title: Single cell sequencing of GFP+ Tri-PyMT cells from lung metastasis

overall_design: GFP+ Tri-PyMT cells were sorted from parental culture of Tri-PyMT cells by flow cytometry and injected into SCID mice to let lung metastasis outgrowth. 15 days after injection, tumor bearing lungs of 3 mice were harvested and prepare for single cell suspensions. GFP+ cells were sorted for single cell RNA sequencing.


title: Nociceptor neurons affect cancer immunosurveillance

status: Public on Aug 10 2022

city: montreal

first: sebastien

last: Talbot

state: quebec

address: 2900 Boulevard Edouard-Montpetit,

department: pharmacology

country: Canada

institute: university of Montreal

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Human lung tissue explants infected with influenza B virus

status: Public on Aug 31 2022

city: Münster

first: Klaus

last: Schughart

address: Von-Esmarch-Str 56

department: Virology

country: Germany

institute: University Münster

summary:
RNAseq analysis of human lung tissue explants infected with Influenza B virus
Results: Differentially expressed genes after infection
Project: Hulu_FluB_2022

overall_design: Lung mRNA profiles of lung tissue explants from three donors after infection with influenza B virus were compared to mock treated controls.

first: Linda

last: Brunotte

first: Duygu Merve

last: Caliskan


title: The impact of inflammation-induced tumor plasticity during myeloid transformation

status: Public on Jul 12 2022

city: New York

first: Anna

last: Yeaton

state: New York

address: 550 1st ave

department: Pathology

country: USA

institute: NYU Langone

summary: Single-cell RNA-seq analysis of mouse bone marrow immune populations isolated from Tet2 HR mutant and Tet2 Wildtype mice.

overall_design:
Single-cell RNA-seq of total bone marrow cells from mice.

Multiple samples were sequenced within a library (WBM_X), with a unique hashtag for each sample. Hashtag demultiplexing was performed using HTODemux() from the Seurat package.

sample ids HRO.5 and HRO.6 refer to TET2-mutated chronic samples

first: Geraldine

last: Cayanan

first: Iannis

last: Aifantis

first: Maria

last: Guillamot-Ruano


title: Differential Whole Genome CRISPR Screening Strategy for SARS-CoV-2 Spike and VSV-G Mediated Pseudoviral Entry

status: Public on Jun 27 2022

city: Brookings

first: Adam

middle: D.

last: Hoppe

state: South Dakota

address: 1055 CAMPANILE AVE BOX 2202, Avera Health and Science

department: Chemistry and Biochemistry

institute: South Dakota State University

summary: The coronavirus disease 2019 (COVID-19) has caused over 6 million deaths worldwide and disrupted the global economy. The causative agent for this disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes mild to lethal respiratory infections. Understanding the cellular host factors that promote and inhibit for SARS-CoV-2 infection is important for identifying virial countermeasures.  Moreover, new methods are needed to be to identify host genes influencing specific steps of viral infections. Here, we developed a CRISPR whole genome screen against SARS-CoV-2 spike enveloped retro-pseudoviruses with a GFP reporter to specifically identify host genes that facilitate viral entry.  By including two counter screen strategies, this approach can be used to distinguish host genes affecting the pseudoviral reporter from those unique to envelope-mediated entry. First, an alternate envelope, VSV-G allowed identification of shared genes associated with retro-transcription, integration and reporter expression. Second, a recently developed Cre-Gag fusion pseudovirus bypassed retro transcription and integration by directly activating a floxed GFP reporter. Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and LDLR, respectively and distinguished genes associated with retroviral reporter expression from envelope mediated entry. Overall, this work provides a new strategy for screening genes influencing envelope mediated entry without the complexity of live-viral screens which are complicated with large numbers of genes associated with all aspects of viral pathogenesis and replication.  This approach should be of use for identifying genes contributing to and inhibiting SARS-CoV-2 entry and provide a platform for the analysis of newly emerging viruses.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: CRISPR-Cas9 whole genome screen against SARS-CoV-2 spike-mNeonGreen, VSV-G-mNeonGreen and VSV-G-Cre pseudovirus for identifying host factors

first: Md

last: Saddam Hossain

first: Jason

middle: G

last: Kerkvliet

middle: D


title: Whole genome microarrays for E12.5 mouse lens epithelium and fibers

status: Public on Jun 21 2022

city: Newark

first: Deepti

last: Anand

state: Delaware

address: 351 Wolf Hall, University of Delaware

department: Biological Sciences

institute: University of Delaware

summary: E12.5 mouse lens epithelium and fiber cells were collected using Leica LMD 6000 Laser microdissection system. Total RNA was isolated from epithelium and fiber cells using Qiagen RNeasy kit

overall_design: Microarray datasets from wild type E12.5 mouse lens epithelium and fiber cells

first: Salil

middle: A

last: Lachke

first: David

last: Beebe


title: Epigenomic and chromosomal architectural reconfiguration in developing human frontal cortex and hippocampus

status: Public on Oct 07 2022

city: Los Angeles

first: Chongyuan

last: Luo

state: CA

address: 695 Charles E Young Dr S

department: Human Genetics

institute: UCLA

summary: Human frontal cortex and hippocampus play key roles in learning and cognition, and are representative structures of the six-layer neocortex and the more ancient allocortex, respectively. Here we investigated the epigenomic and 3D-chromatin conformational dynamics of human frontal cortex and hippocampus development using more than 53,000 joint single-nucleus profiles of chromatin conformation and DNA methylation (sn-m3C-seq) generated from mid-gestational, late-gestational, infant and adult human brains. We found that the remodeling of DNA methylation predominantly happens during late-gestational to early-post-natal development, and is temporally uncoupled from the dynamics of chromatin conformation. We reconstructed regulatory hierarchies of cortical and hippocampal cell differentiation and maturation and identified brain regional-specific regulatory signatures. The single-cell 3D-regulatome approach further enabled the cell-type-specific dissection of neuropsychiatric risk loci during key neurodevelopmental stages and in adult human brains.

overall_design:
Single-nucleus joint profiling of human frontal cortex and hippocampus in developing human brains

Raw data will be uploaded to a controlled access database due to patient privacy issues.

first: Yi

last: Zhang

first: Kevin

last: Abuhanna

first: Matthew

last: Heffel

first: Jingtian

last: Zhou

first: Dong-Sung

last: Lee

first: Kangcheng

last: Hou

first: Oier

last: Pastor Alonso

first: Terence

last: Li

first: Martin

middle: J

first: Fangming

last: Xie

first: Eran

last: Mukamel

first: Bogdan

last: Pasaniuc

first: Joseph

last: Ecker

first: Jesse

last: Dixon

first: Tomasz

last: Nowakowski

first: Mercedes

last: Paredes


title: Synthetic expansion of brain regulatory T cells effectively prevents neuroinflammatory disease II

status: Public on Jun 10 2022

city: Cambridge

first: Adrian

last: Liston

address: Babraham Research Campus

country: United Kingdom

institute: The Babraham Institute

summary: An adaptive immunological component to neuroinflammatory diseases is increasingly being recognized. The recent identification and characterization of a small population of regulatory T cells (Tregs) resident in the brain allows for the potential therapeutic exploitation. As the most potent known anti-inflammatory mediators, Tregs have the capacity to reduce inflammation and promote tissue repair, with a key limitation being the small number present in brain tissue. Here we demonstrate that transgenic ‘micro-dosing’ of IL-2 production to the brain allows the efficient expansion of the brain-resident Treg population. This approach, using expression of IL-2 within the normal physiological range but redirected to the brain, allows a 10-fold accumulation of Treg numbers within the brain without any increase in the Treg numbers in circulating or peripheral tissues. No alteration was observed in the frequency or transcriptional profile of non-Treg populations within the brain, and, apart from the numerical expansion, brain-resident Tregs were unaltered in their transcriptional profile. Mice with expanded brain-resident Treg numbers were protected from neuroinflammation and damage in mouse models of traumatic brain injury, stroke and Multiple Sclerosis, with substantial reductions in inflammation and histological damage, and improvements in clinical measurements. To produce a treatment with translational potential, we engineered a viral vector delivery system, capable of specific production of IL-2 in the brain. This treatment approach provided a titratable expansion of brain Tregs, and effective protection against neuroinflammatory disease, with potential translation to the patient context.

overall_design: 8 samples in total. 4 control replicates and 4 experimental replicates for brain CD45+CD11b+ cells ,  2 control replicates and 2 experimental replicates for brain  CD4 control replicates and 2 experimental replicates for blood  CD4


title: Distinct gene expression by expanded clones of quiescent memory CD4+ T cells harboring intact latent HIV-1 proviruses

status: Public on Jun 03 2022

city: NEW YORK

first: Thiago

middle: Y

last: Oliveira

address: 1230 YORK AVE

department: Immunology, Virology and Microbiology

institute: The Rockefeller University

summary: Antiretroviral therapy controls but does not cure HIV-1 infection due to a reservoir of rare CD4 + T cells harboring latent proviruses. Little is known about the transcriptional program of latent cells. Here we report a novel strategy to enrich clones of latent cells carrying intact, replication-competent HIV-1 proviruses from blood based on their expression of unique T cell receptors. Latent cell enrichment enabled single cell transcriptomic analysis of 1,050 CD4 + T cells belonging to expanded clones harboring intact HIV-1 proviruses from 6 different individuals. The analysis revealed that most of these cells are T effector memory cells that are enriched for expression of HLA-DR, HLA-DP, CD74, CCL5, Granzymes A and K, cystatin F, LYAR and DUSP2. We conclude that expanded clones of latent cells carrying intact HIV-1 proviruses persist preferentially in a distinct CD4 + T cell population opening new possibilities for eradication.

overall_design: Gene expression profiling analysis of RNA-seq data for human CD4 T cells from HIV-1 infected, aviraemic and ART treated individuals

first: Georg

middle: H

last: Weymar

first: Yotam

last: Bar-On

first: Christian

last: Gaebler

first: Victor

last: Ramos

first: Harald

last: Hartweger

first: Gaëlle

last: Breton

first: Marina

last: Caskey

first: Lillian

middle: B

last: Cohn

first: Mila

last: Jankovic

first: Michel

middle: C

last: Nussenzweig


title: RNA sequencing of 16D CRPC cells treated with enzalutamide over varying periods of time

status: Public on May 12 2022

first: Andrew

middle: Scott

last: Goldstein

address: 610 Charles E Young Dr East

department: Urology

institute: University of California, Los Angeles

summary: The goal of this study was to determine how androgen receptor inhibition alters transcriptional programs in castration-resistant prostate cancer cells. 16D castration-resistant prostate cancer cells were grown in the presence of 10 micromolar enzalutamide for 24, 48, 96, 144 hours or for more than 2 months (long-term). Analysis shows that androgen receptor target genes are reduced with enzalutamide while metabolic genes are also differentially expressed.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 16D CRPC cells treated with enzalutamide for 24, 48, 96, or 144 hours, or for more than two months (long-term)

middle: S

first: Preston

last: Crowell


title: Single cell sequencing of Tri-PyMT cells [10X Genomics]

status: Public on Sep 01 2022

first: Dingcheng

last: Gao

state: NY

address: 1300 York Ave

department: CT Surgery

institute: Weill Cornell Medical College

summary: Using an EMT lineage-tracing model, we identified tumor cells in the transitioning phases of both EMT and MET. Further analyses revealed that the ribosome biogenesis (RiBi) pathway was specifically elevated in these transitioning cells, regardless of their destined phenotypical fates.

