title: Evaluating the practical aspects and performance of commercial single-cell RNA sequencing technologies

first: Anna

last: Elz

address: 1201 Eastlake Ave E

institute: Fred Hutchinson Cancer Center

summary: The rapid development of updated and new commercially available single-cell transcriptomics platforms is providing a range of experimental options.  Cost, sensitivity, throughput, flexibility, and ease of use influence that selection.  We performed a comprehensive comparison of single-cell transcriptomic approaches using multiple standardized PBMCs. In evaluating seven recently available kits that interrogate whole transcriptome mRNA and two including TCR sequencing, we report on standard single-cell sensitivity metrics including gene saturation, UMI detection, cell recovery, sequencing efficiency, and cell subset resolution.  We also discuss workflow throughput, sample requirements, timing, cost, and labor as critical factors to consider based on project goals. In addition to variable experimental constraints imposed by each platform, our findings highlight differences in cell recovery and sensitivity, which we found to significantly influence cell subset resolution. This work provides a basis by which users can balance performance and practical considerations when selecting an RNA single-cell sequencing platform.

overall_design: For our first experiment,  Benchmark 1 (BM1), we used two healthy PBMC donor samples in replicate, to compare seven platforms: 10x Genomics’ Chromium NextGEM 3′ v3.1 (labeled “NextGEM 3’” in figures and results),  GEM-X 3’ v4 (GEM-X 3’), and Fixed RNA Profiling (Flex), Parse’s Evercode WT v.3 (Parse v3), Scale Biosciences Single Cell RNA kit (Scale), and Fluent Biosciences PIP Seq T20 3′ v4 (Fluent v4) and PIPseq V T20 3’ (Fluent V).  Each of these kits target 3′ and whole transcriptome RNA from single cells.  All platforms were run in parallel on the same day except for Scale and Fluent PIPseq V assays, which were processed separately using aliquots from the same donors’ leukapheresis samples (Figure 1a). For our second experiment, Benchmark 2 (BM2), we compared platforms that generate TCR repertoire profiling (Supplemental Figure 1a).  For this, we sorted T-cells based on CD3 expression from 4 different healthy donor PBMCs, with overlap of two BM1 donors, to run on 10x Genomic’s Chromium NextGEM 5′ v2 (NextGEM 5′), GEM-X 5′ v3 (GEM-X 5’) and Parse Evercode v.2 TCR (Parse v2).

first: Derrik

last: Gratz

first: Annalyssa

last: Long

first: David

last: Sowerby

first: Azi

last: Hadadianpour

first: Evan

last: Newell


title: Non-viral CISH locus-specific integrated IL-15-armored CAR-NK cells achieve potent anti-tumor efficacy via adaptive NK cell differentiation

status: Public on May 26 2025

city: Guangzhou

first: guozhu

last: xie

address: Shatainan Road No.1023-1063

country: China

institute: Southern Medical University

type: Expression profiling by high throughput sequencing

summary: Chimeric antigen receptor (CAR)-NK therapy has emerged as a highly promising alternative to CAR-T cell therapy, due to its remarkable efficacy and safety in hematological tumors. However, the efficacy of CAR-NK cells currently remains limited in solid tumors. Here, we successfully developed CISH locus-specific integrated CAR-NK cells using a non-viral mini-circular single-stranded DNA (mcssDNA)-based CRISPR/Cas9 targeted genome editing (mcssDNA/CRISPR/Cas9) technology via a single-step electroporation procedure. The developed CISH-knockout (CISH-KO) CAR-NK cells exhibited the enhanced proliferation and cytotoxicity against hepatocellular carcinoma (HCC) compared with conventional lentivirus-transduced CAR-NK (LV-CAR-NK) cells. Single-cell RNA sequencing analysis revealed an important subset of adaptive NK cells that were highly enriched in CISH-KO CAR-NK cells compared to LV-CAR-NK cells and showed the up-regulated JAK/STAT, energy metabolism and activating receptor signaling. Furthermore, non-viral CISH locus-specific integrated IL-15-armored CAR-NK cells were generated and achieved persistent tumor regression and a significant survival advantage in a lung metastatic mouse model of HCC. Collectively, our results demonstrate that non-viral CISH locus-specific integrated CAR-NK cells can be generated by a simplified, single-step manufacturing procedure and achieve potent efficacy against HCC, thus providing an innovative technology for CAR-NK cell therapy against solid tumors.

overall_design: The cells are stained with 0.4% trypan blue to assess cell viability under microscopic observation. Cells with greater than 80% viability are qualified for library construction process. Library construction is performed by using DNBelab C Series High-throughput Single-cell RNA Library Preparation Set V3.0 (MGI,China). The single-cell suspension, oil, beads are added into C4 scRNA slide and instrument in order. Following this, the droplets are broken by vacuum pump, releasing the mRNA-bead complexes. The cDNA synthesis is conducted at the suitable temperature. Then the cDNA and Oligo products are amplified and purified. The products are subjected to concentration and size distribution test to make sure in a good range. The Oligo library is constructed and then subjected to amplification, index ligation and purification for circularization. The cDNA is fragmentated, end repair and A tailing to form cDNA products. Then the cDNA is ligated to adaptor at the suitable temperature for a fixed period of time and then purified. The cDNA with adaptor is amplified via PCR and then purified. The cDNA and Oligo products are respectively denatured into single stranded. The reaction system and program for circularization are respectively configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS). The cDNA library was sequenced by PE 47+100 and Oligo library using PE 32+42.

first: Guozhu

last: Xie

first: Yao

last: Sun


title: Full-length circRNAs in IMR-90 and A549 cell lines using nanopore sequencing

status: Public on May 27 2025

city: Beijing

first: Shaodong

last: Huang

address: Peking University Health Science Center, Beijing, China

department: Department of Biomedical Informatics

institute: Peking University

type: Non-coding RNA profiling by high throughput sequencing

summary: To identify full-length circRNAs in lung normal and cancer cell lines, we enriched circRNAs by rRNA depletion, poly(A) tailing and Rnase R treatment and amplified circRNAs by rolling circular RT. We constructed library according to the protocol of Fangqing Zhao team's. Nanopore is used for full-length sequencing.

overall_design: Full-length circRNAs in IMR-90 and A549 was identified using nanopore sequencing

first: Yulong

last: Li

first: Yiran

last: Liu

first: Lina

last: Bu

first: Shu

last: Meng

first: Dongyu

last: Zhao


title: CHD7 Regulates Definitive Endodermal and Mesodermal Development from Human Embryonic Stem Cells [ATACseq]

city: Storrs

first: Laijun

last: Lai

state: Connecticut

address: 1390 Storrs Road

country: USA

institute: University of Connecticut

type: Genome binding/occupancy profiling by high throughput sequencing

summary: CHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders, including CHARGE (Coloboma, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital hypoplasia, and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus, liver, pancreas, and intestine. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7-/-), heterozygous mutant (CHD7+/-), and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers, mesoderm and ectoderm. We found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. To determine the underlying mechanisms, we compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq). We found that loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.  Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.

overall_design: We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7-/-), heterozygous mutant (CHD7+/-), and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into definitive endoderm (DE) and the other two germ layers, mesoderm and ectoderm in vitro. We also compared global chromatin accessibility among the hESC-DE cells by the assay for transposase-accessible chromatin with sequencing (ATAC-seq).


title: Optimized culturing yields high success rates and preserves molecular heterogeneity, enabling personalized screening for high-grade gliomas

city: Rotterdam

first: Martine

last: Lamfers

address: Dr. Molewaterplein 40

department: Neurosurgery

country: Netherlands

institute: Erasmus MC

summary: Robust and reliable and in vitro models are essential in the discovery of new treatment options for high-grade glioma (HGG), however, establishing successful patient-derived cell cultures has posed significant challenges. We established glioma stem-like cell (GSC) cultures from 114 consecutive HGG specimens, obtained via traditional surgical resection and/or ultrasonic aspiration, using fully-dissociated single cells (single cell-derived, SCD) and partially dissociated tissue fragments (3D-derived, 3DD). Copy number profiling assessed genetic similarities, while single-cell RNA sequencing evaluated transcriptomic heterogeneity. Proof-of-concept personalized drug screening was performed using a panel of approved anti-cancer agents. Higher success rates in establishing GSC cultures were obtained from ultrasonic aspirates (SCD and 3DD) and 3DD surgical samples compared to SCD cultures from surgical samples. Combining these approaches in parallel yielded a 96% success rate. In rare cases, copy number variations in original tumor tissue were lost in culture, leading to discernible changes in cell morphology. Single-cell sequencing revealed greater heterogeneity of transcriptomic clusters in ultrasonic aspiration-derived cultures compared to resection-derived cultures. Our study's protocol supported the screening of 20 anti-cancer agents within a clinically-relevant timeframe in 16 of 18 consecutive HGG samples. Our optimized protocol therefore provides a reliable tool for generating HGG cell cultures which capture the tumors’ molecular characteristics and supports precision medicine applications.

