damage response genes, also active during meiosis, are upregulated in these tumors and are required for the ploidy reduction division. This study suggests that polyploidy and associated cell-cycle variants are critical for increased tumor cell heterogeneity and genome instability during cancer progression.

overall_design: Examination of gene expression from 3 control samples and 3 ImR tumor samples

first: Deeptiman

last: Chatterjee

first: Wu-Min

last: Deng


title: RNA-Sequencing Characterisation of the Renal Cortical Response to Diet-Induced Weight Loss and Medical Therapy in the Zucker Diabetic Sprague Dawley Rat Model of Diabetic Kidney Disease

status: Public on Mar 14 2022

city: Dublin

first: William

middle: P.

state: - Select State/Province -

address: Diabetes Complications Research Centre, Conway Institute, University College Dublin

country: Ireland

institute: University College Dublin

summary: We report results from renal cortical transcriptomic analysis of responses to diet-induced weight loss plus medical therapy in the Zucker Diabetic Sprague Dawley (ZDSD) rat model of diabetic kidney disease.

overall_design: 3 study groups: Sprague Dawley rats, SHAM-operated Zucker Diabetic Sprague Dawley (ZDSD) rats, and ZDSD rats treated with diet-induced weight loss plus medical therapy (ramipril, metformin, liraglutide, rosuvastatin, fenofibrate).

first: Carel

last: le Roux

first: Neil

last: Docherty


title: 14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis [single-cell RNA-seq]

status: Public on Mar 10 2022

city: NEW YORK

first: WENHUO

last: HU

email: huwenhuo@gmail.com, huw@mskcc.org

state: NEW YORK

address: 1275 YORK AVENUE

department: HOPP

institute: MEMORIAL SLOAN KETTERING CANCER CENTER

summary: To unravel the relative abundance and distribution of PGRN, TNFR2, and 14-3-3ε mRNA transcripts in different chondrocyte subpopulations, we performed single-cell RNA-seq (scRNA-seq) in chondrocytes isolated from human OA and normal cartilage.

overall_design: scRNA-Seq were performed on the single cell suspension isolated from 3 human normal and 4 human OA cartilage

first: Wenyu

last: Fu

first: Yujianan

first: Wenhuo

last: Hu

first: Chuanju

last: Liu


title: Optimization of Class I Histone Deacetylase JPS0s Reveals HDAC1/2 Degradation is Critical to Induce Apoptosis and Cell Arrest in Cancer Cells

status: Public on Mar 09 2022

city: Leicester

first: Shaun

middle: Michael

last: Cowley

address: Henry Welcome Building, Leicester LE1 7RH

department: Molecular and Cell Biology

institute: University of Leicester

summary: Class I Histone Deacetylase (HDAC) enzymes 1, 2 & 3 organize chromatin as the catalytic subunits within seven distinct multi-protein co-repressor complexes and are established drug targets. We report optimization studies of benzamide based Von Hippel-Lindau (VHL) E3-ligase Proteolysis Targeting Chimeras (JPS0s), and for the first time describe transcriptome perturbations resulting from these degraders. By modifying the linker and VHL ligand we identified JPS0s 7, 9 & 21 with submicromolar DC50 values for HDAC1 and/or HDAC3 in HCT116 cells. A hook effect was observed for HDAC3 that could be negated by modifying the position of attachment of the VHL ligand to the linker. The more potent HDAC1/2 degraders correlated with greater total differentially expressed genes and enhanced apoptosis in HCT116 cells. We demonstrate that HDAC1/2 degradation by JPS0s correlates with enhanced global gene expression and apoptosis, important for the development of more efficacious HDAC therapeutics with reduced side effects.

overall_design: Determining differential gene expression profiles upon treatment of HCT116 cells with JPS0s targeting class I HDACs

first: India

last: Baker

first: Josh

last: Smalley

first: James

last: Hogdkinson

first: John

last: Schwabe


title: Profiling axonal transcriptome of mouse hippocampal neurons in response to force

status: Public on Mar 07 2022

city: Pisa

first: Vittoria

last: Raffa

state: Please Select

address: S.S. 12 Abetone e Brennero, 4

department: Department of Biology

institute: Università di Pisa

summary: Magnetically-actuated iron oxide nanoparticles (MNP) have emerged as a technology to chronically expose axons of developing neurons to extremely low mechanical forces. This force was found to induce axon outgrowth. Axon-Seq has been performed to profile local gene expression in response to the stimulus.