overall_design: Tri-PyMT cells in culture (10th generation) were sorted into RFP+, GFP+ and Double+ cells. Same numbers of subpopulations were remixed and submitted for single cell sequencing with 10X Genomics system.

first: Yi

last: Ban


title: The transcriptome, enhancer landscape and GR binding profile in primary mouse hepatocytes treated with glucagon and corticosterone

status: Public on May 09 2022

city: Rehovot

first: Ido

address: POB 12

department: Institute of Biochemistry, Food Science and Nutrition

country: Israel

institute: The Hebrew University of Jerusalem

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: We treated primary mouse hepatocytes with glucagon, corticosterone or both in a dual treatment. The control is the non-treated group. Each group has 3 replicates (Except in GR ChIP-seq where each group has 2 replicates)

first: Meital

last: Charni-Natan

first: Dana

last: Goldberg


title: Decay of heterochromatin domains at selective gene loci in aging mouse brain cells

status: Public on Sep 13 2022

city: La Jolla

first: Bing

last: Ren

address: 9500 Gilman Drive

institute: University of California, San Diego

summary: Aging is characterized by a progressive decline of organismal function and increase in vulnerability to diseases. Loss of heterochromatin during aging has been observed in a variety of organisms, however, understanding its role in aging-related phenotypes has been limited by the lack of information about the specific cell types and gene loci affected by this type of epigenetic changes. To address this bottleneck, we probed dynamic changes in chromatin landscape at single cell resolution in the forebrain, hippocampus, heart, skeletal muscle and bone marrows from 3-month, 10-month and 18-month-old mice, and characterized age-associated changes in chromatin accessibility at over 300,000 candidate regulatory elements across 34 major cell types. We report that excitatory neurons displayed profound changes in a specific set of heterochromatin domains during aging, evidenced by an increase in chromatin accessibility, a decrease in the heterochromatin marker H3K9me3, and de-repression of non-coding RNA species. These results suggest that heterochromatin maintenance are impaired in aging excitatory neurons, and this may be associated with increased repetitive element activity and neurodegenerative diseases.

overall_design: We collected the dorsal hippocampus, frontal cortex, heart, leg muscles and bone marrow from male C57BL/6JN mice in three age groups, namely 3-month, 10-month, and 18-month, and performed single-nucleus ATAC-seq to profile  open chormatin changes during aging. We also performed single-nucleus RNA-seq and paired-tag for H3K9me3 on dorsal hippocampus and frontal cortex.

first: Yanxiao

last: Zhang

first: Maria

middle: L

last: Amaral


title: Next Generation Sequencing Quantitative Analysis of RA-UIP, IPF-UIP, and Non-UIP Patient Lung Biopsy Transcriptomes

status: Public on May 31 2022

city: Rochester

first: Asha

last: Nair

address: 200 1ST ST SW

institute: Mayo Clinic

summary:
Purpose: The goal of this study is to compare transcriptome profiling (RNA-seq) of lung tissue biopsies derived from patients with idiopathic pulmonary fibrosis (IPF or IPF-UIP), or rheumatoid arthritis-associated interstitial diseases (RA-ILD or RA-UIP), or non-usual interstitial pneumonia (non-UIP) controls.
Methods: Total RNA was extracted using the RNeasy kit (Qiagen) from fresh frozen lung tissue obtained from the Lung Tissue Repository Consortium (LTRC). RNA samples were then sent to the Genome Analysis Core at Mayo Clinic for sequencing using conventional Illumina mRNA-Seq protocols. A total of 23,295 genes were probed using this platform.
Results: RNA-seq data revealed considerable transcriptomic differences between the groups. A heat map generated with the most differentially expressed genes (P<0.05) revealed two distinct gene expression profiles among the three groups. The heat map for the 3 RA-UIP and 20 IPF patients showed a similar gene expression pattern, which contrasted significantly from the non-UIP control group.
Conclusions: The RNA-Seq data analysis shows similarities and differences between gene expression profiles of RA-UIP and IPF, suggesting both shared and differential disease-causing mechanistic pathways. Transcriptomic data distinguishes the RA-UIP group from IPF via JAK2-related JAK/STAT signaling pathways.

overall_design: Lung tissue biopsy mRNA profiles of RA-ILD, IPF, and non-UIP controls.

first: Robert

last: Vassallo

first: Shaohua

last: Wang


title: Sperm epigenetic clock predicts pregnancy outcomes in the general population

status: Public on Jul 06 2022

city: Amherst

last: Pilsner

state: Massachusetts

address: 686 N PLEASANT

department: Environmental Health Sciences

institute: University of Massachusetts Amherst

summary: We developed two novel sperm epigenetic clocks by applying Super Learner, an ensemble machine learning algorithm, to predict age from sperm EPIC array DNA methylation data via individual CpG sites and differentially methylated regions (DMRs). Overall, our cox model showed that one-year increase in our  developed sperm epigenetic age (SEA) was associated with up to 15% reduction in couples time-to-pregnancy (TTP).

overall_design: A population-based prospective design involving participants that were enrolled in the Longitudinal Investigation of Fertility and the Environment (LIFE) Study. It sought to determine the association between sperm epigenetic age (SEA) with time-to-pregnancy (TTP) among couples (n =379) trying for pregnancy.

first: J

middle: R

first: Hachem

last: Saddiki

first: Brian

middle: W

last: Whitcomb

first: Alexander

last: Suvorov

first: Germaine

middle: M

last: Louis

first: Sunni

last: Mumford

first: Enrique

middle: F

last: Schisterman

first: Oladele

last: Oluwayiose

first: Laura

last: Balzer


title: Identification of the RNA-protein interactions of PAIP1, which plays a novel role in alternative mRNA splicing (RIP-seq)

status: Public on May 05 2022

city: Wuhan

first: Dong

last: Chen

state: Hubei

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

department: Center for Genome Analysis

country: China

institute: ABLife, Inc.

summary: Poly (A) binding protein interacting protein 1 (PAIP1) has been shown to causally contribute to the development and progression of cancer. However, the biological function of PAIP1 in tumorigenesis remains largely unexplored. Here, we used RNA immunoprecipitation to map the interactions between PAIP1 and RNA on a genome wide level. A genome wide analysis showed that PAIP1 binding is specifically enriched in exon, intron and 3’ untranslated regions. The genes bound by PAIP1 were strongly associated with cancer-related pathways and we found that PAIP1 knockdown mediates the statistically significant regulation of alternative splice events. These data suggest that PAIP1 may influence alternative splicing and, using a VEGF-A minigene, we showed that overexpression of PAIP1 inhibits the expression of exon 6. Further analysis of PAIP1 showed that interacting proteins participate in splice factors, spliceosomes and exon junction complex assembly. Our work has demonstrated that PAIP1 has specific RNA binding properties that help to coordinate the isoform-specific expression of coding genes. These genes are associated with cancer, which suggests that PAIP1 is an important regulator of key cancer processes.

overall_design: transcrptional analysis of PAIP1 konck-down and control in HeLa cells

first: Jianfeng

last: Zheng


title: Identification of the RNA-protein interactions of PAIP1, which plays a novel role in alternative mRNA splicing


title: Single-cell transcriptomic profiling provides insights into disease-related processes in human hypertrophic cardiomyopathy

status: Public on Apr 21 2022

city: Utrecht

first: Eva

last: van Rooij

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary:
Background – Hypertrophic cardiomyopathy (HCM) is a common genetic cardiac disorder that is characterized by the presence of left ventricular hypertrophy with histological evidence of myocyte disarray and fibrosis. Pathologic DNA variants in sarcomeric genes are seen in 50–60% of families, but the mechanistic origins of hypertrophy and disarray remain poorly understood. More molecular insights could contribute to a better understanding of these origins and improve therapeutic strategies for HCM.
Methods – To identify gene expression patterns relevant to HCM pathology, we quantified intercellular heterogeneity in cardiomyocyte gene expression by performing single-cell RNA sequencing (scRNA-seq) on myectomy tissue from HCM patients.
Results – Septal myectomy samples provide a unique opportunity to access diseased human heart tissue for scRNA-seq. We analysed gene expression of individual cardiomyocytes for three HCM patients, who each showed a similar presence of cardiomyocyte subpopulations. This similarity indicated these subpopulations emerge from biological processes, and not from experimental variability. Moreover, our data revealed functional links between genes important to HCM. A subpopulation characterized by elevated titin (TTN) expression showed increased expression of genes involved in muscle contraction and development. A subpopulation of Natriuretic Peptide A (NPPA)-enriched cells showed elevated expression of Natriuretic Peptide B (NPPB). Further analysis of gene co-expression revealed five groups of genes with tightly correlated expression (regulons), one consisting of mostly sarcomeric genes and another containing some well-known stress markers. These correlations were confirmed in 97 additional HCM myectomy samples and allowed us to de novo identify transcription factors related to sarcomeric gene expression and stress. Finally, using forward scatter, we charted functional implications of regulon expression and showed a link between the stress-marker regulon and cell size. Histology samples independently confirmed that regulon member skeletal alpha actin (ACTA1) correlates with cell size.
Conclusion – We have shown that single-cell sequencing on myectomy samples from HCM patients identified new functional links between genes, transcription factors, and a functional link between genes and cell size. This highlights the relevance of cardiomyocyte heterogeneity, and additionally provides a wealth of novel insights into molecular events at the base of HCM, which potentially contribute to understanding disease progression.

overall_design:
Single-cell RNA-sequencing on 5 septal myectomy samples from patients with hypertrophic cardiomyopathy

FACS sorting index data are not available for patients 1-3.

first: Joep

middle: E

last: Eding

first: Anne

last: de Leeuw

first: Maya

last: Wright Clark

first: Martijn

last: Wehrens

first: Cornelis

last: Boogerd

first: Bas

last: Molenaar

first: Petra

last: van der Kraak

first: Diederik

last: Kuster

first: Jolanda

last: van der Velden

first: Michelle

last: Michels

first: Aryan

last: Vink


title: The two-component system ChvGI responds to osmotic upshift and peptidoglycan damage in Caulobacter crescentus.