overall_design: To assess copy number variation (CNV) profiles of 31 patient tissues and their derived cell cultures, 200 ng of DNA from each sample was loaded according to the manufacturer’s instructions on an Illumina Global Screening Array (GSAMD-24v1) chip

first: Cassandra

last: Posthoorn-Verheul

first: Federica

last: Fabro

first: Ioannis

last: Ntafoulis

first: Chelsea

last: den Hollander

first: Iris

middle: S

last: Verploegh

first: Rutger

last: Balvers

first: Trisha

middle: V

last: Kers

first: Jessica

last: Hoogeveen

first: Judith

last: van der Burg

first: Bert

last: Eussen

first: Annelies

last: de Klein

first: Kate

middle: J

last: Feller

first: Miao-Ping

last: Chien

first: Clemens

middle: M

last: Dirven

first: Sieger

last: Leenstra

middle: L


title: Single-cell transcriptomics in the lungs of CLEC5A-deficient mice with CLP-induced polymicrobial sepsis

status: Public on May 24 2025

city: Shenyang

first: Chunfeng

last: Liu

state: Liaoning

address: No.36 SanHao Street

institute: Shengjing Hospital of China Medical University

summary: Sepsis is a life-threatening condition with multi-organ failure caused by a dysregulated host response to infection. Among different organs susceptible to infection, the lung is the main one leading to sepsis 4. Patients with severe sepsis are at a high risk of developing acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which occurs in up to 50% of patients. Myeloid C-type lectin domain family 5 member A (CLEC5A) is a pattern recognition receptor that participates in host defense against infection. Blockage of CLEC5A increased survival of mice with polymicrobial sepsis or endotoxemia, and protected the lungs against septic infection. To explore the underlying cellular and molecular mechanisms of CLEC5A in sepsis-induced lung injury, a comprehensive single-cell transcriptomics of CLEC5A-/- lungs was generated by scRNA-seq. Mice were subjected to cecal ligation and puncture (CLP), and the lungs were collected 12 h post-surgery. Single cell suspensions were prepared from lung tissues of three CLEC5A-/- mice and three WT littermates, and pooled cells from these mice were subjected to scRNA-seq processing using the 10×Genomics Chromium system. A total of 55,177 single cells, including 29,306 cells from WT lungs and 15,871 cells from CLEC5A-/- lungs, were obtained after removal of multiplets and filtration of low-quality cells. Total cells were grouped into 34 distinct clusters by Uniform Manifold Approximation and Projection, and a total of 14 distinct cell types were identified, including granulocytes, T cells, B cells, alveolar-macrophages (AM), monocytes, macrophages, natural killer T cells (NKT), mesothelium, natural killer cells (NK), endothelial cells, fibroblasts, dendritic cells (DC), epithelial cells, and smooth muscle cells (SMC).The scRNA-seq analysis implied the potential role of CLEC5A in endothelial cells regarding crosstalk with immune cells in septic lungs. Deletion of CLEC5A inhibited endothelial barrier dysfunction induced by sepsis, and decreased leukocyte-endothelial adhesion and transendothelial migration. The role of CLEC5A in pulmonary endothelial cells and the barrier function is identified for the first time, which contributes to the regulation of immunity/inflammation in the lungs under septic infection. This work extends the therapeutic potential of targeting CLEC5A in sepsis-induced endothelial barrier dysfunction and lung injury.

overall_design: Lung tissues were obtained from CLEC5A-/- and  WT mice with CLP-induced polymicrobial sepsis, and then single-cell suspensions were harvested for scRNA-seq and transcriptomic analysis.

first: Tiening

last: Zhang

first: Xinmei

last: Huang

first: Julie

first: Ri

last: Wen

first: Yongping

first: Yuhang

last: Yang

first: Tao

first: Yue

last: Zheng

first: Anyu

last: Chen

first: Penghui

last: Hao

first: Xiaoxu

last: Tong

first: Ni


title: Transcriptomic profiling of mouse skeletal muscle in wildtype and Tas1r2 knockout mice across age and obesity conditions

status: Public on May 23 2025

city: Columbus

first: Kendall

last: King

state: OH

address: 370 West 9th Ave.

institute: The Ohio State University

summary:
This study aimed to better understand the effects of Tas1r2 loss-of-function on skeletal muscle gene expression across physiological states. Transcriptomic profiling was performed on skeletal muscle from wildtype and Tas1r2 knockout male mice under young, aged, and obese conditions.
RNA was obtained from left leg solei of male mice

overall_design: Thirty samples from mouse soleus and quadriceps muscle were profiled using the Affymetrix Clariom S Mouse microarray platform. Samples include wildtype and Tas1r2 knockout male mice across three biological contexts: young (3–4 months), aged (18–20 months), and leptin-deficient obese (Lep^ob/ob). Each group includes multiple biological replicates (n = 3–6 per group). The dataset includes both control (wildtype) and experimental (Tas1r2 knockout) samples within each age and obesity group. This submission represents the first release of a broader study investigating gene expression in skeletal muscle under different physiological and Tas1r2 functions.

first: Joan

last: Serrano

first: George

last: Kyriazis


title: R-2-hydroxyglutarate-mediated inhibition of KDM4A disrupts telomere integrity

city: Montréal

first: Frédérick

middle: Antoine

last: Mallette

state: Québec

address: 5415 boul. de l'Assomption

country: Canada

institute: Université de Montréal / Hôpital Maisonneuve-Rosemont

summary: Isocitrate dehydrogenases 1 and 2 are mutated in more than 70% of low-grade glioma. This promotes the production of R-2-hydroxyglutarate (R-2HG) instead of α-ketoglutarate (αKG) and R-2HG will inhibit α-ketoglutarate enzymes like the lysine demethylase KDM4A. We hypothesized that KDM4A inhibition through R-2HG contribute to gliomagenesis. In our study, we showed that KDM4A inhibition leads to telomere defects. By ChIP-seq experiments, we demonstrated that KDM4A localized on telomeric repeats and that its depletion increased the level of H3K9me3 on telomeric chromatin.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for KDM4A in HeLa cells

middle: A

first: Laurence

last: Gagné

first: Christina

last: Sawchyn

first: Marie-Ève

last: Lalonde


title: Integrating spatial and single-cell transcriptomics to characterize mouse long bone fracture healing process

status: Public on May 25 2025

first: Lutian

last: Yao

address: 155 Nanjing st

department: Department of Orthopedic

institute: the First Affiliated Hospital of China Medical University

summary: Bone fracture healing is a complex and dynamic processhat requires a detailed understanding of the cellular interactions crucial for successful tissue regeneration. We employs optimized spatial transcriptomics to precisely delineate the locations and interactions of these cells within a mouse femur fracture model at day 0 before fracture and days 5, 15 post-fracture. We improved RNA quality significantly by optimizing our decalcification method using Morse's solution, coupled with the use of the Visium CytAssist platform and integrated analyses through the Seurat, CARD, and Monocle frameworks. This approach allowed us to accurately localize critical cell populations, such as periosteum progenitor cells, and identify pivotal transcription factors that regulate their activation and differentiation into chondrocytes or osteogenic cells. We particularly focused on the transformation from mesenchymal progenitor cells (MPCs) to regenerative MPCs (rMPCs), revealing how these cells recruit macrophages near the fracture line during early healing stages and their involvement in fracture healing. Furthermore, using CellChat, we explored potential receptor-ligand pathways that mediate these cellular interactions. This spatial-temporal mapping and molecular characterization substantially deepen our understanding of the cellular and molecular processes involved in fracture healing, highlighting spatial transcriptomics as a robust approach for elucidating the fundamental mechanisms governing bone regeneration.

overall_design: We conducted high-quality spatial transcriptomic sequencing of long bone periosteal cells at different time points (Day 0, 5 and 15), enabling a more precise exploration of their temporal changes and roles in fracture repair.


title: Zileuton protects against arachidonic acid/5-lipoxygenase/leukotriene axis-mediated neuroinflammation in experimental traumatic brain injury

city: Shanghai

first: Fang

last: Yuan

address: Yi Shan Road

institute: Shanghai Jiao Tong University School of Medicine

summary: Traumatic brain injury (TBI) is a leading cause of death and disability globally. Several studies have shown that 5-lipoxygenase (5-LOX) inhibition reduces leukotriene (LT) release and the inflammatory response, attenuating the development of respiratory diseases, myocardial infarction, and ischemic cerebral injury. However, its role in the pathophysiology of TBI remains unclear. In this study, controlled cortical impact injury was induced to construct a mouse model of TBI. Pericontusional brain tissue samples from sham and TBI mice at 7 days after injury were used for RNA-seq analysis. Altered gene enrichment following TBI, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, was quantified through real-time polymerase chain reaction (RT-PCR). Immunocytochemistry, Western blotting, and single-cell sequencing experiments were also performed to analyze 5-LOX protein expression. Arachidonic acid (AA) was detected through liquid chromatography mass spectrometry/mass spectrometry. Enzyme-linked immunosorbent assay was used to detect LTB4 release after TBI with or without zileuton treatment. Brain damage, blood–brain barrier disruption, and neuronal apoptosis were detected through histological examination. Neurological outcomes were determined through rotarod and fear conditioning tests. TBI induced significant upregulation of genes related to the AA metabolic pathway, particularly the AA/5-LOX/LT axis, as verified by RT-PCR. AA and LTB4 production increased significantly after TBI. The expression levels of Pla2g4a, which hydrolyses phospholipids to release AA, and 5-LOX, which in turn act downstream to convert AA to LT, were dramatically upregulated up to 7 days after TBI. 5-LOX accumulated in the cytoplasm of activated ameboid microglial cells. In vivo, 5-LOX inhibition with zileuton blocked LT release and reduced microglial activation and the production of inflammatory cytokines, including Il-1β, Ccl7, Spp1, Ccr1, Ccl2, and Il-10. Zileuton also reduced TBI-induced lipid ROS and neuronal cell apoptosis, ameliorating brain damage compared to the vehicle group and improving neurological outcomes after TBI. Mechanically, TBI-induced LT upregulation may stimulate BV2 microglial activation through the ERK, NF-κB, and Akt pathways. Our findings demonstrated the role of 5-LOX in TBI and its potential as a therapeutic target in TBI treatment.