overall_design: Mouse hippocampal neurons were incubated with a growth medium modified with MNPs. Two conditions were analysed (cell cultures treated with a magnetic field or a null magnetic field).

first: Alessandro

last: Falconieri

first: Ugo

last: Borello


title: DNA methylation dynamics in primary human monocytes exposed to thyroid hormone ex vivo

status: Public on Mar 08 2022

city: Parkville

first: Boris

last: Novakovic

state: Victoria

address: Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road

country: Australia

institute: Murdoch Children's Research Institute

type: Methylation profiling by genome tiling array

summary: Trained immunity is a phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to certain microbial components, altering their responses to future exposures. This study profiled the genome-wide DNA methylation profile of human monocytes exposed to thyroid hormone for 24 hours.

overall_design: Human primary monocytes were isolated using Ficoll and two rounds of Percoll gradient separation. Baseline monocytes were collected after 1 hour in culture (attached) and 24 hours samples were collected for media only, LPS only, Thyroid hormone (T3), or T3+LPS.

first: Rebecca

last: Shepherd

first: Richard

last: Saffery


title: An AI-guided invariant signature places MIS-C with Kawasaki disease in a continuum of host immune responses

status: Public on Mar 20 2022

city: La Jolla

first: Chisato

last: Shimizu

address: 9500 Gilman Dr.

country: USA

institute: UCSD

summary: A significant surge in cases of multisystem inflammatory syndrome in children (MIS-C, also called Pediatric Inflammatory Multisystem Syndrome - PIMS) has been observed amidst the COVID-19 pandemic. MIS-C shares many clinical features with Kawasaki disease (KD), although clinical course and outcomes are divergent. We analyzed whole blood RNA sequences, serum cytokines, and formalin fixed heart tissues from these patients using a computational toolbox of two gene signatures, i.e., viral pandemic (ViP), and a subset of it called, severe (s)ViP signatures that were developed in the context of SARS-CoV-2 infection in COVID-19 and a 13-transcript signature previously demonstrated to be diagnostic for KD. Our analyses revealed that KD and MIS-C are on the same continuum of the host immune response as COVID-19 but diverge with two different cardiac phenotypes. The ViP signatures helped unravel the nature of the host immune response (IL15-centric) in MIS-C and KD, reveal unique targetable cytokine pathways in MIS-C, place MIS-C farther along in the spectrum in severity compared to KD and pinpoint key clinical (reduced cardiac function) and laboratory (thrombocytopenia and eosinopenia) parameters that can be useful to track severity.

overall_design: Analysis of whole blood RNA sequences from the patients with Kawasaki disease using a computational toolbox of two gene signatures,

first: Jane

last: Burns


title: Tertiary Lymphoid Structure Raises Survival and Immunotherapy in HPV- HNSCC

status: Public on Mar 03 2022

first: Hao

last: Li

address: 237 Luoyu Road

department: Shool and Hospital of Stomatology

country: China

summary: The presence of B cells in tumors have been recently reported to predict the better prognosis of patients with cancer. B cells were found primarily in so-called tertiary lymphoid structures (TLSs) in tumor. TLSs are ectopic lymphoid aggregates that form at sites of micro-secondary lymphoid organs, including a B cell follicle with a network of follicular dendritic cells (FDCs) surrounded by T cell zone composed of CD4+ T follicular helper (Tfh) cells and CD8+ T cells and high endothelial venules (HEVs). The presence of TLS is associated with positive outcomes in several human malignancie and More importantly, TLSs enriched in B cells have been proven beneficial for response to ICB in multiple types of cancer. Here, we establish tobacco-associated HNSCC mouse model with TLS enrichment by overexpression of LIGHT in tumor to evaluate the TLS influence in HPV- HNSCC and immunotherapy in HPV- HNSCC.