status: Public on Apr 11 2022

city: Namur

first: Regis

last: Hallez

address: 61 Rue de Bruxelles

country: Belgium

institute: UNamur


title: Nociceptor neurons affect cancer immunosurveillance [RNA-seq 2]

status: Public on Aug 10 2022

first: sebastien

last: Talbot

address: 2900 Boulevard Edouard-Montpetit,

institute: university of Montreal

summary: Sequencing Neuron, melanoma and CD8 when culture alone or co-culture with one another

overall_design: Naive DRG neurons (TRPV1cre::QuASR2-eGFPfl/wt), B16F10-mCherry-OVA, and OVA-specific cytotoxic CD8+ T-cells were cultured alone or in combination. After 48h, the cells were harvested, FACS-purified, and RNA-sequenced.

first: Sebastien


title: Multimodal single cell sequencing of human diabetic kidney disease implicates chromatin accessibility and genetic background in disease progression [cut-and-run and bulk ATAC-seq to support single cell data]

status: Public on Jul 28 2022

city: St. Louis

first: Yoshiharu

last: Muto

state: MO

address: 660. S Euclid Ave., CB 8118

department: Medicine / Nephrology

institute: Washington University in St. Louis

summary:
Purpose: Identification of glucocorticoid recepor binding sites on human kidney proximal tubular cells to better understand epigenetic alterations  in diabetic kidney diseases
Methods: CUT&RUN assay libraries for cultured cells or human kidneys were generated with the CUTANA kit (EpiCypher, 14-1048). 500,000 cells were mixed and incubated with Concanavalin A (ConA) conjugated paramagnetic beads. Antibodies to the glucocorticoid receptor were added to target samples and IgG was added to negative controls. The remaining steps were performed according to manufacturer’s instructions. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, E7645S) with manufacturer’s instructions, including minor modifications indicated by CUTANA described above. CUT&RUN libraries were sequenced on a NovaSeq instrument (Illumina, 150 bp paired-end reads). For bulk ATAC-seq, 50,000 nuclei were transposed in 25 μL of ATAC-seq transposition mix [12.5 μL 2× Illumina Tagment DNA (TD) buffer; 10.5 μL nuclease-free water; 2.0 μL Tn5 transposase (Illumina/Nextera, FC-121-1030)] and incubated at 37°C for 1 h on a thermomixer. The transposed DNA was purified with QIAGEN MinElute kit (28004) and amplified with dual indexes.

overall_design: Cut-and-run for glucocorticoid receptor binding sites on normal human kidney cortex or immortalized renal proximal tubule epithelial cells (hTERT-RPTEC, ATCC). Bulk ATAC-seq on primary RPTEC (Lonza) or hTERT-RPTEC.

first: Parker

last: Wilson

first: Benjamin

last: Humphreys


title: Identification of the RNA-protein interactions of PAIP1, which plays a novel role in alternative mRNA splicing (RNA-Seq)

first: Dong

last: Chen

first: Jianfeng

last: Zheng


title: PolyA+ RNA-Seq profiling of Drosophila melanogaster late third instar larval ovary, testis and sex transformed gonads as a result of tra and dsx perturbation.

status: Public on Jun 16 2022

city: Bethesda

first: Brian

last: Oliver

state: MD

address: 50 South Drive

department: LCDB

institute: NIDDK, NIH

summary: We designed this experiment to investigate the transcriptional changes in gonads as a result of sex transformation. Here we performed transcriptional profiling of the ovary transformed into testis from the tra loss of function (XX_tra_lof), testis transformed into ovary from the tra gain of function (XY_tra_gof) and ovary transformed into testis in dsxM gain of function (XX_DsxM_gof/lof) Drosophila melanogaster third instar larvae in biological quadruplicates. In addition, as controls we sequenced ovaries and testes from the female and male wildtype larvae respectively. We constructed polyA+ libraries of the gonads, cleaned off the fatbody and performed 50 bp, stranded single-end RNA-Seq.

overall_design: We dissected late third instar larvae and pooled (see Extraction protocol for details) 25 ovaries, 15 testes, 25 ovaries transformed into testes from the tra loss of function (XX_tra_lof), 25 testes transformed into ovaries from the tra gain of function (XY_tra_gof) and 25 ovaries transformed into testes from dsxM gain of function (XX_DsxM_gof/lof) larvae in biological quadruplicates. Within a sample type, pools of gonads were collected at the same time, but different replicates and genotypes were collected on different days. We extracted RNA from the samples stored at and constructed libraries simultaneously for all samples in a 96 well plate.

first: Sharvani

last: Mahadevaraju


title: The two-component system ChvGI responds to osmotic upshift and peptidoglycan damage in Caulobacter crescentus [RNA-seq]

summary: Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. ChvG/ChvI (ChvGI) is a TCS conserved in γ-proteobacteria and is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to activate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and confirmed that ChvI regulates cell envelope architecture at different levels by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. Furthermore, we identified genes with osmoregulatory properties and confirmed that osmotic upshift is a signal triggering ChvG-dependent phosphorylation of ChvI. In addition, we challenged chv mutants with other cell envelope related stress and found that targeting with antibiotics the transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Moreover, these antibiotics activate expression of the chvIG-hprK operon in ChvI-dependent and independent ways. ChvI phosphorylation is also shown to be activated upon antibiotic treatment with vancomycin. Finally, we observed that the sensor kinase ChvG fused to a fluorescent protein relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer.

overall_design: Determination of ChvI regulon in Caulobacter crescentus WT and DchvI cells grown in complex media PYE supplemented with 6% sucrose

first: Alex

last: Quintero-Yanes

first: Aurélie

last: Mayard

first: Régis


title: Liver iNKT transcriptional profiling

status: Public on Apr 22 2022

city: Barcelona

first: Pere

last: Santamaria

address: C/Rosselló 149-153

country: Spain

institute: IDIBAPS

summary: Invariant NKT (iNKT) cells comprise a heterogeneous group of terminally differentiated, non-circulating, tissue-resident T-lymphocytes that recognize glycolipids, including alpha-galactosylceramide (aGalCer), in the context of CD1d.   Here, we show that murine and human liver-resident aGalCer/CD1d-binding iNKTs correspond to a novel Zbtb16+/Tbx21+/Gata3+/Maflow/Rorc– subset that exhibits profound plasticity. Repetitive encounters of these cells with intravenously-delivered aGalCer/CD1d-coated nanoparticles (NPs) trigger their differentiation into immunoregulatory, IL-10/IL-21-producing, Mafhigh cells expressing a T-regulatory type 1 (TR1)-like transcriptional signature.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We defined the transcriptional profile of iNKT cells from liver of untreated NOD.c3c4, NOD and B6 mice. We compared the transcriptional profile of aGalCer/CD1d-NP-treated and untreated NOD.c3c4 mice. Finally, we also characterized the transcriptional profile of human liver iNKT cells.

last: Santamaria Vilanova

first: Patricia

last: Solé Sánchez


title: Duplications disrupt chromatin architecture and rewire GPR101-enhancer communication in X-linked acrogigantism [RNA-Seq]

status: Public on Feb 23 2022

city: Pieve Emanuele

first: Giampaolo

last: Trivellin

state: Lombardy

address: Via Rita Levi Montalcini 4

country: Italy

institute: Humanitas University

summary: X-linked acrogigantism (X-LAG) is the most severe form of pituitary gigantism and is characterized by aggressive growth hormone (GH)-secreting pituitary tumors that occur in early childhood. X-LAG is associated with chromosome Xq26.3 duplications (the X-LAG locus typically includes the VGLL1, CD40LG, ARGHEF6, RBMX, and GPR101 genes) that lead to massive pituitary tumoral expression of GPR101, a novel regulator of GH secretion. In this work, we aimed to unravel the mechanism by which the duplications lead to marked pituitary misexpression of GPR101 alone. Using 4C-seq, we characterized the normal chromatin structure at the X-LAG locus. Using GPR101, RBMX, and VGLL1 viewpoints, we showed that GPR101 is located within a topologically associating domain (TAD) delineated by a tissue-invariant border that separates it from centromeric genes and regulatory sequences. Then, we showed that the duplications in multiple patients led to ectopic interactions that crossed the invariant TAD border, indicating the existence of a similar and consistent mechanism of neo-TAD formation in X-LAG. Rewiring GPR101-enhancer interaction within the new regulatory unit is likely to cause the high levels of aberrant expression of GPR101 in X-LAG pituitary.

overall_design: Human 4C-seq profiles derived from 6 blood-derived cells of individuals with X-linked acrogigantism and 3 healthy independent controls.

first: Martin

last: Franke

first: Adrian

last: Daly

first: Leonor

last: Palmeira

first: Amit

last: Tirosh

first: Antonio

last: Stigliano

first: Eszter

last: Trifan

first: Fabio

last: Faucz

first: Dayana

last: Abboud

first: Patrick

last: Petrossians

first: Juan

last: Tena

first: Eleonora

last: Vitali

first: Andrea

last: Lania

first: José

last: Gómez-Skarmeta

first: Albert

last: Beckers

first: Constantine

last: Stratakis


title: Characterization of pdgfrb reporter expressing cells from the adult zebrafish heart ventricles by single cell RNA sequencing

status: Public on Feb 19 2022

first: subir

last: kapuria

address: 4650 Sunset Blvd.

department: Cardiothoracic surgery

institute: Children's Hospital Los Angeles

summary: Platelet-derived growth factor (PDGF) signaling regulates perivascular cell or mural cell association with the blood-vessel endothelial cells. Mural cells express the receptor of the signaling, pdgfrb. Besides mural cells the outer epithelial layer of the heart, epicardium and epicardial derived cells express pdgfrb. In this dataset we characterized pdgfrb  expressing cells in adult zebrafish heart ventricles in AB wildtype fish, pdgfrb loss-of-function mutant fish. Since pdgfrb is activated in injured zebrafish heart, we aslo compared pdgfrb expressing cells in injured (amputated, 7 days post amputation) heart ventricle with similar cells in uninjured hearts.

overall_design: We FACS sorted Tg(pdgfrb:EGFP) reporter expressing cells from the adult heart ventricles of the wildtype, pdgfrb mutant Zebrafish. We also FACS sorted these cells from adult uninjured and injured (7 days post amputation) heart ventricles and performed single cell RNA sequencing. All zebrafish used here were of AB strain.