overall_design: In this study, we conducted an RNA-seq analysis of pericontusional brain tissue samples from sham and TBI mice at 7 days post-injury. We observed substantial enrichment of genes associated with the AA pathway, particularly the AA/5-LOX /LT axis; such genes were upregulated after TBI. Then, we performed whole-genome gene expression analysis from TBI and Zileuton treated mice at 7 days after TBI.


title: Utx (Kdm6a) promotes differentiation non-catalytically in somatic self-renewing epithelia [CUT&RUN]

status: Public on May 22 2025

city: Philadelphia

first: Brian

middle: C.

last: Capell

state: PA

address: 421 Curie Boulevard Biomedical Research Building 1020 Biomedical Research Building 1020

department: Dermatology

institute: University of Pennsylvania

summary: To investigate the role UTX (KDM6A) in stratifying epithelia

overall_design: Kdm6a (Utx) was deleted in the keratin-14 expressing tissues of mice via Krt14-Cre recombination

first: Gina

last: Pacella

first: Nina

last: Kuprasertkul

first: Lydia

last: Bao

first: Sijia

first: Carina

last: D'souza

first: Stephen

last: Prouty

first: Amy

last: Anderson

first: Alexandra

last: Maldonado-Lopez

first: Morgan

last: Sinkfield

first: Cyria

last: Olingou

first: John

middle: T

last: Seykora

middle: C


title: Barcoded monoclonal embryoids are a potential solution to confounding bottlenecks in mosaic organoid screens

city: Seattle

first: Chengxiang

last: Qiu

state: WA

address: William H. Foege Hall, 3720 15th Ave NE

department: Genome Sciences

institute: University of Washington

summary: Genetic screens in organoids hold tremendous promise for accelerating discoveries at the intersection of genomics and developmental biology. Embryoid bodies (EBs) are self-organizing multicellular structures that recapitulate aspects of early mammalian embryogenesis. We set out to perform a CRISPR screen perturbing all transcription factors (TFs) in murine EBs. Specifically, a library of TF-targeting guide RNAs (gRNAs) was used to generate mouse embryonic stem cells (mESCs) bearing single TF knockouts. Aggregates of these mESCs were induced to form mouse EBs, such that each resulting EB was “mosaic” with respect to the TF perturbations represented among its constituent cells. Upon performing single cell RNA-seq (scRNA-seq) on cells derived from mosaic EBs, we found many TF perturbations exhibiting large and seemingly significant effects on the likelihood that individual cells would adopt specific fates, suggesting roles for these TFs in lineage specification. However, to our surprise, these results were not reproducible across biological replicates. Upon further investigation, we discovered cellular bottlenecks during EB differentiation that dramatically reduce clonal complexity, curtailing statistical power and confounding interpretation of mosaic screens. Towards addressing this challenge, we developed a scalable protocol in which each individual EB is monoclonally derived from a single mESC. In a proof-of-concept experiment, we show how monoclonal, genetically barcoded EBs enable us to better quantify the consequences of TF perturbations as well as “inter-individual” heterogeneity across EBs harboring the same genetic perturbation. Looking forward, monoclonal EBs and EB-derived organoids may be powerful tools not only for genetic screens, but also for modeling Mendelian disorders, as their underlying genetic lesions are overwhelmingly constitutional (i.e. present in all somatic cells), yet give rise to phenotypes with incomplete penetrance and variable expressivity.

overall_design: To explore the function of individual TFs in cell fate determination, a monoclonal CRISPRcut or CRISPRi mouse ES line was transduced at low multiplicity of infection with a lentiviral CROP-seq library of gRNAs targeting TFs and containing GFP and puromycin selection markers. After optional puromycin selection to titrate the fraction of perturbed cells, ES cells were self-aggregated into mosaic embryoid bodies (EBs). After 21 days, GFP-positive cells were subjected to scRNA-seq and gRNA enrichment PCR. In a subsequent experiment, we aimed to overcome the mosaic screen bottleneck by generating a pool of monoclonal EBs through sorting and seeding piggyFlex mESCs at low density on mouse embryonic fibroblasts. Individual mESCs grew into colonies for 5 days followed by lifting with Collagenase IV treatment and gentle agitation. Colonies, now clonal aggregates, were then differentiated into EBs on low adherent plates for 8 days. We further perturbed TFs in these monoclonal EBs and profiled them using scRNA-seq.

first: Sam

last: Regalado

first: Silvia

last: Domcke

first: Jean-Benoît

last: Lalanne

first: Beth

last: Martin

first: Aidan

last: Keith

first: Maddy

last: Duran

first: Cole

last: Trapnell

first: Jay

last: Shendure


title: Nat10 regulates the homeostasis of pluripotency and 2C state in mESCs by N4-acetylcytidine mRNA modification

city: Tianjin

first: Guofeng

last: Feng

address: 94 Weijin Road

institute: Nankai University

summary: Mouse embryonic stem cells (mESCs) were isolated from the inner cell mass (ICM), which can prevent an excessive number of cells from transitioning to the 2-cell state and retain only 1%–5% of cells transiently expressing 2C genes such as Zscan4 and ERVs, and 95%–99% of normally expressing pluripotent transcription factors such as Oct4 and Nanog. The homeostasis state is always regulated by epigenetics especially heterochromatin modifications. However, it is still unclear what a specific factor can regulate this homeostasis state. Here we report that N-acetyltransferase 10 (Nat10), the writer for N4-acetylcytidine (ac4C) modification of mRNA, can serve as a new heterochromatin associated protein which can regulate the homeostasis of 2C genes and the expression of pluripotency genes in mESCs through a dual role. Mechanistically, Nat10 deficiency decreased ac4C modification on Oct4 and Esrrb mRNAs, resulting in decreased pluripotency. At the same time, ac4C modification on the heterochromatin component Kap1 mRNA is also reduced, which indirectly leads to a decrease in the binding of the H3K9me3 histone modification to the 2C gene, thereby removing the repression of the 2C genes and allowing the 2C genes to be expressed at high levels. These findings elucidate a novel role of Nat10 in mESCs and reveal new mechanisms linking the 2C program and pluripotency to ac4C modifications in mESCs.

overall_design: The RNA is first extracted from the Nat10 CON and Nat10 KD mESCs by TRIzol, the RNA is quality controlled, and the concentration and purity of the RNA is detected. Total RNA was subjected to immunoprecipitation with the GenSeq® ac4C RIP Kit (GenSeq Inc.) by following the manufacturer’s instructions. Briefly, RNA was randomly fragmented to about 200 nt by RNA Fragmentation Reagents. Protein A/G beads were coupled to the ac4C antibody by rotating at room temperature for 1 h. The RNA fragments were incubated with the bead-linked antibodies and rotated at 4 ℃ for 4 h. After incubation, the RNA/antibody complexes were washed for several times, and then, captured RNA was eluted from the complexes and purified. RNA libraries for IP and input samples were then constructed with GenSeq® Low Input Whole RNA Library Prep Kit (GenSeq, Inc.) by following the manufacturer’s instructions. Libraries were qualified using Agilent 2100 bioanalyzer (Agilent) and then sequenced in a NovaSeq platform (Illumina).


title: H2BK119R cause genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe

city: nanjing

first: Guangchun

last: Lu

address: wenyuan road

institute: Nanjing Normal University

summary: To investigate the different of gene expression between H2BK119R and wild strain, and then, we performed the gene expression analysis from the RNA-Seq data.

overall_design: Gene expression analysis of the H2BK119R strain was compared with the wild-type.

first: Gang

first: Liu

last: Li


title: ATAC-seq for “HDAC1 controls the generation and maintenance of effector-like CD8+ T cells during chronic viral infection”

city: Vienna

first: Wilfried

last: Ellmeier

address: Lazarettgasse 19

country: Austria

institute: Medical University of Vienna

summary: CD8+ T cell exhaustion is a complex process involving the differentiation of persistently activated CD8+ T cells into functionally distinct cell subsets. Here, we investigated the role of the key epigenetic regulator histone deacetylase 1 (HDAC1) in the differentiation of exhausted T (Tex) cells during chronic viral infection. We uncovered that HDAC1 controls the generation and maintenance of effector-like CX3CR1+ Tex cells in a CD8+ T cell-intrinsic manner. Deletion of HDAC1 led to expansion of an alternative Tex subset characterized by high expression of T cell exhaustion markers, and this was accompanied by elevated viremia. HDAC1 bound to and facilitated an open chromatin state of effector-like signature gene loci in progenitor Tex cells, thereby priming cell fate specification toward the CX3CR1+ Tex subset. Our study un-covers a selective role for HDAC1 in CX3CR1+ Tex subset differentiation, which is essential for controlling viral load during chronic infection.

overall_design: Naïve WT and HDAC1-cKO P14 T cells (CD90.2+) were intravenously transferred into CD90.1+ congenic mice, which were infected with the LCMV Cl13 strain one day after the transfer. Eight days later, splenocytes of 4-5 female mice, where either WT or HDAC1-cKO cells had been transfered, were isolated,  and early-Texprog (CD90.2+CD8a+Tim3-Ly108+) as well as non-early Texprog (CD90.2+CD8a+Tim3+Ly108-) cells were sorted into 1x PBS. After spinning cells down at 500g for 5min at 4°C, the cell pellets were resuspended in tagmentation-mix and incubated for 30min at 37°C with shaking (300rpm). The reaction was stopped by incubating the samples on ice. For DNA isolation, the MinElute PCR Purification Kit (Qiagen) was used according to the manufacturer’s instructions and eluted into 12 μl of elution buffer.