overall_design: 4MOSC1-con as control group: 3, 4MOSC1-LIGHT (overexpression of LIGHT) group: 3

first: Su-Wen

last: Zhu

first: Jun-Jie

last: Zhou

first: De-Run

last: Chen

first: Jie

first: Wu-Yin

last: Wang

first: Meng-Jie

last: Zhang

first: Zhi-Jun

last: Sun


title: Effect of Omomyc treatment on human TNBC MDAMB231 cell line

city: Barcelona

first: Mariano

last: Zacarias-Fluck

address: Natzaret, 115-117

department: Preclinical and Translational Research Program

country: Spain

institute: Vall d'Hebron Institute of Oncology (VHIO)

summary: Analysis of MDAMB231 cells treated with 12.8 µM Omomyc or vehicle (PBS) for three days.

overall_design: Samples were prepared at least in triplicates. Control samples were treated with the vehicle

first: Daniel

last: Massó-Vallés

first: Laura

last: Soucek


title: DNA methylation and transcriptional dynamics in primary human monocytes exposed to thyroid hormone ex vivo

status: Public on Mar 06 2022

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

overall_design: Refer to individual Series


title: Triple-negative breast cancer cell lines treated with mebedazole (an FDA-approved anti-parasitic).

status: Public on Feb 28 2022

city: Baltimore

first: Natalie

last: Joe

state: MD

address: 1550 Orleans Street, Suite 2-103

institute: Johns Hopkins University School of Medicine

summary: The main purpose of this project is to identify gene expression changes between treated triple negative breast cancer cells (w/ MBZ) vs. non-treated cells (w/o MBZ). We have submitted two different cell ilnes, and those overlapping pathways will be important to elucidate the mechansim of MBZ. Cell Lines = SM or MD Drug treatment = 0M or 1M

overall_design: In order to determine the overall effect of MBZ treatment in triple-negative breast cancer (TNBC), TNBC cell lines (MDA-MB-231 and SUM159) were treated either with 1 uM MBZ or DMSO vehicle control for 72 hours and RNA was extracted.

first: Daniele

last: Gilkes


title: Transcriptional dynamics in primary human monocytes exposed to thyroid hormone ex vivo

first: Boris

last: Novakovic

summary: Trained immunity is a phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to certain microbial components, altering their responses to future exposures. This study profiled the transcriptome of human monocytes exposed to thyroid hormone for 24 hours.

overall_design: Human primary monocytes were isolated using Ficoll and two rounds of Percoll gradient separation. Baseline monocytes were collected after 1 hour in culture (attached). Samples for media only, LPS only, Thyroid hormone (T3), or T3+LPS exposures were collected at 4 hours and 24 hours post stimulation.

first: Rebecca


title: Control and maintenance of β-cell identity and function by calcineurin/NFATc2 and ROS/PI3K signaling during metabolic and inflammatory stress

status: Public on Mar 01 2022

city: Dallas

first: Nicole

last: Baldwin

address: 3434 Live Oak St

institute: Baylor Research Institute


title: RNA-Seq to identify genes regulated by NFATc2 expression in islet stress response and function

summary: The downstream CN target NFATc2 is highest among enriched regulators of top induced genes in human islets exposed to high glucose and IL-1β. Thus, we used a conditional transgenic β-cell specific NFATc2 knock out (NFATc2βKO) mouse model to analyze requirements of NFATc2 to regulate genes in response to metabolic and inflammatory stress. Transcript analysis of NFATc2βKO islets exposed to high glucose and IL-1β revealed NFATc2-dependent downregulation of genes known to promote β-cell differentiation and upregulation of β-cell disallowed genes. Collectively, these results suggest that CN/NFATc2 signaling is required for expression of genes that maintain β cells in a differentiated state, and its inactivation allows genome-wide transcriptional changes that promote β-cell dedifferentiation when exposed to metabolic and inflammatory stress.

overall_design: islets were isolated from two wildtype and two NFATc2βKO mice. Islets from each mouse were either untreated or subjected to high glucose and cytokine cocktail for 2 hr. All treatments and knockout mice samples were compared to wildtype controls.