first: Subir

last: Kapuria

first: Ching-Ling

last: Lien


title: Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells [scATAC-seq]

status: Public on May 26 2022

city: Paris

first: Andràs

last: Pàldi

state: Ile de France

address: 19 rue Chaligny

department: Centre de Recherche de Saint Antoine

country: France

institute: Ecole Pratique des Hautes Etudes

summary: When human cord blood derived CD34+ cells are induced to differentiate, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. In particular, the cells passe through a transitory phase known as “multilineage primed” state. These cells are characterized by a mixed gene expression profile, different in each cell, with the co-expression of many genes characteristic for concurrent cell lineages. The aim of our study is to understand the mechanisms of the establishment and the exit from this transitory state. We investigated this issue using single-cell RNA sequencing and ATAC-seq. Two phases were detected. The first phase is a rapid and global chromatin decompaction that makes most of the gene promoters in the genome accessible for transcription. It results 24h later in enhanced and pervasive transcription of the genome leading to the concomitant increase in the cell-to-cell variability of transcriptional profiles. The second phase is the exit from the multilineage-primed phase marked by a slow chromatin closure and a subsequent overall downregulation of gene transcription. This process is selective and results in the emergence of coherent expression profiles corresponding to distinct cell subpopulations. The typical time scale of these events spans 48 to 72 hours. These observations suggest that the non-specificity of genome decompaction is the condition for the generation of a highly variable multilineage expression profile. The non-specific phase is followed by specific regulatory actions that stabilize and maintain the activity of key genes, while the rest of the genome becomes repressed again by the chromatin re-compaction. Thus, the initiation of differentiation is reminiscent of a trial-and-error process that associates the spontaneous generation of gene expression diversity to subsequent regulatory actions that maintain the activity of some genes while the rest of the genome sinks back to the repressive closed chromatin state.

overall_design: Evolution of single cell mRNA profile and DNA accessibility profile of HSC after cytokine stimulation

first: Romuald

last: Parmentier

first: Laëtitia

last: Racine


title: Duplications disrupt chromatin architecture and rewire GPR101-enhancer communication in X-linked acrogigantism [4C]

first: Giampaolo

last: Trivellin

first: Martin

last: Franke

first: Adrian

last: Daly

first: Leonor

last: Palmeira

first: Amit

last: Tirosh

first: Antonio

last: Stigliano


title: Patch-Seq Analysis of Dbx1-derived Respiratory Neurons of the preBötzinger Complex in Neonatal Mice

status: Public on Feb 10 2022

city: Williamsburg

first: Prajkta

last: Kallurkar

state: VA

address: 540 Landrum Dr, Integrated Science Center 3

institute: William & Mary

summary: Inspiratory breathing movements depend on neurons in the preBötzinger complex (preBötC) whose progenitors express the transcription factor Dbx1 (henceforth Dbx1 neurons). We use the Patch-Seq technique and next-generation sequencing to measure the transcriptomes of Dbx1 preBötC neurons. The Dbx1 preBötC neurons can generate both, the rhythm and the rudimentary motor output pattern for inspiratory breathing. We used electrophysiological properties of Dbx1 preBötC neurons to categorize them as putative rhythm- and pattern-generators and then aspirated their cellular contents for scRNA sequencing.

overall_design: Patch-Seq on putative rhythm- and pattern-generating Dbx1-derived inspiratory neurons of the preBötzinger Complex in neonatal mice (P0-P2)

first: Maria

last: Picardo

first: Yae

middle: K

last: Sugimura

first: Gregory

last: Smith

first: Margaret

last: Saha

first: Christopher

last: Del Negro


title: Duplications disrupt chromatin architecture and rewire GPR101-enhancer communication in X-linked acrogigantism

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

overall_design: Refer to individual Series


title: RNA and surface epitope sequencing of single cells involved in spondyloarthritis

status: Public on Mar 07 2022

city: San Francisco

first: Wilson

last: Liao

address: 2340 Sutter St.

department: Dermatology

institute: University of California, San Francisco

summary: Early diagnosis of psoriatic arthritis (PSA) and differentiation of this disease from similar but distinct arthritides, such as ankylosing spondylitis (AS), is important for successful therapeutic intervention but currently remains challenging due, in part, to the scarcity of non-invasive biomarkers. In this study, we perform single cell profiling of transcriptome and cell surface protein expression on the peripheral blood immunocyte populations of individuals with PSA, individuals with AS, individuals with cutaneous psoriasis (PSO) alone, and healthy individuals to identify gene and protein features differentially expressed between these groups across 30 immune cell types and to further evaluate the diagnostic potential of these features using a machine learning classification approach.

overall_design: RNA and surface epitope sequencing of peripheral blood mononuclear cells in 28 patients with psoriatic arthritis (PSA), 10 patients with ankylosing spondylitis (AS), 24 patients with cutaneous psoriasis only (PSO), 14 psoriasis patients with unclear PSA diagnosis (PSX), and 29 healthy subjects. StudyInfo_20220124.xlsx describes the samples, as well as raw and processed data used for separate studies of (1) AS and healthy subjects and (2) PSA, PSO, PSX, and healthy subjects, as well as a description of the columns in the corresponding CellMetadata files of each study. CellMetadata_AS_20220103.tsv indicates the subset of cells used in the study of AS and healthy subjects.  CellMetadata_PSA_20220103.tsv indicates the subset of cells used in the study of PSA, PSO, PSX, and healthy subjects.

first: Jared

last: Liu

first: Samuel

last: Alber

first: Sugandh

last: Kumar

first: Julie

last: Hong

first: Zhi-Ming

last: Huang

first: Diana

last: Paez

last: Castillo

last: Calvo

first: Hsin-Wen

last: Chang

first: Daniel

last: Cummins

first: Mimi

last: Chung

last: Yeroushalmi

first: Erin

last: Bartholomew

first: Marwa

last: Hakimi

first: Chun

last: Ye

first: Tina

last: Bhutani

first: Mehrdad

last: Matloubian

first: Lianne

last: Gensler


title: Transcriptional Coupling of Distant Regulatory Genes in Living Embryos

status: Public on Feb 01 2022

city: Princeton

first: Mike

last: Levine

state: NJ

address: South Dr

department: Lewis Sigler Institute

institute: Princeton University

summary: The prevailing view of metazoan gene regulation is that individual genes are independently regulated by their own dedicated sets of transcriptional enhancers. Past studies reported long-range gene-gene associations, but their functional significance in regulating transcription remains uncertain and controversial. Here we employ quantitative single cell live imaging methods to provide the first demonstration of co-dependent transcriptional dynamics of genes separated by large genomic distances in living embryos. We find extensive physical and functional associations of distant paralogous genes, including co-regulation by shared enhancers and co-transcriptional bursting over distances of nearly 250kb. Regulatory inter-connectivity depends on promoter-proximal tethering elements and perturbations in these elements uncouple transcription and alter the bursting dynamics of distant genes, suggesting a role of genome topology in the formation and stability of co-transcriptional hubs. Transcriptional coupling of distant genes throughout the Drosophila genome underlies a broad spectrum of conserved developmental processes, suggesting a general strategy for long-range integration of gene activities.

overall_design: MicroC experiments were conducted in biological duplicates. The control line was yw. Experimental lines were CRISPR-mediated deletions of specific architectural elements. All experiments were conducted with embryos at the nc14 stage.

first: Xinyang

last: Bing

first: Michal

last: Levo

first: Joao

last: Raimundo


title: Chickpea Roots Undergoing Colonisation by Phytophthora medicaginis do not Display Hormone Accumulation nor Signalling Typically Associated with Model Leaf Hemibiotrophic Diseases

status: Public on Jan 30 2022

city: Sydney

first: Jonathan

middle: Michael

last: Plett

state: New South Wales

address: 50 Bourke street

department: Soil Biology and Genomics

country: Australia

institute: Western Sydney University

summary: As a broad group, hemibiotrophic pathogens cause significant losses within agriculture threatening the sustainability of food systems globally. The complex manner in which these microbes colonize their hosts, including an initial biotrophic phase of colonization followed by a biotrophic-to-necrotrophic switch (BNS) phase and ending with a necrotrophic mode of nutritional acquisition, renders their management more complex than pathogens which display a single nutritional strategy.  Within model plant systems, typically each of these phases is characterized by both common and discrete transcriptional responses as the host modulates physiological processes in concert with changes in pathogen biology.  Plant hormones also play an important role in these stages with classic models showing that salicylic acid accumulates during the biotrophic phase and jasmonic acid/ethylene responses occur during the necrotrophic phase. In this study, we use the interaction between the hemibiotroph Phytophthora medicaginis and the roots of its host Cicer arietinum (chickpea) to define the duration of the different life stages of P. medicaginis during the pathogenesis of chickpea.  Using transcriptional profiling across all three stages of infection we demonstrate that chickpea displayed some similarities in response to P. medicaginis as has been previously documented in other model plant-pathogen hemibiotrophic interactions.  However, our transcriptomic results suggest that chickpea does not conform to the phytohormone response model observed in leaf colonization, nor to that observed for roots of other plant species being colonised by soil-borne hemibiotrophs. We confirm these findings using targeted quantification of salicylic acid and jasmonic acid. The findings from this study demonstrate that a wider spectrum of plant species should be investigated in future to understand the physiological changes in plants during the different stages of colonization by hemibiotrophic soil-borne pathogens before we can better manage these economically important microbes in agronomically relevant conditions.

sample_organism:
Cicer arietinum
Phytophthora medicaginis

overall_design: In total 16 samples were analysed by RNA-sequencing of which 12 samples included 4 replicates each of 3 timepoints 12, 24 and 72 hours post P. medicaginis inoculation in chickpea var. 'Sonali' roots encompassing three phases of infection namely biotrophic, biotrophic to necrotrophic switch and necrotrophic phases, respectively. The remaining four samples included RNA from mock-inoculated chickpea roots inoculated with agar blocks without oomycete mycellium overlaid on cellophane membranes as well as axenically grown P.medicaginis grown on V8 juice media supplemented with CaCO3 before transferring to full stength MS media supplemented with CaCO3 for 12 hours before harvesting.

first: Donovin

last: Coles

first: Sean

last: Bithell

first: Meena

last: Mikhael

first: Willam

last: Cuddy


title: Liver Stromal Cells Restrict Macrophage Maturation and Stromal IL-6 Limits the Differentiation of Cirrhosis-linked Macrophages

status: Public on Jan 31 2022

city: Boston

first: Shenglin

last: Mei

state: MA

address: 10 Shattuck Street

institute: Harvard medical school (DMBI)

summary: Liver disease is the cause of approximately two million deaths globally per year, yet effective therapies are lacking. Iterative tissue injury leads to prolonged inflammation, extensive fibrosis, and the possible progression to deadly chronic-end stage disease, such as cirrhosis and hepatocellular carcinoma (HCC). In this study we investigated the ability of liver stromal cells to effect macrophage maturation and differentiation by using an in vitro human stromal-myeloid coculture systems and single cell RNA sequencing.

overall_design:
Human liver tissue were disaggregated and profiled using the Chromium Single Cell 3’ Library and Gel Bead Kit v2 (10X Genomics) .
In vitro human stromal-myeloid coculture systems


title: Astrocyte immunosenescence in IL-10 and cholesterol signaling in the aged brain impairs microglial regulation following peripheral immune activation

status: Public on Jan 24 2022

city: Durham

first: Shane

middle: Mitchell

last: O'Neil

state: NC

address: 701 W Main St

department: Neuroscience

institute: Duke University

summary: The purpose of this study was to determine the cell-, age-, and time-specific transcriptional profiles of cells within the brain after peripheral immune challenge with lipopolysaccharide.