first: Ramona

last: Rica

first: Monika

last: Waldherr

first: Emi

last: Miyakoda

first: Ana

last: Kutschat

first: Marlene

last: Schülein

first: Jing

last: Zhang

first: Ricardo

last: Orbegozo-Medina

first: Lisa

last: Sandner

first: Valentina

last: Stolz

first: Darina

last: Waltenberger

first: Thomas

last: Krausgruber

first: Christoph

last: Bock

first: Nicole

last: Boucheron

first: Davide

last: Seruggia

first: Shinya

last: Sakaguchi


title: The nucleolar aberrancies that drive ribosome impairment induced by RNA binding proteins are hallmarks of aging

city: Madrid

first: Osvaldo

last: Graña

state: Comunidad de Madrid

address: C/ Melchor Fernández Almagro, 3

department: Structural Biology

country: Spain

institute: CNIO

summary: The nucleolus is a dynamic structure where ribosome subunits are produced. Indeed, nucleoli respond to any change in cellular homeostasis by altering the rate of ribosome biogenesis, thus working as a stress sensor. Consequently,  imbalances in ribosome biogenesis promotes changes in morphology and function and can evoke a nucleolar stress and DNA damage responses. These changes in nucleolar architecture and composition, culminating in impaired ribosome biogenesis, prompt the emergence of the nucleolar stress, which manifests as a contributing factor in aging and cancer. Here, we illuminate the pivotal role that the RNA binding protein Hnrnpk plays in orchestrating nucleolar dynamics and sustaining ribosome function. As a ribonucleoprotein, Hnrnpk governs the chaperoning of nascent transcripts to facilitate their processing and subsequent nuclear export for ribosomal integration. When overexpressed, Hnrnpk induces disruptive alterations in nucleolar structure, resulting in stress-like phenotypes; such as unbalances of molecular components, accumulation and delocalization of nucleolin, and decrease expression and occupation of fibrillarin. Consequentially, these aberrations in nucleolar equilibrium engenders a disruption in ribosome biogenesis and concomitantly halts the protein translation machinery.  Nucleolin (Ncl) haploinsufficiency is correlated with enlarged nucleoli, increased ribosome components, heightened translational rates translation and a concomitant decrease in lifespan. Thus, an Hnrnpk overexpression-dependent surge of Ncl levels stemming may instigate a decline in nucleolar integrity and perturbations in ribosome biogenesis—an event intimately associated with ribosomopathies and the ensuing syndrome of bone marrow failure syndrome.  Intersting, the aging process shares a number of common biological hallmarks with  bone marrow failure. Again,  alterations in Hnrnpk expression, specifically overexpression triggers nucleolar stress, driving cell cycle arrest and senescence of the cells. Our investigations demonstrate that p53, c-Myc and Ncl haploinsufficiency rescue these Hnrnpk-mediated phenotypes. Collectively, the data begin to decipher novel signaling pathway involved in   nucleolar stress - ribosome stress – and p53 driven cell cycle arrest that governs this process.  Together, these findings support the idea that nucleolar disturbances contribute to ribosome impairment, thus catalyzing the genesis of hematopoietic anomalies and aging process. Significantly, this study elucidates Hnrnpk as a previously unrecognized master regulator within these intricate ribosomal mechanisms;  providing a novel window to view the orchestration of nucleolar integrity, ribosome function, and cellular senescence.

overall_design: Hnrnpk overexpression (Synergistic activator mediator system) MEFs (3 x 2 replicates). Empty vector as controls (x3 replicates)

first: Miguel

last: Gallardo

last: Graña-Castro


title: Effect of iron on the infection in G. mellonella larvae

status: Public on May 21 2025

city: Yokohama

first: Imad

last: Abugessaisa

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

department: IMS

country: Japan

institute: RIKEN

summary:
Mycetoma is a neglected, chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains. However, knowledge of the function and formation of the grain is limited. To map the processes leading to M. mycetomatis grain formation, we used a Galleria mellonella larvae infection model and time-course transcriptomic profiling.
In the infected G. mellonella 88.0% of the RNA sequence reads mapped to G. mellonella, only 0.01% mapped to M. mycetomatis. Differential Gene Expression analysis revealed that 3.498 G. mellonella and 137 M. mycetomatis genes were differentially expressed during infection. Most of the enriched GO terms of both host and pathogen are linked to energy pathways, nucleobase metabolic process, and cation and iron transport. Genes related to iron transport were highly expressed by both G. mellonella (transferrin and ferritin) and M. mycetomatis (SidA, SidD and SidI). A protein-protein interaction network analysis of D. melanogaster homologues genes in M. mycetomatis revealed the expression of the entire siderophore biosynthesis pathway throughout infection.
Many host and pathogen genes were differentially expressed during infection. The identification of the importance of iron acquisition during grain formation can be exploited as a potential novel diagnostic and therapeutic strategy for mycetoma.

overall_design: Galleria mellonella larvae were injected with FeCl2 and FeCl3 to a final concentration of 25 µM to create an iron overload, and 332 µM ethylenediamine-N,N'-diacetic acid (EDDA) based on previous findings of Dunphy et al. in order to create an iron deficit [PMID:12770052]. Healthy larvae were first used to determine the toxicity of the different conditions to the larvae. Next, larvae were injected with either FeCl2, FeCl3 or EDDA to final concentrations as stated above. After 30 minutes, the larvae were infected with M. mycetomatis strain MM55 and survival was monitored for 10 days. Additionally, after 4, 24, and 72 hours larvae were sacrificed for RNA isolation and histological examination.

first: Mickey

last: Konings

first: Takeya

last: Kasukawa

first: Wendy

last: van de Sande


title: DOCK2 protects against bacterial sepsis by constraining T helper 1 response

city: Chongqing

first: Ye

last: shusen

address: No.1 Medical College Road, Yuzhong District, Chongqing

institute: Chongqing Medical University

summary: Sepsis is a systemic host response to infection with life-threatening consequence, which ranks among the top 10 causes of death worldwide. Nevertheless, our understanding of the molecular and cellular impact of sepsis remains rudimentary. Here, we identified dedicator of cytokinesis 2 (DOCK2) is a critical downregulating factor for lipopolysaccharide (LPS) signal pathways. DOCK2-deficient mice were highly sensitive to LPS-induced sepsis and Escherichia coli sepsis with increased levels of inflammatory cytokines, especially interferon-g (IFN-g), which were mainly due to hyperresponsive T helper 1 (Th1) cells. Ulteriorly, we verified the vital role of DOCK2-mediated Th1 cells in sepsis by neutralizing both IFN-g and CD4 and found both of which blockade reduced the severity of sepsis in Dock2-/- mice. Mechanically, DOCK2-mediated cell cycle progression and cytokine signaling act in concert to govern peripheral Th1 cell fate. Taken together, our data indicate that DOCK2 acts as a protective role in regulating systemic inflammation and multi-organ injury in bacterial sepsis by constraining Th1 response.

overall_design: A mouse sepsis model was established through LPS induction and Escherichia coli (E. coli) infection. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to detect T helper 1 (Th1) cell subsets and serum pro-inflammatory cytokines in septic mice. Additionally, in vivo neutralization experiments were conducted to block IFN-γ and CD4+ T cells, respectively, to explore the regulatory effect of DOCK2 on septic mice. Finally, the regulatory mechanism of DOCK2 was analyzed using an in vivo RNA-seq system.

first: Shusen

last: Ye

first: Linzi

last: Huang

first: Yuhao

last: Zheng

first: Shanshan

last: Liu

first: Xiangyang

last: Wang

first: Haoyuan

last: Yu

first: Lisi

last: Zhu


title: Timecourse RNA-seq analysis of Madurella mycetomatis grain development in Galleria mellonella

summary: Mycetoma is a neglected, chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains. However, knowledge of the function and formation of the grain is limited. To map the processes leading to M. mycetomatis grain formation, we used a Galleria mellonella larvae infection model and time-course transcriptomic profiling.  In the infected G. mellonella 88.0% of the RNA sequence reads mapped to G. mellonella, only 0.01% mapped to M. mycetomatis. Differential Gene Expression analysis revealed that 3.498 G. mellonella and 137 M. mycetomatis genes were differentially expressed during infection. Most of the enriched GO terms of both host and pathogen are linked to energy pathways, nucleobase metabolic process, and cation and iron transport. Genes related to iron transport were highly expressed by both G. mellonella (transferrin and ferritin) and M. mycetomatis (SidA, SidD and SidI). A protein-protein interaction network analysis of D. melanogaster homologues genes in M. mycetomatis revealed the expression of the entire siderophore biosynthesis pathway throughout infection.  Many host and pathogen genes were differentially expressed during infection. The identification of the importance of iron acquisition during grain formation can be exploited as a potential novel diagnostic and therapeutic strategy for mycetoma.

overall_design: Galleria mellonella larvae were infected with M. mycetomatis genome strain Madurella mycetomatis. At 4h, 24h, 72h and 168h post-inoculation, RNA was extracted from larval content. RNA-Seq  libraries were prepared for time-course transcriptomic profiling and analysis.

first: Ri-Ichiroh

last: Manabe

first: Tsugumi

last: Kawashima

first: Ahmed

middle: H

last: Fahal


title: RNA-seq profiles of Madurella mycetomatis mm55 cultured pathogen

summary: Mycetoma is a neglected, chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains.