first: Carly

last: Darden

first: Michael

last: Lawrence


title: RNA-Seq to identify genes expressed by islets in response to metabolic and inflammatory stress and the role of CN/NFAT signaling in this response

summary: To identify genes expressed in islets in response to metabolic and inflammatory stress, we performed total RNA-Seq on isolated human islets exposed to IL-1β in the presence of high glucose. Gene ontology (GO) analysis of upregulated transcripts indicated enrichment of genes associated with cytokine signaling and proinflammatory responses. Multiple cytokines and chemokines were induced, and several key regulators of oxidative and ER stress responses were upregulated. The results revealed acute induction of proinflammatory transcripts and upregulation of genes involved in oxidative and ER stress in human islets in response to IL-1β and high glucose. Blockade of CN/NFAT signaling by FK506 pretreatment resulted in suppression of genes required for islet cell differentiation, development, and function.

overall_design: Islets from a human donor were collected and experimental treatments done in duplicate. Treatment samples were normalized to the untreated control samples.


title: DNA methylation and transcriptomic signatures in patients with peanut- and multi-food allergy


title: NPM1c hijacks the active trancription in leukemia development [CUT&RUN]

status: Public on Feb 27 2022

city: Houston

first: Xiaotian

state: TX

address: 6431 Fannin St

department: Biochemistry and Molecular Biology

institute: The University of Texas Health Science Center at Houston

summary: Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of functions including ribosome biogenesis, mRNA processing, and maintenance of genomic stability1-4. In acute myeloid leukemia (AML), the terminal exon of NPM1 is often mutated (~30% of adult AMLs), resulting in the change of the nucleolar localization signal into a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c)2,3,5. AMLs carrying this mutation have aberrant expression of the HOXA genes, whose overexpression leads to leukemogenic transformation6-8. Recently, it was shown that depletion or re-localization of the NPM1c protein into the nucleus causes downregulation of the HOXA genes, leading to the speculation that NPM1c directly regulates their transcription9-11. Here, we show that NPM1c binds to a subset of active gene promoters marked with high levels of H3K27ac in NPM1c leukemia cell lines and primary leukemia blasts, including well-known leukemia-driving genes such as posterior HOXA, HOXB, and MEIS1/PBX3 genes as well as novel targets IRX5 and NKX2-3. The binding of NPM1c on chromatin sustains active transcription of key target genes by maintaining high local-concentration of transcriptional complexes, including the Super Elongation Complex (SEC) and Menin/MLL1. NPM1c also maintains the active chromatin landscape by inhibiting the activity of histone deacetylases (HDACs). Depletion of NPM1c causes histone deacetylation and the silencing of key leukemic genes, leading to cell differentiation and growth arrest. The export protein XPO1 plays a key role by tethering NPM1c onto chromatin. The combination of XPO1 inhibitors (e.g., Selinexor and Eltanexor) with the Menin inhibitor MI-3454 has a synergistic effect on inducing differentiation in both NPM1-mutated leukemia cell lines and PDX model. Together, these findings reveal the neomorphic function of NPM1c as transactivator for leukemic gene expression and open up potential avenues for therapeutic intervention.

overall_design: XPO1 and NPM1c's chromatin binding is assayed by using Cut&Run with the 500nM dTag-13 treatment at different time points in OCI-AML3 Degron2 cells. Samples were taken at 3hr, 6hr, 24hr, 4 days after wash-off. Various other transcriptional complex - RNA polemerase 2, Menin were assayed by using Cut&Run with the 500nM dTag-13 treatment at different time points in OCI-AML3 Degron2 cells. Samples were taken at 1hr,3hr, 6hr, 24hr. XPO1 and NPM1c's chromatin binding has also been examined in primary human NPM1c+ acute myeloid leukemia blast as well as NPM1c knock-in of HOXB8 cell lines.


title: Ocular proteomic and transcriptomic changes with aging in response to lensectomy in a rabbit model of lensectomy with intraocular lens insertion

status: Public on Feb 26 2022

city: Wauwatosa

first: Iris

last: Kassem

state: Wisconsin

address: 8701 W Watertown Plank Rd

department: Ophthalmology

institute: Medical College of Wisconsin

summary: Previous work has established the use of a juvenile rabbit model and demonstrated an increased abundance of inflammatory and coagulation cascade proteins in the aqueous humor (AH) following lensectomy with IOL implantation at the time of surgery (Young et al., 2020).Expanding on this work, we seek to establish how the abundance of inflammatory and coagulation proteins in the anterior chamber changes with increasing age following lensectomy with IOL insertion in this rabbit animal model, seeking to explain the difference in post-operative scarring seen between children and adult humans.