overall_design: Hippocampal transcriptional profiles of adult and aged BALB/c mice with i.p. saline or 4h or 24h after i.p. LPS.

first: Emma

last: Hans

first: Starr

last: Jiang

first: Lynde

last: Wangler

middle: P

last: Godbout


title: SNP genotyping of dogs Patagonian Sheepdogs

status: Public on Jan 15 2022

first: Heidi

middle: G

last: Parker

address: 50 South Dr. Rm 5347

department: CGB

institute: NHGRI

type:
SNP genotyping by SNP array
Genome variation profiling by SNP array

summary: Comparison of SNP profiles of the Patagonain Sheepdog with European breeds confirms Scottish origin and position basal to modern herding breeds.

overall_design: DNA was collected from working Patagonian sheepdogs. SNPs were genotyed using the Illumina CanineHD SNP chip. These data were combined with previously published SNP genotypes and analyzed using distance measures, haplotype sharing, and admixture analyses.

first: Natasha

last: Barrios

first: César

last: González-Lagos

first: Marcelo

middle: A

last: Gómez

first: Elaine

last: Ostrander


title: The gut microbiota affects the social network of honeybees

status: Public on Jan 05 2022

city: Lausanne

first: Joanito

last: Liberti

address: Biophore

country: Switzerland

institute: University of Lausanne

summary: RNA-sequencing of gut and different brain regions of gnotobiotic honeybees

overall_design: The microbiota composition in the gut of bees was manipulated to produce four treatments: bees were either (i) kept microbiota-depleted (MD) or colonized with (ii) a gut homogenate (CL), (iii) a synthetic community of 13 gut microbiota strains (CL_13), or (iv) two strains of Bifidobacterium asteroides (CL_Bifi). The four colonization treatments were performed at first day of adult emergence of bees collected at the pupal stage from ten different hives. We sampled the gut of each of 40 bees, as well as the brain, which was further dissected into the mushroom bodies (MB), the antennal lobes and subaesophageal ganglion (AL) and the optic lobes (OL). Each sample was sequenced twice (across two lanes) on a Illumina HiSeq 4000 sequencer.

first: Tomas

last: Kay

first: Andrew

last: Quinn

first: Lucie

last: Kešnerová

first: Erik

middle: T

last: Frank

first: Amélie

last: Cabirol

first: Thomas

middle: O

last: Richardson

first: Philipp

last: Engel

first: Laurent

last: Keller


title: RNA-seq Analysis of intestinal tubes from the Unfed and Fed Xenopus laevis tadpoles

city: Kumamoto

first: Asako

last: Shindo

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

country: Japan

institute: Kumamoto University

summary: In this study, we assessed nutrient-dependent organ morphogenesis in Xenopus laevis. We performed the RNA-seq analysis of the gastrointestinal (GI) tract for both fed and unfed tadpoles to identify feeding-dependent inter-organ transmitters secreted from GI tract post-prandially during development. We found that one of the GI hormones, glucose-dependent insulinotropic polypeptide (GIP), is required for thyroid morphogenesis.

overall_design:
Intestinal mRNA profiles of the unfed and fed Xenopus laevis tadpoles (10 dpf to 16 dpf, raised at 19 ℃)
contributor: Bioengineering Lab (https://gikenbio.com/)


title: Integrative Low-input Transcriptomic and Cistromic Analyses Identify CREB Target Genes in Cystic Epithelial Cells [CUT&RUN, ChIP-Seq]

status: Public on Jan 01 2022

city: Tianjin

first: Zhiheng

last: Liu

state: Tianjin

address: No.22 Qixiangtai Road

department: Tianjin Research Center for Basic Medical Science

institute: Tianjin Medical University

summary: Transcription factors (TFs) play crucial roles in kidney development and disease by recognizing specific DNA sequences to control gene expression programs. The kidney’s cellular heterogeneity poses substantial challenges to identifying the genomic binding sites and direct target genes of TFs in vivo. We apply the CUT&RUN (cleavage under targets and release using nuclease) technique, together with transcriptomic analysis, to identify cAMP-response element-binding protein (CREB) target genes in cystic epithelial cells of autosomal dominant polycystic kidney disease (ADPKD). Our results reveal that CREB binds to and activates ribosomal biogenesis genes, and that inhibition of CREB retards cyst growth in ADPKD mouse models. Our findings demonstrate that CUT&RUN is a powerful method for genome-scale profiling and identifying direct targets of TFs from small numbers of specific kidney cells.

overall_design: CUT&RUN of p-CREB, H3K4me3 and H3K27ac were performed in freshly isolated Dolichos biflorus lectin (DBA) positive cells from WT or ADPKD mice kidneys. CUT&RUN were compared with ChIP-seq in WT 9-12 cells.

first: Yupeng

last: Chen


title: Single-cell analysis reveals the progress in skin repair after radiation in rats.

status: Public on Jan 19 2022

city: suzhou

first: zhang

middle: shu

last: yu

address: No. 199, Ren'ai Road

department: soochow university

institute: Soochow University

summary: This study aims to compare the cell heterogeneity and mRNA expression between irradiated and non-irradiated rat skin tissues at the single cell level. In this study, the skin was isolated from rats with radiation or non-irradiated rats. Male Sprague Dawley (SD) rats (6 weeks of age) were used. For irradiation, the hair on the gluteal region of the rats was shaved using a razor. The animals were immobilized using adhesive tape on a plastic plate to minimize motion during radiation exposure. A 3-cm-thick piece of lead was used to shield the animals and localize the radiation field (3-4 cm). Irradiation was performed on the treatment area at a dose rate of 1000 cGy/min using a 6-MeV electron beam accelerator. Irradiated skin was taken on day 7, 14, 28 and 60 for RNA-seq, respectively. The control group of skin were collected from four rats without radiation and mixed for RNA-seq.

overall_design: Single cell RNA-seq analysis of skin tissue from irradiated and non-irradiated parts of SD rats at different time periods. ScRNA sequencing libraries were further generated by the 10x Chromium Single Cell 3′ Library & Single Cell 3’ v3 Gel Beads (10x Genomics). Briefly, gel beads with cell barcodes and skin cell suspension were wrapped in liquid droplets by a microfluidic platform (10x Genomics). A droplet containing single cells was collected and then lysed to generate Gel Bead-In Emulsions (GEMs).

first: Shuyu

last: Zhang

first: Daojiang

last: Yu

first: Tao

last: Yan


title: Integrative Low-input Transcriptomic and Cistromic Analyses Identify CREB Target Genes in Cystic Epithelial Cells [RNA-Seq]

overall_design: RNA-seq were performed in freshly isolated DBA positive cells from mice treated with vehicle or CREB inhibitor 666-15.


title: Specification of CNS macrophage subsets occurs postnatally in defined anatomical niches

status: Public on Jan 26 2022

city: Freiburg

first: Roman

last: Sankowski

address: Breisacher Str. 64

department: Institute of Neuropathology

institute: University of Freiburg Medical Center

summary: All tissue resident macrophages of the central nervous system (CNS), including parenchymal microglia as well as CNS-associated macrophages (CAMs) such as meningeal (mMF) and perivascular macrophages (pvMF) are part of the CNS endogenous innate immune system that acts as the first line of defense during infections or trauma. It has been suggested that microglia and all CAM subsets are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAMs subsets in situ are poorly understood. Using novel fate mapping systems, single-cell profiling and cell-specific mutants, we show that only mMF and microglia share a common prenatal progenitor. In contrast, pvMF originate from perinatal mMF only after birth in an integrin-dependent manner. Furthermore, the establishment of pvMF critically requires the presence of vascular smooth muscle cells. In summary, our data reveal a novel precisely timed process in distinct anatomical niches for the establishment of CNS macrophage subsets.

overall_design: A1/A2 cells were FACS-sorted, from extraembryonic YS at E9.5 into a 384-well (Bio-Rad Laboratories, Munich, Germany) plate containing lysis buffer and barcodes. The gating strategies for FACS-sorting, related to the datasets shown in Fig. 2a-d, Extended Data Fig. 5b-d, were shown in Extended Data Fig. 5a.

first: Marco

last: Prinz

first: Takahiro

last: Masuda

first: Lukas

last: Amann


title: Acute myeloid leukemia (AML) patient blasts and healthy hematopoietic stem and progenitor cell (HSPC) 3'RNA-Sequencing Data

status: Public on Dec 31 2021

city: La Jolla

first: Dong-Er

address: 3855 Health Sciences Drive #0815

institute: UC San Diego Moores Cancer Center

summary: Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. By performing 3’RNA sequencing on acute myeloid leukemia (AML) patient samples and healthy hematopoietic stem and progenitor cells (HSPCs), we show that patient cells exhibit global 3’ untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in poly(A) site usage. Among APA regulators, FIP1L1 expression correlated with the degree of APA dysregulation and knockdown of this RNA-binding protein (RBP) reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3’UTR lengthening and downregulation of fusion oncoprotein AML1-ETO. In non-t(8;21) cells, knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study gives mechanistic insight into the role of APA in AML pathogenesis and provides evidence that targeting global APA patterns can overcome the differentiation block of AML patients.

overall_design: Data are 3'RNA-sequencing (3'READS) of CD34+ healthy HSPC controls and CD34+ bead-enriched AML blasts from AML patient samples. For analysis, two different healthy HSPC samples were compared to eight patient samples.

first: Amanda

last: Davis

first: Dong


title: Multipopulational transcriptome analysis of beef cattle at arrival to identify and further validate candidate biomarkers for predicting clinical bovine respiratory disease

status: Public on Dec 13 2021

city: Canyon

first: Matthew

middle: Adam

last: Scott

state: TX

address: 3201 Russell Long Blvd

department: Large Animal Clinical Sciences

institute: Texas A&M University

summary:
Objective: Classify and further validate BRD-associated genes and mechanisms found to be differentially expressed at facility arrival in post-weaned beef cattle from multiple independent populations. Our approach was to profile and compare at-arrival whole blood transcriptomes of post-weaned beef cattle that failed to develop symptoms associated with clinical BRD and cattle that ultimately were diagnosed and treated for clinical BRD within the first 28 days of facility arrival, further stratifying diseased animals into severity cohorts.
Methods: Whole blood RNA profiles were generated via NGS approach, using an Illumina Novaseq 6000. Individual samples were processed using an adaptation of the Revised Tuxedo Suite protocol (Tuxedo 2), then analyzed in R using edgeR with likelihood-ratio testing. Genes were considered differentially expressed with an FDR cutoff of 0.05. Downstream analysis involved PCA, gene ontology term enrichment, pathway analysis, protein-protein interaction networking, and IPA.
Results: 132 differentially expressed genes were identified between all three cohorts. Biological processes and pathways primarily involved neutrophilic activity, antimicrobial killing, cell cornification, and inflammatory signaling/regulation.
Conlusions: This study corroborated genes and pathways identified in our previous work, while providing novel genes and pathways for future analysis.