overall_design: M. mycetomatis genome strain MM55 was cultured on Sabouraud Dextrose Agar for two to three weeks at 37⁰C. Mycelium was harvested, sonicated at 28 micron (Soniprep 150, Beun de Ronde, the Netherlands) and incubated at 37⁰C in RPMI-media supplemented with  0.35 g/L L-glutamine, 1.98 mM 4-morpholinepropanesulfonic acid (MOPS) and 100 mg/L chloramphenicol.  After two weeks, the mycelia were separated and washed by vacuum filtration (Nalgene, Abcoude, the Netherlands using a 22 micron filter (Whatman). The mycelial biomass was scraped from the filter, the wet weight was determined and a suspension was prepared containing 100 mg mycelial biomass per ml phosphate-buffered saline (PBS). The suspension was sonicated at 28 micron for 2 minutes (Soniprep 150, Beun de Ronde, the Netherlands), the resulting homogeneous suspension was washed once in PBS and again diluted to a concentration of 4 mg per 40 µL PBS, corresponding to 600-850 CFU/larvae.


title: Analysis of Madurella mycetomatis grain development in Galleria mellonella

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

sample_organism:
Galleria mellonella
Madurella mycetomatis

overall_design: Refer to individual Series


title: Nuclear functions of CFAP20 in transcription and replication [TTchem-DRB-seq]

status: Public on May 20 2025

city: Leiden

first: Martijn

middle: S.

last: Luijsterburg

state: zuid-holland

address: Einthovenweg 20

department: Human Genetics

institute: LUMC

summary: Fine-tuning DNA replication and transcription co-occurrence is vital to avoid collisions between their machineries. This is especially relevant near promoters, where RNAPII initiates transcription and often arrests, forming R-loops. Arrested RNAPII poses a roadblock for DNA replication, which evolutionarily initiates near promoters. The mechanisms that salvage arrested RNAPII during elongation to avoid conflicts with incoming replisomes remain unaddressed. In this study, employing genome-wide and proteomic approaches, we identify and characterize CFAP20 as part of a protective pathway that rescues arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops specifically near promoters, displaying defects in replication origin firing and fork progression. Co-depletion of the Mediator coactivator complex or removal of RNAPII engaged with R-loops rescues these replication phenotypes. Therefore, we propose that CFAP20 salvages arrested RNAPII under conditions of high Mediator-driven transcription to promote RNAPII elongation, thereby preventing collisions with co-directional replisomes.

overall_design:
To explore the role of CFAP20 in transcription elongation, we mapped nascent transcription after release from DRB treatement, using TTchem-DRB-seq in RPE1 cells being either wild type (WT) or CFAP20-KO.

NOTE: The records have been updated for data processing steps, contributor lists, replacement/addition of processed data files on May 30, 2025.

first: Sidrit

last: Uruci

first: Daphne

middle: E

last: Boer

first: Paul

middle: W

last: Chrystal

first: Maxime

last: Lalonde

first: Andreas

last: Panagopoulos

first: George

last: Yakoub

first: Idil

last: Kirdök

first: Klaas

last: de Lint

first: Melanie

last: van der Woude

first: Tiemen

last: Wendel

first: Sem

last: Brussee

first: Annelotte

middle: P

last: Wondergem

first: Nila

middle: K

last: van Overbeek

first: Jolanthe

last: Lingeman

first: Mats

last: Ljungman

first: Alexander

last: van Oudenaarden

first: Haico

last: van Attikum

first: Alfred

last: Vertegaal

first: Sylvie

last: Noordermeer

first: Rob

last: Wolthuis

first: Matthias

last: Altmeyer

first: Stephan

last: Hamperl

first: Vincent

last: Tropepe

first: Jeroen

last: van den Berg

first: Diana

last: van den Heuvel


title: PIEZO1 mediates mechanical reprogramming of neutrophils for proangiogenic specialization in the lung

status: Public on May 19 2025

first: Jing

last: Wang

address: No. 227 South Chongqing Road, Shanghai

summary: Neutrophils are the most abundant immune cells that constantly patrol or marginate inside vascular beds to support immune homeostasis. The extent to which neutrophils undergo reprogramming in response to the changes in vascular architecture and the resultant biological implications of such adaptations remain unclear. Here, we performed intravital imaging and transcriptional profiling to investigate neutrophil behavior across different tissues. Our findings revealed that neutrophils had significant deformability and spontaneous calcium signaling while navigating through the narrow pulmonary vessels. Pulmonary neutrophils exhibited unique transcriptional profiles and were specialized for proangiogenic functions. We found that the mechanosensitive ion channel PIEZO1 was essential for neutrophil reprogramming. Deletion of Piezo1 in neutrophils ablated the lung-specific proangiogenic transcriptional signature and impaired capillary angiogenesis in both physiological and pathological conditions. Collectively, these data show that mechanical adaptation of neutrophils within the pulmonary vasculature drives their reprogramming in the lungs and promotes pulmonary vascular homeostasis.

overall_design: RNA-seq profiling of lung, peripheral blood and bone marrow from wt mouse, RNA-seq profiling of lung and peripheral blood from wt and piezo1 cko mouse, RNA-seq profiling of lung from wt and PMN deletion mouse

first: Jin


title: Nuclear functions of CFAP20 in transcription and replication

first: Martijn

last: Luijsterburg


title: Sequential emergence and contraction of epithelial subtypes in the prenatal human choroid plexus revealed by a stem cell model

city: Irvine

first: Edwin

last: Monuki

state: CA

address: 845 Health Science Road

department: Experimental Pathology

institute: University of California Irvine

summary: Despite the major roles of choroid plexus epithelial cells (CPECs) in brain homeostasis and repair, their developmental lineage and diversity remain undefined. In simplified differentiations from human pluripotent stem cells, derived CPECs (dCPECs) display canonical properties and dynamic motile multiciliated phenotypes that interacted with Aβ uptake. Single dCPEC transcriptomes over time correlates well with human organoid and fetal CPECs, while pseudotemporal and cell cycle analyses highlight the direct CPEC origin from neuroepithelial cells. In addition, time series analyses define metabolic (type 1) and ciliogenic dCPECs (type 2) at early timepoints, followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contract. These temporal patterns were confirmed in independent derivations and mapped to prenatal stages using human tissues. In addition to defining the prenatal lineage of human CPECs, these findings suggest new dynamic models of ChP support for the developing human brain.

overall_design: Four time points and two replicates per time point were harvested. Following single cell dissociation, derived CPEC cultures were FACS sorted for live single cells and subjected to 10x Chromium capture for ~10,000 cell per sample and Illumina sequencing for ~50,000 reads/cell. Datasets were processed with SoptSC.

first: Haley

last: Masters

first: Shuxiong

first: Christina

last: Tu

first: Quy

last: Nguyen

first: Yutong

last: Sha

first: Matthew

last: Karikomi

first: Pamela

last: Fung

first: Benjamin

last: Tran

first: Cristina

last: Martel

first: Nellie

last: Kwang

first: Michael

last: Neel

first: Olga

middle: G

last: Jaime

first: Victoria

last: Espericueta

first: Brett

last: Johnson

first: Kai

last: Kessenbrock

first: Qing

last: Nie


title: H2BG52 D67 P102 onco-mutants cause genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe

first: Guangchun

last: Lu

summary: To investigate the different of gene expression between H2BG52 D67 P102 onco-mutants and wild strain, and then, we performed the gene expression analysis from the RNA-Seq data.

overall_design: Gene expression analysis of the H2BG52 D67 P102 onco-mutants strain was compared with the wild-type.

first: Gang

last: Feng


title: EOMES safeguards TSC chromatin accessibility by directing BRG1 to TSC-associated loci [RNA-seq]

city: Oxford

first: Alexandra Maria

last: Bisia

address: S Parks Rd

department: Sir William Dunn School of Pathology

country: United Kingdom

institute: University of Oxford

summary: EOMES is an essential transcription factor (TF) for murine trophoblast stem cell (TSC) maintenance. Despite that, the details of EOMES' function at the molecular level remain obscure. Here, we carried out rapid immunoprecipitation and mass spectrometry of endogenous protein (RIME) to identify TSC-specific protein interactors on EOMES. In addition to other established TFs involved in TSC maintenance, we found that EOMES interacts with several chromatin remodeller subunits, including BRG1. By exploiting an Eomes-degron system, we acutely depleted EOMES protein in TSCs and in parallel inhibited BRG1 function with small molecule BRM014. EOMES depletion and BRG1 inhibition resulted in reduced accessibility at largely overlapping genomic regions associated with TSC-related loci. Additionally, EOMES depletion results in misregulation of genes essential for TSC maintenance and function, as well as genes encoding cytoskeletal, cell-cell interaction and matricellular components.

overall_design: Investigation of gene regulation by acute EOMES depletion in Eomes-degron TSCs by administration of dTAG-13, by BRG1 inhibition by administration in BRM014, or by DMSO vehicle control, followed by RNA-seq.

first: Alexandra

first: Maria-Eleni

last: Xypolita

first: Elizabeth

last: Bikoff

last: Robertson

first: Ita

last: Costello


title: EOMES safeguards TSC chromatin accessibility by directing BRG1 to TSC-associated loci [CUT&RUN]

overall_design: Investigation of EOMES chromatin occupancy by Cleavage UnderTargets and Release Using Nuclease (CUT&RUN) with a rabbit polyclonal anti-Eomes antibody and an anti-rabbit IgG control antibody.


title: CUT&RUN for “HDAC1 controls the generation and maintenance of effector-like CD8+ T cells during chronic viral infection”

first: Wilfried

last: Ellmeier

overall_design: 10 WT and mice with T cell-specific HDAC1-deficiency (all age-matched) were infected with LCMV cl13 samples and their spleens were isolated on d8 post infection. T cells were enriched and cells were sorted for viability, TCRb, CD8, CD44, PD-1 and either Ly108 (early-TexProg) or Tim3 (early-non-TexProg). Nuclei from both early-TexProg and early-non-TexProg cells were isolated and CUT&RUN was performed for the following targets: HDAC1, H3K27ac, Runx3, H3K27me3, IgG.