overall_design: Comparing the transcription profile of the cornea and iris/ciliary body (CB) in juvenile and adult rabbits and specifically looking at proteins related to coagulation and inflammation and how they change with age in the anterior segment following lensectomy with IOL insertion

first: Jonathan

last: Young

first: Niamh

last: Wynne

first: Khalid

middle: Y

last: Al-Kirwi

first: Amanda

middle: R

last: Buchberger

first: Christine

last: Skumatz

first: Ross

last: Collery


title: Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay

status: Public on Feb 17 2022

city: Bar Harbor

first: Kousuke

last: Mouri

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: MPRAduo dataset contains two types of sequencing reads: (i) reads to associate test elements and barcode tags and (ii) reads to count barcode tags from cDNA and plasmid samples to calculate expression level. Benchmark library was tested in GM12878 with 2 runs: SE library (run1) and ES library (run2). The library for whole genome REST binding site (WGREST) was tested in GM12878, HepG2, K562, and SK-N-SH. All tag counting was done with 4 biological replicates.

first: Hannah

last: Dewey

first: Ryan

last: Tewhey


title: DNA methylation and transcriptomic signatures in patients with peanut- and multi-food allergy (RNA-Seq)

summary: This study profiled the epigenomes and transcriptomes of total nCD4T cell populations from adolescents with peanut-only (single food allergy- SA) and multi-food allergy (MA) at quiescence and following activation with anti-CD3/antiCD28

overall_design: mRNA sequencing of total nCD4T cell populations isolated using flow cytometry, and activated with anti-CD3/CD28 for 72 hours from single-food allergic, multi-food allergic, and non-allergic adolescents.

first: Melanie

last: Neeland

first: Samira

last: Imran

first: Kirsten

last: Perrett

first: Richard

last: Saffery


title: Synthetic lethality of paclitaxel and afatinib in MDA-MB-231 cells

status: Public on Apr 02 2022

city: New York

first: Won-Min

last: Song

state: New York

address: One Gustave L. Levy Place, Box 1123

institute: Icahn school of medicine at Mount Sinai

summary: We performed mono- or combo-therapies of paclitaxel and afatinib and performed RNA-seq to compare the perturbed transcriptome in MDA-MB-231 cells

overall_design: 4 replicates of afatinib, paclitaxel, afatinib + pacliataxel and DMSO treated samples

first: Pei-ling

last: Chia


title: ZnT8 Loss of Function Mutation Increases Resistance of Human Embryonic Stem Cell-Derived Beta Cells to Apoptosis in Low Zinc Condition

status: Public on Feb 11 2022

city: Tarrytown

first: Sandra

last: Kleiner

address: 777 Old Saw Mill River Rd

institute: Regeneron Pharmaceuticals, Tarrytown, New York, USA

summary: The rare SLC30A8 mutation encoding a truncating p.Arg138* variant (R138X) in zinc transporter 8 (ZnT8) is associated with a 65% reduced risk for type 2 diabetes. To determine whether ZnT8 is required for beta cell development and function, we derived human pluripotent stem cells carrying the R138X mutation and differentiated them into insulin-producing cells. We found that human pluripotent stem cells with homozygous or heterozygous R138X mutation and the null (KO) mutation have normal efficiency of differentiation towards insulin-producing cells, but these cells show diffuse granules that lack crystalline zinc-containing insulin granules. Insulin secretion is not compromised in vitro by KO or R138X mutations in human embryonic stem cell-derived beta cells (sc-beta cells). Likewise, the ability of sc-beta cells to secrete insulin and maintain glucose homeostasis after transplantation into mice was comparable across different genotypes. Interestingly, sc-beta cells with the ZnT8 KO mutation showed increased cytoplasmic zinc, and cells with either KO or R138X mutation were resistant to apoptosis when extracellular zinc was limiting. These findings are consistent with a protective role of zinc in cell death and with the protective role of zinc in T2D.

overall_design: Gene expression difference between R138X, SLC30A8 NULL compared to control samples.