overall_design:
48 whole blood mRNA samples of stocker cattle at facility arrival were sequenced with an Illumina Novaseq 6000.
Disease severity cohorts:
"Healthy" animals never showed clinical signs throughout the course of that year's study.
"Treated_1" showed clinical signs after arrival and were only treated with injectable antimicrobials.
"Treated_2+" showed clinical signs after arrival and were treated more than one time with injectable antimicrobials and/or were euthanized due to clinical decline.
The antimicrobials used to treat clinical respiratory disease were florfenicol, ceftiofur, and enrofloxacin.


title: FIP1L1 knockdown in Kasumi-1 AML cells

overall_design: RNA-sequencing and 3'RNA-sequencing (3'READS) of Kasumi-1 cells transduced with two independent FIP1L1-targeting shRNAs or a control shRNA. For RNA-sequencing, 4 biological replicates were prepared for each condition. For 3'READS, 3 biological replicates were prepared for shControl and shRNA #2; 4 biological replicates were prepared for shRNA #1.


title: Cell-Autonomous Requirement of Shp2 and β-Catenin for Myc-Driven Hepatocarcinogenesis Revealed by Single Cell RNA-Sequencing

status: Public on Dec 01 2021

first: Yan

last: Liang

address: 9500 Gilman Drive, BRFII Room 1220 Bay B

institute: UC San Diego

summary: We applied single-cell RNAseq analysis to characterize hepatic cell types, including hepatocytes (Hep) and non-parenchymal cells (NPC), in mouse liver, during Myc-induced hepatocellular carcinoma (HCC) development.

overall_design: Hepatocytes (Hep) and Non-parenchymal cells (NPCs) were isolated from wild type (WT) and hepatocyte-specific Shp2 knockout (SKO) livers, at day 0 (0D-Ctrl) before HTVi, 10 days after Myc injection (10D-Myc), 10 days after empty vector injection (10D-Vector), and 4 weeks after Myc injection (4W-Myc).

first: Wendy

middle: S

first: Min

last: Zong

first: Kota

last: Kaneko

first: Kun

first: Gen-Sheng

last: Feng


title: sci-ATAC-seq identifies unique neuronal adhesion molecules and common transcription factor networks in high-grade localized prostate cancers

status: Public on Nov 04 2021

city: Happy Valley

first: Alex

middle: Daniel

last: Chitsazan

state: OR

address: 2720 S Moody Ave

institute: Oregon Health and Science University

summary: Flash-frozen prostate tumors collected from patients via radical prostatectomy were analyzed using single-cell ATAC-seq (sci-ATAC-seq). Unique cis- and trans-regulatory signatures between low- and high-grade localized prostate tumors are uncovered.

overall_design: Nuclei was extracted from frozen prostate tumors using a dounce homogenizer. FFPE blocks were cretaed with the rest of the tumor for tissue sectioning. Extracted nuclei were tagmented with barcoded transposomes using a 96-well plate. A secondary set of barcodes were introduced after the tagmentation reaction via PCR. As a result of this two-step barcoding, individual nuclei acquire a unique set of indeces in a high-throughput way.

first: Sebnem

last: Eksi

middle: D

first: Andrew

last: Adey

first: Paul

last: Spellman


title: The two-component system ChvGI responds to osmotic upshift and peptidoglycan damage in Caulobacter crescentus [ChIP-seq]

status: Public on Apr 11 2022

city: Namur

first: Regis

last: Hallez

address: 61 Rue de Bruxelles

institute: UNamur

summary: Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. ChvG/ChvI (ChvGI) is a TCS conserved in γ-proteobacteria and is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to activate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and confirmed that ChvI regulates cell envelope architecture at different levels by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. Furthermore, we identified genes with osmoregulatory properties and confirmed that osmotic upshift is a signal triggering ChvG-dependent phosphorylation of ChvI. In addition, we challenged chv mutants with other cell envelope related stress and found that targeting with antibiotics the transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Moreover, these antibiotics activate expression of the chvIG-hprK operon in ChvI-dependent and independent ways. ChvI phosphorylation is also shown to be activated upon antibiotic treatment with vancomycin. Finally, we observed that the sensor kinase ChvG fused to a fluorescent protein relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer.

overall_design: Examination of ChvI whole genome binding/occupancy (ChIP-Seq) in WT of Caulobacter crescentus grown in synthetic minimal media M2G

last: Quintero-Yanes

first: Aurélie

last: Mayard

first: Régis


title: CD244 expression represents functional decline of murine hematopoietic stem cells after in vitro culture

status: Public on Nov 01 2021

first: Kenichi

last: Miharada

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

overall_design: Refer to individual Series


title: Characterization of sequence determinants of enhancer function using natural genetic variation [RNA-seq]

status: Public on Jan 17 2022

city: Los Angeles

first: Marty

last: Yang

state: CA

address: 615 Charles E Young Dr S

department: Biological Chemistry

institute: University of California, Los Angeles

summary: Sequence variation in enhancers, a class of cis-regulatory elements that control cell type-specific gene transcription, contributes significantly to phenotypic variation within human populations. Enhancers are short DNA sequences (∼200 bp) composed of multiple binding sites (4-10 bp) for transcription factors (TFs). The transcriptional regulatory activity of an enhancer is encoded by the type, number, and distribution of TF binding sites that it contains. However, the sequence determinants of TF binding to enhancers and the relationship between TF binding and enhancer activity are complex, and thus it remains difficult to predict the effect of any given sequence variant on enhancer function. Here, we generate allele-specific maps of TF binding and enhancer activity in fibroblasts from a panel of F1-hybrid mice that have a high frequency of sequence variants. We identified thousands of enhancers that exhibit differences in TF binding and/or activity between alleles and use these data to define features of sequence variants that are most likely to impact enhancer function. Our data demonstrate a critical role for AP-1 TFs at many fibroblast enhancers, reveal a hierarchical relationship between AP-1 and TEAD TF binding at enhancers, and delineate the nature of sequence variants that contribute to AP-1 TF binding. These data represent one of the most comprehensive assessments to date of the impact of sequence variation on enhancer function in chromatin, with implications for identifying functional cis-regulatory variation in human populations.

overall_design: Genome-wide maps of H3K27ac, H3K4me1/2, H3K4me3, AP-1, CTCF, TEAD, chromatin accessibility, chromatin-associated RNA-seq in starved and serum-stimulated primary mouse embryonic fibroblasts from F1-hybrid mice.

first: Emi

last: Ling

first: Michael

last: Greenberg

first: Thomas

last: Vierbuchen


title: RNAseq analysis of NKG2A-knockout versus wildtype expanded human NK cells

status: Public on Sep 01 2021

city: Maastricht

first: Roel

last: Klein Wolterink

address: Universiteitssingel 50

department: Internal Medicine

institute: Maastricht University

summary: Checkpoint blockage has revolutionized cancer treatment. NKG2A is an inhibitory receptor expressed by cytotoxic lymphocytes, including NK cells. In contrast to other checkpoint inhibitory antibodies, anti-NKG2A antibodies have shown only limited success. Here, we designed a Cas9-based strategy to delete KLRC1 from human NK cells. Electroporation of KLRC1-targeting Cas9-RNP efficiently eliminated NKG2A expression from primary human NK cells. NKG2A-deficient NK cells showed normal proliferation, only minor transcriptional changes related to enhanced NK cell activation and maintained their phenotype and licensing status. Genetic deletion of NKG2A fully bypassed HLA-E inhibition and further enhanced NK cell activity against various tumor cell lines, thereby outperforming anti-NKG2A antibodies. In combination with antibody-coating of tumor cells to induce antibody-dependent cellular cytotoxicity, genetic deletion of NKG2A independently promoted cytotoxicity. Thus, Cas9-mediated targeting of NKG2A is an effective way to target this important inhibitory checkpoint. This technique is easily amenable to adoptive cell therapy in the clinical setting, where NKG2A deletion will promote anti-tumor responses and may help NK cells to better infiltrate and persist in an inhibitory tumor microenvironment.

overall_design: 6 paired (wildtype versus NKG2A-knockout) expanded human NK cell samples were collected 7 days after electroporation of KLRC1-targeting RNP. Total RNA was isolated and subjected to RNA re-sequencing on the DNBseq platform.

first: Ying

last: Gong

first: Wilfred

last: Germeraad

first: Gerard

last: Bos


title: DIS3L2 regulates male germ cell mesiosis in mice

status: Public on Jul 15 2021

city: Bethesda

first: Zhengpin

last: Wang

address: National Institutes of Health, 9000 Rockville Pike

department: Laboratory of Cellular and Developmental Biology

institute: NIDDK, NIH

summary: Male germ cell meiosis is essential for generating haploid spermatozoa in mice. Here, we investigate the essential role of DIS3L2 in male germ cell meiosis in mice. Conditional inactivation of DIS3L2 in spermatocytes with Stra8-cre transgenic mice have severely impaired meiotic progression, which results in defective meosis and spermatogenesis associated with a Sertoli cell-only syndrome and adult sterility. RNA-seq analysis reveales that Dis3l2 deficiency causes significant dysregulation of the expression of transcripts in mutant testes. Meiosis-assocaited genes are significantly decreased in the absence of DIS3L2. Therefore, we show that DIS3L2 ribonuclease plays a critical role in germ cell meiosis during spermatogenesis in mice.

overall_design: RNA-seq was performed using postnatal day 12 (P12) testes between control and Dis3 cKO mice in order to identify the differentially expressed genes.

first: Guoyun

last: Yu

first: Jurrien

last: Dean


title: Using small RNA-Seq to identify differential small RNA content of bacterial outer membrane vesicles (OMVs) secreted by Pseudomonas aeruginosa in response to tobramycin exposure

status: Public on Sep 12 2021

city: Hanover

first: Zhongyou

last: Li

state: NH

address: 504 Remsen

institute: Geisel School of Medicine at Dartmouth

type: Non-coding RNA profiling by high throughput sequencing

summary: Characterization of the sRNA content of OMVs harvested from Pseudomonas aeruginosa strain PA14 LB cultue with and without tobramycin (1ug/mL)

overall_design: control OMVs (ctrl-OMVs) and tobramycin OMVs (Tobi-OMVs) from 3 separate preparations.