first: Ramona

last: Rica

first: Monika

last: Waldherr

first: Emi

last: Miyakoda

first: Ana Patricia

last: Kutschat

first: Marlene

last: Schülein

first: Jing

last: Zhang

first: Ricardo Alfredo

last: Orbegozo-Medina

first: Lisa

last: Sandner

first: Valentina

last: Stolz

first: Darina

last: Waltenberger

first: Thomas

last: Krausgruber

first: Christoph

last: Bock

first: Nicole

last: Boucheron

first: Davide

last: Seruggia

first: Shinya

last: Sakaguchi


title: scRNA-seq for “HDAC1 controls the generation and maintenance of effector-like CD8+ T cells during chronic viral infection”

overall_design: Viable naïve (CD90.2+CD8a+CD44–) and LCMV-specific (CD90.2+CD8a+CD44+GP33-tet+) WT and HDAC1-cKO CD8+ T cells from murine splenocytes were isolated and analyzed using scRNA-seq. Samples were hashtagged for multiplexing and sequenced in two runs (two biological replicates).

first: Ana

first: Ricardo


title: EOMES safeguards TSC chromatin accessibility by directing BRG1 to TSC-associated loci [ATAC-seq]

first: Alexandra Maria

last: Bisia

overall_design: Investigation of chromatin accessibility regulation by acute EOMES depletion in Eomes-degron TSCs by administration of dTAG-13, by BRG1 inhibition by administration in BRM014, or by DMSO vehicle control, followed by ATAC-seq.

first: Alexandra

first: Ita

last: Costello

first: Elizabeth

last: Bikoff

last: Robertson

first: Maria-Eleni

last: Xypolita


title: HDAC1 controls the generation and maintenance of effector-like CD8+ T cells during chronic viral infection

first: Wilfried

last: Ellmeier

summary: CD8+ T cell exhaustion is a complex process involving the differentiation of persistently acti-vated CD8+ T cells into functionally distinct cell subsets. Here, we investigated the role of the key epigenetic regulator histone deacetylase 1 (HDAC1) in the differentiation of exhausted T (Tex) cells during chronic viral infection. We uncovered that HDAC1 controls the generation and maintenance of effector-like CX3CR1+ Tex cells in a CD8+ T cell-intrinsic manner. Dele-tion of HDAC1 led to expansion of an alternative Tex subset characterized by high expression of T cell exhaustion markers, and this was accompanied by elevated viremia. HDAC1 bound to and facilitated an open chromatin state of effector-like signature gene loci in progenitor Tex cells, thereby priming cell fate specification toward the CX3CR1+ Tex subset. Our study un-covers a selective role for HDAC1 in CX3CR1+ Tex subset differentiation, which is essential for controlling viral load during chronic infection.

overall_design: The individual series contain raw and processed data for scRNA-seq, ATAC-seq and CUT&RUN analysis of murine CD8+ Tcells 8 days p.i. with LCMV Cl13. Wildtype as well as HDAC1-cKO cells were analyzed, for ATAC-seq and CUT&RUN they were separated into progenitor (Tprog/early-Texprog) and non-progenitor (Tterm/early-non-Texprog) subsets. scRNA-seq data additionally includes naive CD8+ T cells.

first: Ramona

last: Rica

first: Monika

last: Waldherr

first: Emi

last: Miyakoda

last: Kutschat

first: Marlene

first: Jing


title: Pathogenic PPP2R5D variants alters the transcriptome of patient iPSC-derived glutamatergic neurons

city: Boston

first: Randee

last: Young

state: MA

address: 4 Blackfan st

department: Pediatrics

institute: Boston Children's Hospital

summary: The pathogenic PPP2R5D E198K and E420K variant alters the transcriptome of patient iPSC-derived cortical neurons

overall_design: Patient-iPSC derived iNGN2 glutamatergic neurons and isogenic controls were collected from three independent differentiations for each line (corE198K, E198K, corE420K, E420K) at DIV14, and total mRNA was extracted. cDNA libraries were made for each individual neuronal sample using Illumina TruSeq Stranded mRNA Library Prep Kit and 20 million pair-end reads were sequenced on an Illumina NovoSeq 6000.

first: Wendy

last: Chung


title: Utx (Kdm6a) promotes differentiation non-catalytically in somatic self-renewing epithelia [ChIP-seq]

first: Brian

last: Capell

summary: To investigate the role UTX (KDM6A) in stratifying epithelia

overall_design: Kdm6a (Utx) was deleted in the keratin-14 expressing tissues of mice via Krt14-Cre recombination

first: Gina

last: Pacella

first: Nina

last: Kuprasertkul

first: Lydia

last: Bao

first: Sijia

last: Huang

first: Carina

last: D'souza

first: Stephen

last: Prouty

first: Amy

last: Anderson

last: Maldonado-Lopez

first: Morgan

last: Sinkfield

first: Cyria

last: Olingou

first: John

last: Seykora


title: Utx (Kdm6a) promotes differentiation non-catalytically in somatic self-renewing epithelia [RNA-seq]

first: Alexandra


title: The response of Melanoma cell line A375 to cytotoxic T lyphocyte killing after serum starvation and MKL1 overexpression

city: Nanjing

first: Miaomiao

last: Hou

address: No. 12 Jiangwang Temple, Xuanwu District

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

summary: Changes in cellular state has dramatic impact on cancer cell responsiveness to cytotoxic T lyphocyte (CTL) killing. In this study we tested serum starvation and increasing cytoskeleton of cancer cells. Serum starvation significantly  upregulated the expression of genes involved in cholesterol biogenesis, which might be due a dramatic reduction in cellular cholesterol level. Indeed, cholestrol reduction sensitized the human melanoma cell line A375 for  CTL killing. However, increasing the cytoskeleton of cancer cells did not cause a change in cancer cell responsiveness to CTL killing, suggesting cholesterol-associated membrane composition other than cellular rigidity plays a more important role in determining cancer cell responsiveness to CTL killing.

overall_design: A375 cells were cultured under normal condition (10% FBS) and serum-starved condition (0.1%) for 48 hours and harvested for RNA-seq analysis of gene expression. A375 cells were overexpressed with control vector, MKL1, or constititively active MKL1 (caMKL1) for 48 hours and harvested for RNA-seq analysis of gene expression.

first: Dimin

last: Li

first: Xiao

last: Hu


title: Functional polymer-dependent 3D culture enhances the immunomodulating potential of human bone marrow mesenchymal stromal cells

city: Seoul

first: Jung-Woong

last: Kim

address: 84 Heuksuk-ro Dongjak-ku

department: Life Science

country: South Korea

institute: Chung-Ang University

summary:
Human mesenchymal stromal cells (MSCs) hold great regenerative medicine potential due to their pluripotency and paracrine functions. However, MSCs lose stemness and immunomodulatory capabilities in two-dimensional (2D) culture, which differs from the natural MSC niche. We investigated methods to enhance MSC characteristics by employing an in vivo-like culture using functional polymers. Implementing FP003 polymer-based 3D culture, we observed increased expression of stemness markers (Oct4, Nanog), in hMSCs compared to 2D culture on regular plastic. This 3D environment also enhanced expression COX2 and HO1, known for their immunomodulatory functions. MSCs cultured in 3D exhibited higher secretion of PGE2 and effectively suppressed TNF- release from LPS-stimulated splenocytes, surpassing 2D-cultured MSCs.
To explore therapeutic potential in vivo, we administered 3D-cultured MSCs via intravenous injection in a mouse model of neuroinflammation. Remarkably, this approach significantly reduced the expression of Iba1 and GFAP compared to 2D-cultured MSC injection. RNA-seq analysis revealed upregulated adhesion-related genes, including ITGA2, in MSCs cultured in 3D. Ectopic ITGA2 expression notably enhanced immunomodulatory function. In summary, our findings demonstrate that an in vivo mimetic 3D culture provides a beneficial microenvironment for MSCs, enhancing their immunomodulatory function through ITGA2 expression. Engineered MSCs can thus be utilized as an effective cell therapy source.

overall_design: In order to investigate the characteristics of MSCs cultured in a three-dimensional (3D) setting, a comparison was made between the distribution of 3D-cultured MSCs in a medium containing the polymer (FP003) and 2D-cultured MSCs in a medium without the polymer.


title: CAGE profiles of Madurella mycetomatis mm55 cultured pathogen

first: Imad

last: Abugessaisa

first: Mickey

last: Konings

first: Ri-Ichiroh

last: Manabe

first: Tsugumi

last: Kawashima

first: Takeya

last: Kasukawa

first: Ahmed

last: Fahal

first: Wendy

last: van de Sande


title: Rewired m6A methylation of promoter antisense RNAs in Alzheimer’s disease regulates neuronal gene expression in the 3D nucleome

city: Houston

first: Benxia

state: Texas

address: 431 Fannin

institute: University of Texas Health Science Center at Houston

type: Methylation profiling by high throughput sequencing

summary: N6-methyladenosine (m6A) is an abundant internal RNA modification that can impact gene expression at both post-transcriptional and transcriptional levels. However, the landscapes and functions of m6A in human brains and neurodegenerative diseases, including Alzheimer's disease (AD), are under-explored. Here, we examined RNA m6A methylome using total RNA-seq and meRIP-seq in middle frontal cortex of post-mortem brains from individuals with or without AD, which revealed m6A alteration on both mRNAs and various noncoding RNAs. Notably, many promoter-antisense RNAs (paRNAs) displayed cell-type-specific expression and changes in AD, including one produced adjacent to MAPT that encodes the Tau protein. MAPT-paRNA is highly expressed in neurons, and m6A positively controls its expression. In iPSC-derived human excitatory neurons, MAPT-paRNA does not impact the nearby MAPT mRNA, but instead promotes expression of hundreds of neuronal and synaptic genes, and is protective against excitotoxicity. Analysis of single nuclei RNA-DNA interactome in human brains supports that brain paRNAs interact with both cis- and trans-chromosomal target genes to impact their transcription. These data reveal landscapes and functions of noncoding RNAs and m6A in brain gene regulation and AD pathogenesis.