first: Lina

last: Sui

first: Anthony

last: Romer

first: Qi

last: Su

first: Yurong

last: Xin

first: Jinrang

last: Kim

first: Dieter

last: Egli


title: Single cell-type transcriptome profiling reveals genes that promote nitrogen fixation in the infected and uninfected cells of legume nodules

status: Public on Jan 18 2022

city: Hefei

first: Longlong

last: Wang

state: Anhui

address: No. 130, Changjiang West Road

department: School of Agronomy

institute: Anhui Agricultural University

summary: In this study, we have manually separated two main types of cells in mature nodules, known as infected cells (ICs) and uninfected cells (UCs). Smart-Seq2 mediated single-cell RNA sequencing reveals that 939 genes are significantly differentially expressed between ICs and UCs, including transcription factors, transporters, and catalytic enzymes. our data provide valuable resources for investigating genetic components responsible for the differentiated but coordinated cellular process between ICs and UCs. It also lays the foundation for improving the SNF efficiency in legume crops and for reconstructing the SNF system in non-legume crops.

overall_design: Single-cell transcriptome profiling of the infected cells and uninfected cells in mature root nodules at 4 weeks post-inoculation.

first: Yu

last: Zhou

first: Runhui

last: Li

first: Jianjun

last: Liang

first: Tao

last: Tian

first: Jie

last: Ji

first: Runzhou

last: Chen

first: Yumiao

first: Qiuling

last: Fan

first: Guogui

last: Ning

first: Robert

last: Larkin

first: Manuel

last: Becana

first: Deqiang

last: Duanmu


title: RNA-seq of Acinetobacter baumannii ATCC 17978 Lab-WT: under blue light

status: Public on Jan 31 2022

city: Seoul

first: Jihye

last: Yang

address: Anam-ro 145

department: Department of Environmental Science and Ecological Engineering

institute: Korea University

summary: Comparison for gene expression of Acinetobacter baumannii Lab-WT under blue light irradiation

overall_design: 1 control (dark control) and 1 sample (blue light irradiated, 6W/m²), 12h at 23 °C


title: Inflammatory monocytes promote granuloma control of Yersinia infection

status: Public on Feb 07 2022

city: Philadelphia

first: Rina

last: Matsuda

state: PA

address: 3800 Spruce Street

institute: University of Pennsylvania

summary: Granulomas are organized immune cell aggregates formed in response to chronic infection or antigen persistence. The bacterial pathogen Yersinia pseudotuberculosis (Yp) blocks innate inflammatory signalling and immune defence, inducing neutrophil-rich pyogranulomas (PGs) within lymphoid tissues. Here we uncover that Yp also triggers PG formation within the murine intestinal mucosa. Mice lacking circulating monocytes fail to form defined PGs, have defects in neutrophil activation and succumb to Yp infection. Yersinia lacking virulence factors that target actin polymerization to block phagocytosis and reactive oxygen burst do not induce PGs, indicating that intestinal PGs form in response to Yp disruption of cytoskeletal dynamics. Notably, mutation of the virulence factor YopH restores PG formation and control of Yp in mice lacking circulating monocytes, demonstrating that monocytes override YopH-dependent blockade of innate immune defence. This work reveals an unappreciated site of Yersinia intestinal invasion and defines host and pathogen drivers of intestinal granuloma formation.

overall_design: Total 9 samples. Both non-granuloma and granuloma samples were collected from three Yersinia-infected mice. Uninfected samples were collected from three uninfected mice.

first: Daniel

last: Sorobetea

first: Stefan

last: Peterson

first: James

last: Grayczyk

first: Indira

last: Rao

first: Elise

last: Krespan

first: Matthew

last: Lanza

first: Charles-Antoine

last: Assenmacher

first: Matthias

last: Mack

last: Beiting

first: Enrico

last: Radaelli

first: Igor

last: Brodsky


title: Evidence for CD04+ T Cell Methylation Signatures of Network-oriented SOCS3, ITGAL, and NR4A2 Genes with Hemodynamics in Patients with Pulmonary Arterial Hypertension (Array2 DataSet)

city: Naples

first: Claudio

last: Napoli

state: Italy

address: P.za Miraglia, 2

institute: University of Campania "L. Vanvitelli"

summary: We hypothesized that methylation dynamics in circulating CD04+ T cells may underlie a precise transcriptional reprogramming that associates with the cardiopulmonary hemodynamic profile of affected patients. We aimed at profiling DNA methylome of circulating CD04+ T cells from patients with PAH and controls by integrating network medicine and hemodynamic parameters.