first: Katja

last: Koeppen

first: Bruce

last: Stanton


title: 4C-seq analysis of three-dimensional chromatin interactions at the mouse Shox2 locus during embryonic limb development

status: Public on Jun 30 2021

city: Calgary

first: John

last: Cobb

state: AB

address: 2500 University Dr. NW

department: Biological Sciences

institute: University of Calgary

summary: The SHOX2 transcription factor is required during mouse embryonic development for the formation of the proximal elements of limbs, the humerus and femur. The Shox2 gene is flanked by an extensive gene desert spanning over 500 kilobases (kb) that contains many evolutionarily conserved elements with predicted cis-regulatory activities. However, the transcriptional enhancer potential of the vast majority of these regions have not yet been assessed. Therefore, we have generated a map of the Shox2 regulatory landscape during limb development using chromosome conformation capture techniques (4C-Seq). We report that at least five enhancers, distributed over more than 500 kb interact with the Shox2 gene and control its expression in developing proximal limbs, as confirmed by transgenic mouse assays. Furthermore, by using two of the identified enhancer candidates as 4C-seq viewpoints, we also find evidence that three of these putative enhancers interact with each other as well as the Shox2 gene, perhaps forming a cooperative regulatory complex. We expect this study to provide insight into the regulation of the human SHOX gene, a closely-related homolog of Shox2, that is similarly flanked by a large gene desert. Notably, deletions within the gene desert downstream of human SHOX are implicated as a major cause of the limb deformities characteristic of Léri-Weill dyschondrosteosis.

overall_design: 4C-seq analysis was performed using a viewpoint adjacent to the Shox2 promoter to detect chromatin interactions in proximal limbs from wild-type E12.5 mouse embryos, using two independent replicates . A secondary 4C-seq analysis was performed with the same samples, using sequences of the putative enhancers LHB-A (LE2) and PDE4 (LE4) as viewpoints to confirm their interaction with Shox2.

first: Samuel

last: Abassah-Oppong

first: Eddie

last: Rodríguez-Carballo


title: Integrative Low-input Transcriptomic and Cistromic Analyses Identify CREB Target Genes in Cystic Epithelial Cells

first: Zhiheng

last: Liu


title: Gene expression profiles in maspin-overexpressing RERF-LC-AI and LK-2 lung squamous cell carcinoma cell lines

status: Public on Jul 27 2021

city: Yonago

first: Takahiro

last: Matsushige

state: Tottori

address: 86 Nishi-cho

department: Faculty of Medicine

institute: Tottori University

summary: To investigate the subcellular localization of maspin in lung squamous cell carcinoma (LUSC) and its functional relevance, gene expression profiles were analyzed in RERF-LC-AI and LK-2 cells overexpressing maspin. The data provides insight into the differential function of maspin in LUSC depending on its subcellular localization.

overall_design: Establishment of control and maspin-overexpressing cell lines using RERF-LC-AI and LK-2 cells was performed by transduction with lentiviral vectors. Total RNA from these cells were recovered using Rneasy MINI Kit. To purify the strand-specific mRNA library, the mRNA was reverse transcribed into cDNA using random primers, and then the synthesized double-strand cDNA was fragmented, and the cDNA was subsequently ligated adapters. Transcriptome sequencing in strand-specific mRNA-seq library was performed using illumine NovaSeq 6000 Sequencing System.

first: Tomohiko

last: Sakabe

first: Hirotoshi

last: Mochida

first: Yoshihisa

last: Umekita


title: Generation of Human Islet Cell-Type-Specific Identity Genesets

status: Public on Jun 26 2021

city: Geneva

first: Léon

last: van Gurp

state: Geneva

address: Rue Michel Servet 1

department: Genetic Medicine and Development

institute: University of Geneva

summary: Generation of mature cells with stable functional identities is crucial for developing cell-based replacement therapies. Current global efforts to produce insulin-secreting beta-like cells to treat diabetes are hampered by the lack of tools to reliably assess cellular identity. We conducted a thorough single-cell transcriptomics meta-analysis to generate robust genesets defining the identity of human adult alpha-, beta-, gamma- and delta-cells. After extensive validation, we showed the efficacy of the novel genesets to define changes in islet cell identity, whether during embryonic development or in different experimental setups aimed at developing new functional glucose-responsive insulin-secreting cells, such as through pluripotent stem-cell differentiation or islet cell reprogramming protocols. Finally, we evaluated whether the perturbed metabolic conditions typical of diabetes influence islet cell identity. We observed that alpha-cells from diabetic donors exhibit an altered phenotype. In conclusion, these novel genesets represent valuable tools that robustly benchmark gain and loss in islet cell identity traits.

overall_design: Human islets from 3 independent non-diabetic donors (RRID: SAMN11633049, SAMN11963659, SAMN12227196) were dissociated and labelled with cell-surface antibodies. Cells were sorted on a Moflo Astrios (Beckman Coulter) system. Populations gated as gamma- or delta-cells in 2D12 vs. 2B4 plots were further gated in a CD9 vs. FSC-H plot. For gamma-cells, all CD9+ cells were complemented to 15000 cells with CD9- cells. For delta-cells, 15000 CD9+ and FSC-HLOW cells were sorted. Each population was processed as an independent experiment using the Chromium single cell gene expression protocol v3 (10x Genomics). A median of 55000, 100bp paired-end reads per cell were sequenced using Illumina Hiseq4000. Data were mapped against the Homo sapiens transcriptome reference 38 (GRCh38) using 10x Genomics Cell Ranger v3.0.2.

first: Leon

last: Fodoulian

first: Daniel

last: Oropeza

first: Kenichiro

last: Furuyama

first: Eva

last: Brú Tarí

first: Iván

last: Rodríguez

first: Fabrizio

last: Thorel

first: Pedro

middle: L

last: Herrera


title: RFX7 DNA binding landscape in RPE-1

status: Public on Jun 17 2021

city: Jena

first: Martin

last: Fischer

state: Thuringia

address: Beutenbergstraße 11

department: Computational Biology

institute: Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)

summary: Identification of the DNA binding landscape of the transcription factor regulatory factor X 7 (RFX7) in Nutlin-3a and DMSO control treated RPE-1 cells.

overall_design: RFX7 ChIP-seq in Nutlin-3a and DMSO control treated RPE-1 cells

first: Konstantin

last: Riege

first: Steve

last: Hoffmann


title: Gene expression data of murine hematopoietic stem cells before and after in vitro culture

first: Kenichi

last: Miharada

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

institute: Kumamoto University

summary:
Isolation of long-term reconstituting hematopoietic stem cell (HSC) has been possible by utilizing flow cytometry with a combination of multiple antibodies against cell surface markers. However those cell surface phenotypes do not represent functional HSC after in vitro culture.
We compared gene expression profiling of phenotypic HSC (CD48-KSL cells) before and after in vitro culture, and discovered that cultured HSC express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSC can be divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. Induction of CD244 may partially be triggered by induction of endoplasmic reticulum (ER) stress, as chemically induced ER stress signal increased CD244+ subpopulation while ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which were correlated to the output of reconstitution assay. These data suggest CD244 positivity is a potent marker to exclude non-functional HSC after in vitro culture thereby useful to elucidate mechanism of functional decline of HSC during ex vivo treatment.

overall_design: CD48- c-Kit+ Sca-I+ Lineage- (CD48-KSL) cells were sorted from bone marrow of 10 weeks old or 18 months old C57BL/6 SJL mice. A part of young HSCs were then cultured in Stemspan SFEM medium supplemented with 100 ng/ml of murine stem cell factor (mSCF) and 100 ng/ml of human thrombopoietin (hTPO) with or without 100 μM of tauroursodeoxycholic acid (TUDCA) or 100 μM of tauroursodeoxycholic acid (TUDCA) , and 14-days later CD48-KSL fraction was re-sorted.

first: Valgardur

last: Sigurdsson

first: Visnja

last: Radulovic


title: Acute myeloid leukemia (AML) patient blasts and healthy hematopoietic stem and progenitor cell (HSPC) RNA-sequencing Data

first: Dong-Er

last: Zhang

summary: Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) chromosomal translocation is among the most common subtypes of AML and produces the AML1-ETO (AE) oncogenic fusion gene. AML1-ETO expression is critical to for t(8;21) AML leukemogenesis and maintenance. Post-transcriptional regulation of gene expression is often mediated through transcript 3'-untranslated regions (UTR). AML1-ETO uses the 3’UTR of the ETO gene, which is not normally expressed in hematopoietic cells. Therefore, the mechanisms regulating AML1-ETO via the 3’UTR are attractive therapeutic targets. In this study, we examine the regulation of AML1-ETO via the 3’UTR. We demonstrate that AML1-ETO primarily uses a 3.7kb isoform of the ETO 3’UTR in both t(8;21) patients and cell lines. Using a luciferase assay approach, we identify an AML1-ETO 3’UTR fragment between 2.8 and 3.4kb which is negatively regulated, increases expression upon inhibition of microRNA biogenesis, and contains a putative let-7 microRNA target site. We show that let-7b directly represses AML1-ETO through this site. An analysis of The Cancer Genome Atlas AML dataset shows that let-7b and let-7 family miRNA expression is significantly lower in t(8;21) AML than other AMLs. Finally, we demonstrate that let-7b-5p inhibits the proliferation of t(8;21) AML cell lines, rescues expression of AML1-ETO target genes, and promotes differentiation. Our findings establish AML1-ETO as a let-7b target gene and suggest that let-7 based therapeutics may be applied to t(8;21) AML.

overall_design: RNA-sequencing of CD34+ healthy HSPC controls and CD34+ bead-enriched AML blasts from AML patient samples. For analysis, five healthy HSPCs were sequenced and compared to eight AML patient samples, subdivided into four t(8;21) and four non-t(8;21) AML patients.

first: Amanda

last: Davis

last: Johnson


title: RFX7 DNA binding landscape in HCT116

summary: Identification of the DNA binding landscape of the transcription factor regulatory factor X 7 (RFX7) in Nutlin-3a and DMSO control treated HCT116 cells.

overall_design: RFX7 ChIP-seq in Nutlin-3a and DMSO control treated HCT116 cells


title: Large scale analysis of leaf gene expression upon treatment of apple seedlings with acibenzolar-S-methyl or water

status: Public on Jun 11 2021

city: Angers

first: Alexandre

last: Degrave

address: André Lenôtre

institute: Agrocampus Ouest

summary: Investigation of whole genome gene expression level changes in the youngest expanded leaves of open-pollinated Golden Delicious seedlings either sprayed with reverse osmosis water or acibenzolar-S-methyl (ASM).  Treatment was performed by spraying sprayed to runoff (with a pressurized hand sprayer) with the commercial product Bion 50 WG (Syngenta, Basel, Switzerland; 50% of ASM) prepared in reverse osmosis water at a final concentration of 0.4 g/L. The youngest developped leaf of each seedling was sampled 3 days after the treatment.

overall_design: A total of 8 samples were associatied as pairs to perform 4 competitive hybridisations, including dye switch. Biological replicates are from 4 independantly grown apple seedling series. These comparisons allowed to determine the impact of ASM on apple gene expression.  This is a dual channel array processed as a single-channel analysis.