overall_design: we first characterized RNA m6A methylome from individuals diagnosed with AD and from cognitively normal age-matched counterparts individuals. We uncovered a large number of m6A-modified RNAs in the human brain, including both mRNAs and multiple categories of ncRNAs, many of which are previously unannotated. RNA m6A methylome in AD displays alteration and correlation with expression levels of mRNA and ncRNAs

first: Wenbo


title: Nuclear functions of CFAP20 in transcription and replication [ChIP-seq]

first: Martijn

last: Luijsterburg

overall_design:
To explore spatial connections between transcription and replication at the genome-wide level, as well as the role of CFAP20 in these processes, we mapped RNA polymerase II (RNAPII) and CFAP20  occupancy using chromatin immunoprecipitation (ChIP)-seq, nascent transcription by bromouridine (BrU)-seq and TTchem-seq, and R-loops by DNA-RNA hybrid immunoprecipitation (DRIP)-seq in RPE1 cells being either wild type (WT), CFAP20-KO, CFAP20-KO rescued with CFAP20-GFP, or CFAP20/CCNC-dKO.
NOTE: The records have been updated for data processing steps, contributor lists, replacement/addition of processed data files, additional sample records on May 30, 2025.

first: Sidrit

last: Uruci

first: Daphne

last: Boer

first: Paul

last: Chrystal

first: Maxime

last: Lalonde

first: Andreas

last: Panagopoulos

first: George

last: Yakoub

first: Idil

last: Kirdök

first: Klaas

last: de Lint

first: Melanie

last: van der Woude

first: Tiemen

last: Wendel

first: Sem

last: Brussee

first: Annelotte

last: Wondergem

first: Nila

last: van Overbeek

first: Jolanthe

last: Lingeman

first: Mats

last: Ljungman

first: Alexander

last: van Oudenaarden

first: Haico

last: van Attikum

first: Alfred

last: Vertegaal

first: Sylvie

last: Noordermeer

first: Rob

last: Wolthuis

first: Matthias

last: Altmeyer

first: Stephan

last: Hamperl

first: Vincent

last: Tropepe

first: Jeroen

last: van den Berg

first: Diana

last: van den Heuvel

middle: S


title: Laboratory mice engrafted with natural gut microbiota possess a wildling-like phenotype

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

country: Germany

institute: University clinics Freiburg

summary: Conventional laboratory mice housed under specific pathogen-free (SPF) conditions are the standard model in biomedical research. However, in the past years, many published results involving rodents have been considered irreproducible, raising concerns about the suitability of mice as model organisms. Emerging evidence indicates that variations in SPF microbiota contribute significantly to data variability across different laboratories. Despite efforts to standardize the microbiota, current microbial consortia lack the complexity and resilience needed to replicate interactions in free-living mammals.  We present a standardizable and feasible approach for transplanting natural gut microbiota from wildlings into conventional laboratory mice. After engraftment, these TXwildlings adopt a structural and functional wildling-like microbiota and host physiology toward a more mature immune system, with characteristics similar to those of adult humans. We anticipate that using wild mouse-derived microbiota as standard for laboratory mouse models will improve the reproducibility and generalizability of basic and preclinical biomedical research.

overall_design: To find out how the host phenotype of TXwildlings and TXlab mice changed after microbiome transplantation, we analyzed colon, liver, lung, and lymphnode tissue of these animals. Data are from 2 independent experiments with five mice per group.

first: Solveig

last: Runge

first: Benedikt

last: Hild

last: Rosshart

first: Melnaie

last: Boerries


title: Integrating Reproductive States and Social Cues in the control of Sociosexual Behaviors

city: Beijing

first: Kun

last: Li

address: HaiDian

institute: Tsinghua University

summary: Female sociosexual behaviors, essential for survival and reproduction, are adaptively modulated by ovarian hormones and triggered in the context of appropriate external social cues. Here we identify primary estrous-sensitive Cacna1h-expressing medial prefrontal (mPFCCacna1h+) neurons that integrate hormonal states with recognition of potential mates to orchestrate these complex cognitive behaviors. Bidirectional manipulation of mPFCCacna1h+ neurons drives opposite-sex-directed behavioral shifts between estrus and diestrus females via anterior hypothalamic outputs. In males, these neurons serve opposite functions compared to estrus females. Miniscope imaging reveals mixed-representation of self-estrous states and social target sex in distinct mPFCCacna1h+ subpopulations, with biased-encoding of opposite-sex social cues in estrus females and males. Mechanistically, ovarian hormone-driven upregulation of Cacna1h-encoded T-type calcium channels underlies estrus-specific activity changes and sexual-dimorphic function of mPFCCacna1h+ neurons. These findings uncover a prefrontal circuit that integrates internal hormonal states and target-sex information to exert sexually bivalent top-down control over adaptive social behaviors.

overall_design:
For pathway-specific TRAP-seq, retrograde viruses rAAV2-EGFP-Rpl10a were injected into the AHNc of wild type mice. The mPFC regions from male and female mice with AHNc virus injection were used for independent TRAP replicates. For cell type-specific TRAPseq, three male and eight female Cacna1h-Cre::EGFP-Rpl10a mice of four distinct estrous cycles were used for independent TRAP replicates.

NOTE: Additional sample records (GSM9014308-GSM9014343) have been included; both raw and processed data have been replaced for GSM8661153, GSM8661154 records on May 29, 2025.

first: Xinli

last: Song


title: Type 2 cytokines pleiotropically modulate sensory nerve architecture and neuroimmune interactions to mediate itch [Mouse ear pinnae]

status: Public on May 15 2025

city: Cambridge

first: Yingnan

last: Han

address: 350 Water Street

institute: Sanofi

summary:
Background: Anti-type 2 cytokine therapies represent promising interventions for chronic itch; however, their precise mechanisms in restoring nerve architecture and mitigating inflammation and pruritus remain incompletely understood. This study aimed to elucidate the mechanistic roles of IL-4, IL-13, and IL-31 in the pathophysiology of itch associated with type 2 inflammatory skin diseases.

Methods: The effect of IL-4, IL-13, and/or IL-31 on neurite outgrowth and/or transcriptomic changes were analyzed in human and mouse dorsal root ganglion (DRG) neuronal cultures. Mouse ear pinnae were processed for histologic, transcriptomic, and proteomic analyses 4 days after intradermal injection of IL-4, IL-13, and/or IL-31. To evaluate functional correlations with neuronal responses, mice were subcutaneously challenged with IL-4, IL-13, and/or IL-31, and scratching behavior was monitored. Association between IL-4/IL-13–IL-4Rα axis and severity of atopic dermatitis (AD) was evaluated through correlative analyses of human DRG transcriptomic changes and AD transcriptomic datasets (GSE130588 and BioMap consortium).

Results: IL-4 and IL-13 promote mouse and human DRG sensory neuron growth, with effects similar to or greater than IL-31. In mice, intradermal IL-4, IL-13, and IL-31 increased epidermal nerve growth; however, only IL-4 and IL-13 induced hyperplasia and immune cell recruitment. Multi-omic analyses revealed that IL-4 and IL-13 have a broader impact on neuroimmune interactions than IL-31. In a murine DRG neuron-eosinophil co-culture, IL-4Rα blockade reduced neurite growth. IL-13 and IL-31 elicited acute scratching, demonstrating their roles as direct pruritogens; IL-4 synergistically enhanced IL-13-induced itch, resulting in greater pruritic responses than IL-31. Additionally, a set of itch-associated genes upregulated by IL-4 and IL-13 and downregulated by dupilumab-mediated IL-4Rα blockade in human DRG neuronal cultures showed positive correlation with AD severity.

Conclusions: These findings establish the IL-4/IL-13–IL-4Rα axis as a key regulator of inflammatory skin nerve innervation, neuroimmune interactions, barrier integrity, and itch response, highlighting its mechanistic role in modulating sensory neuronal function and shaping the inflammatory microenvironment that drives itch pathophysiology.

overall_design: RNA-seq profiling of mouse ear pinnae with intradermal injection of IL-4, IL-13, IL-31, or PBS (n=5 per group)

first: Mithilesh

last: Jha

first: Zhipeng

last: Liu

first: Yannis

last: Hara

first: Ingeborg

last: Langohr

first: Caroline

last: Morel

first: Colleen

last: Maloney

first: Peter

last: Piepenhagen

first: Heming

last: Xing

first: Corneliu

last: Bodea

first: Dinesh

last: Bangari

first: Hamid

last: Mattoo

first: Alexandra

last: Hicks


title: Type 2 cytokines pleiotropically modulate sensory nerve architecture and neuroimmune interactions to mediate itch [Human DRG]

overall_design: RNA-seq profiling of Human DRG neuronal culture treated with PBS, IL-4+IL-13+IgG4k-Isotype, or IL-4+IL-13+dupilumab (n=3 per group)


title: Coordinated Histone Methylation Loss and MYC Activation Promote Translational Capacity Under Amino Acid Restriction

city: Los Angeles

first: Siavash

last: Kurdistani

address: 615 Charles E Young Dr South

department: Biological Chemistry

institute: UCLA

summary: Epigenetic mechanisms underlying cellular adaptation to and recovery from nutrient deficiency are incompletely understood. Here, we report an adaptive epigenetic response to amino acid (AA) restriction that primes cells for rapid recovery. Depriving human cells of AAs led to selective loss of histone H4 lysine 20 monomethylation (H4K20me1) from gene bodies and increased MYC binding at promoters genome-wide, with strongest effects at ribosomal protein and translation initiation genes. Although AA restriction reduced overall protein synthesis, it was accompanied by increased translational capacity through upregulation of protein synthesis genes and accumulation of monomeric ribosomes. Combined SETD8, the H4K20 methyltransferase, depletion and MYC over-expression in nutrient-rich media was necessary and sufficient to recapitulate the transcriptional and functional effects of AA restriction. These findings suggest that cells respond to AA deprivation through coordinated chromatin and transcriptional changes that anticipate nutrient restoration and promote rapid recovery of protein synthesis when AAs are replenished.