overall_design: Identification of differentially methylated CpGsites (dmCpGs) annotated to specific genes able to discriminate circulating CD04+ T cells isolated from PAH and controls.

first: Giuditta

last: Benincasa

first: Bradley

last: Maron

first: Ornella

last: Affinito

first: Michele

last: D’Alto

first: Monica

last: Franzese

first: Paola

last: Argiento

first: Concetta

last: Schiano

first: Emanuele

last: Romeo

last: Bontempo

first: Paolo

last: Golino

first: Liberato

last: Berrino

first: Joseph

last: Loscalzo


title: Transcriptomic Analysis of Classical Monocytes from African American Patients with Systemic Sclerosis

city: Birmingham

first: Peter

middle: Christopher

last: Allen

state: Alabama

address: 1720 University Blvd

department: Graduate Biomedical School

institute: University of Alabama at Birmingham

summary: Systemic sclerosis is a multisystem autoimmune disorder that has an unclear etiology and disproportionately affects African Americans (AA). Additionally, monocytes show heightened activation in SSc, and in AA relative to European ancestry (EA) individuals. Monocytes are thus a good target tissue for elucidating disease mechanisms.  In this study, we sought to identify differentially expressed genes in classical monocytes from AA SSc patients relative to unaffected AA controls.  We performed RNAseq to profile the transcriptome on FACS-isolated classical monocytes (CD14++CD16-) from 16 female AA SSc cases and 18 female AA controls.  We observed modest gene expression differences between cases and controls. The genes harboring the top differentially methylated cytosines are enriched for metabolic processes. The relatively modest differences in gene expression observed between patients and controls is consistent with prior evidence of stronger inflammatory signatures in AA.

overall_design: RNAseq was was used to profile gene expression levels on FACS-isolated classical monocytes (CD14++CD16-) from 16 female AA SSc cases and 18 female AA controls.

first: Devin

last: Absher

first: Paula

last: Ramos


title: Classical Monocytes from African American Patients with Systemic Sclerosis


title: Evidence for CD04+ T Cell Methylation Signatures of Network-oriented SOCS3, ITGAL, and NR4A2 Genes with Hemodynamics in Patients with Pulmonary Arterial Hypertension (RRBS DataSet)

type: Methylation profiling by high throughput sequencing


title: DNA Methylation Analysis in Classical Monocytes from African American Patients with Systemic Sclerosis

summary: Systemic sclerosis is a multisystem autoimmune disorder that has an unclear etiology and disproportionately affects African Americans (AA). Additionally, monocytes show heightened activation in SSc, and in AA relative to European ancestry (EA) individuals. Monocytes are thus a good target tissue for elucidating disease mechanisms.  In this study, we sought to investigate differentially methylated loci in classical monocytes from AA SSc patients and unaffected AA controls.  We used Illumina’s MethylationEPIC BeadChip to profile DNA methylation patterns on FACS-isolated classical monocytes (CD14++CD16-) from 12 female AA SSc cases and 12 female AA controls.  We observed modest DNA methylation differences between cases and controls. The genes harboring the top differentially methylated cytosines are enriched for immune pathways and metabolic processes. The relatively modest differences in DNA methylation observed between patients and controls is consistent with prior evidence of stronger inflammatory signatures in AA.

overall_design: Illumina’s MethylationEPIC BeadChip was used to profile DNA methylation on FACS-isolated classical monocytes (CD14++CD16-) from 12 female AA SSc cases and 12 female AA controls.


title: Evidence for CD04+ T Cell Methylation Signatures of Network-oriented SOCS3, ITGAL, and NR4A2 Genes with Hemodynamics in Patients with Pulmonary Arterial Hypertension

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

overall_design: Refer to individual Series


title: Evidence for CD04+ T Cell Methylation Signatures of Network-oriented SOCS3, ITGAL, and NR4A2 Genes with Hemodynamics in Patients with Pulmonary Arterial Hypertension (Array1 DataSet)