first: Christelle

last: Heintz

first: Romain

last: Warneys

first: Muriel

last: Bahut

first: Marie-Noëlle

last: Brisset


title: Gene expression data of murine hematopoietic stem cells after in vitro culture and anemic stress

city: Kumamoto

summary:
Isolation of long-term reconstituting hematopoietic stem cell (HSC) has been possible by utilizing flow cytometry with a combination of multiple antibodies against cell surface markers. However those cell surface phenotypes do not represent functional HSC after in vitro culture. We compared gene expression profiling of phenotypic HSC (CD48-KSL cells) before and after in vitro culture, and discovered that cultured HSC express mast cell-related genes including Cd244. After in vitro culture, phenotypic HSC can be divided into CD244- and CD244+ subpopulations, and only CD244- cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. Induction of CD244 may partially be triggered by induction of endoplasmic reticulum (ER) stress, as chemically induced ER stress signal increased CD244+ subpopulation while ER stress suppression using a molecular chaperone, TUDCA, decreased CD244+ population, which were correlated to the output of reconstitution assay. These data suggest CD244 positivity is a potent marker to exclude non-functional HSC after in vitro culture thereby useful to elucidate mechanism of functional decline of HSC during ex vivo treatment.
It has been unknown whether HSC respond to acute anemic stress. We compared frequency and differentiation potential of HSC in steady-state vs acute anemic mice, and discovered that HSC rapidly expand upon the anemia induction and change their lineage balance to more erythrocytic.

overall_design: CD244- CD48- c-Kit+ Sca-I+ Lineage- (CD244-CD48-KSL) cells were sorted from bone marrow of 10 weeks old C57BL/6JRj mice. A part of the cells were cultured in Stemspan SFEM medium supplemented with 100 ng/ml of murine stem cell factor (mSCF) and 100 ng/ml of human thrombopoietin (hTPO), and 7-days later CD244- and CD244+CD48-KSL fractions were re-sorted. In another setting, 10 weeks old mice were injected with PBS or 60 mg/kg of phenylhydrazine, or phlebotomized, and 24 hours later CD150+CD48-KSL cells were isolated from the bone marrow.

first: Mark

last: van der Garde

first: Shuhei

last: Koide


title: Single-Base Resolution Mapping Reveals Distinct 5-Formylcytidine in Saccharomyces cerevisiae mRNAs

city: Beijing

first: Zonggui

last: Chen

state: Beijing

address: No.5 Yiheyuan Road Haidian District

department: Academy for Advanced Interdisciplinary Studies

institute: Peking University

summary:
5-Formylcytidine (f5C) is one type of post-transcriptional RNA modifi-cations, which is known at the wobble position of tRNA in mitochon-dria and essential for mitochondrial protein synthesis. Here, we show a method to detect f5C modifications in RNA and a transcriptome-wide f5C mapping technique, named f5C-seq. It is developed based on the treatment of pyridine borane, which can reduce f5C to 5,6-dihydrouracil (DHU), thus inducing C-to-T transition in f5C sites during PCR to achieve single-base resolution detection. Thousands of f5C sites were identified after mapping in Saccharomyces cerevisiae by f5C-seq. Moreover, codon composition demonstrated a preference for f5C within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA.

Reference build: S288C_reference_genome_R64-2-1_20150113

overall_design: f5C-seq performed on Saccharomyces cerevisiae cells; samples Titles with "experiment" means the mRNA was treated with pyridine borane; samples Titles with "input" means the extracted mRNA without the treatment of pyridine borane before library preparation.

first: Y

last: Wang

first: Z

first: X

last: Weng

last: Zhou


title: Drug Combination Signatures for Prediction and Mitigation of Toxicity [single cell]

status: Public on May 29 2021

city: New York

first: Ravi

last: Iyengar

state: NY

address: 1425 Madison Avenue, Room 12-70C

department: Pharmacological Sciences

institute: Icahn School of Medicine at Mount Sinai

summary: Single cell transcriptomic signatures of cardiomyocytes differentiated from hiPSC lines generated from clinically well-characterized, diverse healthy human individuals. We provide single cell mRNAseq data of cardiomyocytes differentiated from 4 hiPSC lines.

overall_design: The overall goal of the drug signatures for prediction and mitigation of toxicity study is to use genomic and proteomic high-throughput measurements as the basis for computational analysis that integrates network analyses with structural constraints and dynamical models in multiple cell types to identify signatures that predict toxicity induced by 55 FDA-approved chemotherapeutic drugs and potential mitigation of this toxicity.  For this purpose, we have generated human induced pluripotent cell (hiPSC) lines from established fibroblast lines of forty clinically healthy racially diverse male and female study participants who ranged in age from 22 and 61 years. One clone from each of the forty hiPSC line was fully characterized for normal karyotype, short-tandem repeat matching to the original fibroblast line (authentication), pluripotency by select pluripotent marker expression via immunocytochemistry and by mRNAseq-based PluriTest analysis, and whole-genome sequencing. In order to establish  singel cell transcriptomic signatures specific to clinically well-characterized healthy individuals, cardiomyocytes were differentiated from four hiPSC lines derived from 2 male and 2 female, age matched (23 and 33 yrs old, and 24 and 36 yrs old, respectively) persons using an established protocol, purified to 95% purity (SIRPA+CD90-) using a metabolic switch protocol, reseeded and treated for 24 hrs with DMSO (14.1mM) before single cell mRNAseq was performed using the 10x Genomics platform.


title: ChIP-seq Analysis for H3K4me3 in Neomycin-induced Lgr5+ Progenitor Cells of Neonatal Mouse Cochlea

status: Public on Jun 05 2021

city: Nanjing

first: Xiangyu

last: Ma

address: Sipailou II

institute: Southeast University

summary: In the study, we detailedly manifested the regulatory roles of histone modifications in neomycin-induced proliferation of Lgr5+ progenitor and HC regeneration by integrated RNA-seq and ChIP-seq analysis. we found 94 differentially expressed genes which are related to differential binding regions via analyzing ChIP-seq and RNA-seq data. we are delighted to find that H3K4me3 can modulate the expressions of 12 genes to involve in cell cycle, neural development, inner ear development, Wnt and Hippo signaling pathways to regulate proliferation and HC regeneration based on the protein interaction network and gene ontology (GO) functional annotation analysis.

overall_design: Examination of H3K4me3 modification in the mouse cochlea Lgr5+  progenitor cells under neomycin-treated and untreated conditions.

first: Shasha

first: Shijie

last: Qin


title: 3D heterospecies spheroids of pancreatic stroma and cancer cells demonstrate key phenotypes of pancreatic ductal adenocarcinoma

status: Public on May 18 2021

city: Stockholm

first: Xinyuan

last: Liu

address: Hälsovägen 7

department: Department of Clinical Science, Intervention and Technology

country: Sweden

institute: Karolinska Institute

summary: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, partly due to the dense desmoplasia and a lack of suitable model systems to study. In the present work, we developed a 3D heterospecies spheroid model to study the microenvironmental interactions between tumor cells and stellate cells which can also be employed to test therapeutic regimens. We set up monospheroids and heterospheroids made up from murine pancreatic stellate cells (mPSCs) and human PDAC cells (Panc1), which allowed for direct isolation of mRNA from a mixed cell population followed by an in silico separation of the RNA-seq reads. Global transcript level changes for cells in heterospheroids versus monospheroids were calculated, followed by gene set enrichment analysis and molecular subtype analysis. We observed an apparent shift of Panc1 from the classical to the squamous/basal-like phenotype upon co-culture with mPSCs. Moreover, mPSCs acquired a different cancer-associated fibroblast-related phenotype upon co-culture with Panc1. We analyzed the tumor cell-specific chemosensitivities towards gemcitabine, paclitaxel and SN38 and compared these to published pharmacotranscriptomic signatures. In conclusion, our heterospecies spheroid model reflected key aspects of PDAC and facilitated the study of intercellular interactions between tumor and stroma while additionally proving to be a good model for studying therapeutic responses.

overall_design: mRNA profiles of Panc1, imPSCc2 monospheroids and Panc1/imPSCc2 heterospheroids cultured under 2 conditions: high serum condition and low serum condition.

first: Beate

last: Gündel

first: Xidan

last: Li

first: Jianping

first: Anthony

last: Wright

first: Matthias

last: Löhr

first: Gustav

last: Arvidsson

first: Rainer

last: Heuchel


title: Transcriptomics of Mycobacterium tuberculosis exposed to zinc limitation, zinc depletion by calprotectin and TPEN, and zinc toxicity

status: Public on Apr 21 2021

city: Honolulu

first: Sladjana

last: Prisic

state: HI

address: 1800 East-West Rd

institute: University of Hawaii

summary: The purpose of this study was to determine the transcriptomes of Mycobacterium tuberculosis m2 6206 (Mtb) exposed to varying concentration of zinc (Zn2+) in vitro that mimic the concentrations of Zn2+ experienced in vivo during infection. Mtb were grown in the chemically defined Sauton's medium without Zn2+ added until expression of Zur-regulated genes were observed, indicating the onset of Zn-limitation. Cells were then treated with with different concentrations of Zn2+ through addition of 500 uM Zn2+, 6uM Zn2+, no added Zn2+, 0.35 mg/mL recombinant calprotectin (CP) or 0.8 µM N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). After 24 hours of exposure to the different [Zn2+] conditions, RNA was harvested, prepared for RNA-seq, and sequenced on the Illumina HiSeq platform with a paired-end (2x150bp) configuration.

overall_design: Transcriptomes of Mtb  exposed to 500 μM Zn2+, 6 μM Zn2+, no added Zn2+, 0.35mg/mL recombinant calprotectin (CP), and 0.8 μM TPEN

first: Allexa

last: Dow

first: Brandi

last: Antonio


title: Large scale analysis of leaf gene expression upon infection of susceptible or resistant apple varietes with different strains of Erwinia amylovora

status: Public on May 25 2021

first: Alexandre

last: Degrave

summary: Investigation of whole genome gene expression level changes in leaves of susceptible (MM106) or resistant (ME010) apple genotypes either non-inoculated, inoculated with the wild type strain of Erwinia amylovora (CFBP1430) or the non-pathogenic mutant strain PMV6023. The mutant strain PMV6023 is affected in the biosynthesis of the type 3 secretion system delivering bacterial effectors into apple cells. Leaves were harvested for RNA extraction before inoculation, 6 hours and 24 hours after inoculation.

overall_design: A total of 28 samples were associatied as pairs to perform 14 competitive hybridisations. Biological replicates are from 2 independantly grown grafted apple scions series, leading to 7 swaps. Three swaps including dye switch were associated to compare leaf samples from susceptible and resistant varieties either before inoculation, 6 or 24 hours after inoculation with the wild type strain CFBP1430. Four additional swaps including dye switch were associated to compare leaf samples harvested within each apple variety, either 6 or 24 hours after inoculation with CFBP1430 or PMV6023 bacterial strains. These latter swaps allowed to determine the impact of type III effector delivery on host gene expression either for susceptible or resistant genotypes, at 6 or 24 hours after inoculation.

first: Matthieu

last: Gaucher

first: Sandra

last: Pelletier

first: Jean-Pierre

last: Renou

first: Marie-Noëlle

last: Brisset