overall_design: mRNA profiles of HeLa cells cultured in complete DMEM medium or in DMEM lacking amino acids.

first: Chen

last: Cheng


title: Regulation of Collecting Lymphatic Vessel Contractile Function by TRPV4 Channels

status: Public on May 16 2025

city: New Orleans

first: Jorge

last: Castorena-Gonzalez

state: LA

address: 1430 Tulane Avenue, MC-8683

department: Pharmacology

institute: Tulane University

summary: Background: Dysregulation of TRPV4-mediated signaling has been associated with inflammation and tissue fibrosis, both of which are key features in the pathophysiology of lymphatic system diseases; however, the expression and functional roles of lymphatic TRPV4 channels remain largely unexplored.  Methods: We generated a single-cell RNA sequencing dataset from microdissected mouse collecting lymphatic vessels to characterize the expression of Trpv4. Using a novel Trpv4fx/fx mouse strain and the Cre-lines Prox1-CreERT2 and LysM-Cre we assessed the role of TRPV4 channels in lymphatic endothelial cells (LECs) and peri-lymphatic myeloid cells, respectively. Confocal microscopy and extensive functional experimentation on isolated and pressurized lymphatics, including measurements of intracellular calcium activity, were employed to validate our scRNAseq findings and to elucidate the underlying mechanisms. Clinical significance was assessed using biopsies from breast cancer-related lymphedema (BCRL) patients.  Results: We characterized the single-cell transcriptome of collecting lymphatic vessels and surrounding tissues. Trpv4 was highly enriched in LECs and in a subset of Lyve1+ macrophages displaying a tissue-resident profile. In clinical samples, BCRL was associated with increased infiltration of macrophages co-expressing LYVE1 and TRPV4. Pharmacological activation of TRPV4 channels led to contractile dysregulation in isolated collecting lymphatics. The response was multi-phasic, including initial vasospasm and subsequent vasodilation and inhibition of contractions, which was associated with the activation of thromboxane A2 receptors (TXA2Rs) in lymphatic muscle cells (LMCs) by secreted prostanoids from TRPV4+ myeloid cells, and increased nitric oxide (and perhaps other vasodilatory prostanoids) from LECs. The TXA2R-mediated vasospasm resulted from increased mobilization of calcium from intracellular stores through IP3 receptors and store-operated calcium entry.  Conclusions: Our results uncovered a novel mechanism of lymphatic contractile dysregulation mediated by the crosstalk between TRPV4-expressing myeloid cells, including LYVE1+ macrophages, and LMCs or LECs. These findings highlight potentially important roles of TRPV4 channels in lymphatic dysfunction associated with inflammation, including secondary lymphedema.

overall_design: Collecting lymphatic vessels and surrounding tissues were microdissected from male and female C57BL/6J (N=4 for each sex). These mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 10-12 weeks of age and utilized for experiments after a 2-week acclimation period. For each sample, single-cell suspensions were prepared from lymphatic tissues pooled from 4 different anatomical regions, i.e., popliteal, inguinal axillary, superficial cervical, and mesentery.

first: Mary

last: Schulz

first: Victoria

last: Akerstrom

first: Kejing

first: Sarah

last: Broyhill

first: Min

last: Li

first: Michelle

last: Lambert

first: Tatia

last: Goldberg

first: Raghu

last: Kataru

first: Jinyeon

last: Shin

first: Stephen

last: Braun

first: Charles

last: Norton

first: Rafael

last: Czepielewski

first: Babak

last: Mehrara

first: Timothy

last: Domeier

first: Scott

last: Zawieja


title: Rifampin mediated decapsulation and enhanced bacterial clearance: a new mechanism of rifamycins

city: Ithaca

first: Jeongmin

last: Song

state: NY

address: 930 Campus Rd

department: Microbiology and Immunology

institute: Cornell University

summary: Many clinically relevant bacterial pathogens are encapsulated, as exemplified by Salmonella enterica serovar Typhi. S. Typhi, the causative agent of the life-threatening systemic disease enteric fever, expresses Vi as the outermost surface glycan that protects the bacteria from host immune responses. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) S. Typhi strains, as well as Vi variants, are widespread globally. Our WGS analyses indicate that almost all S. Typhi clinical isolates are susceptible to rifamycins and azithromycin. Rifampin, even at sub-MIC levels, eliminates the protective capsule Vi, a process referred to as ‘decapsulation’, thereby enhancing bacterial clearance. Antibiotic-mediated decapsulation of S. Typhi appears specific to rifamycins, since azithromycin does not decapsulate S. Typhi. Rifampin mediated decapsulation occurs at the transcriptional level, where both high AT content and specific RpoB residues play a crucial role. Rifampin also effectively decapsulates Vi variants, which accounts for 1 in 5 S. Typhi isolates at the global level. A mechanistic explanation for rifampin mediated decapsulation of S. Typhi appears to be applicable to other encapsulated pathogens, including S. Paratyphi C.

sample_organism: Salmonella enterica subsp. enterica serovar Typhi

overall_design: This includes six RNA-seq samples: three from untreated S. Typhi and three from S. Typhi treated with 2 µg/ml rifampin.

first: Gi Young

last: Lee


title: Organism-wide cellular dynamics and epigenomic remodeling in mammalian aging

city: New York

first: Ziyu

last: Lu

address: 1230 York Avenue

institute: The Rockefeller University

summary: Aging leads to functional decline across tissues, often accompanied by profound changes in cellular composition and cell-intrinsic molecular states. However, a comprehensive catalog of how the population of individual cell types change with age and the associated epigenomic dynamics is lacking. Here, we constructed a single-cell chromatin accessibility atlas consisting of ~7 million cells from 21 tissue types spanning three age groups in both sexes. This dataset revealed 536 main cell types and 1,828 finer‐grained subtypes, defined by unique chromatin accessibility landscapes at ~1.3 million cis‐regulatory elements. We observed widespread remodeling of immune lineages, with increases in plasma cells and macrophages, and depletion of T and B cell progenitors. Additionally, non-immune cell populations, including kidney podocytes, ovary granulosa cells, muscle tenocytes and lung aerocytes, showed marked reductions with age. Meanwhile, many subtypes changed synchronously across multiple organs, underscoring the potential influence of systemic inflammatory signals or hormonal cues. At the molecular level, aging was marked by thousands of differentially accessible regions, with the most concordant changes shared across cell types linked to genes related to inflammation or development. Putative upstream factors, such as intrinsic shifts in transcription factor usages and extrinsic cytokine signatures, were identified. Notably, around 40% of aging‐associated main cell types and subtypes showed sex‐dependent differences, with tens of thousands of chromatin accessibility peaks altered exclusively in one sex. Together, these findings present a comprehensive framework of how aging reshapes the chromatin landscape and cellular composition across diverse tissues, offering a comprehensive resource for understanding the molecular and cellular programs underlying aging and supporting the exploration of targeted therapeutic strategies to address age-related dysfunction.

overall_design: C57BL/6 WT mouse samples from 21 tissue types (brown adipose tissue, bone marrow, bone marrow, cecum, colon, esophagus, eye, heart, intestine, kidney, liver, lung, muscle (quadriceps and gastrocnemius), ovary/uterus, pancreas, back skin, spleen, stomach, thymus, gonodal white adipose tissue, inguinal white adipose tissue, mesenteric white adipos tissue) were collected at three age groups from both sexes (1-month-old, n = 10, 5 male and 5 female; 5-month-old, n = 10, 5 male and 5 female;  21-month-old, n = 12, 6 male and 6 female) for single-cell chromatin accessibility profiling using EasySci-ATAC-seq protocol (PMID: 38036784).

first: Wei

last: Zhou

first: Junyue

last: Cao


title: Megacomplex protects ESR1 from degradation by Fulvestrant in epithelial ovarian cancer

city: Bethesda

first: Laura

last: Elnitski

address: 49 Convent Dr

institute: NIH

summary: Ovarian cancer, a significant contributor to cancer-related mortality, often exhibits limited responsiveness to hormonal therapies targeting the estrogen receptor (ESR1). This study aimed to elucidate the mechanisms behind ESR1 resistance to the therapeutic drug Fulvestrant (ICI182780 or ICI). We identified a 1.3MDa Megacomplex comprising ESR1, FOXA1, and PITX1 using size exclusion chromatography. These factors showed colocalized binding at 6,775 genomic positions in the ovarian cancer cell line, PEO4, including important super-enhancers. Notably, compared to the cytoplasmic version, nuclear ESR1 was minimally degraded by ICI, suggesting a mechanism for drug resistance via the protective confines of the Megacomplex. Combining a small molecule inhibitor of active chromatin and super-enhancers, JQ1, with ICI significantly enhanced ESR1 degradation, especially within the Megacomplex. Molecular analysis showed that this combination disrupted the cyto-nuclear ESR1 shuttling responsible for tumor cell proliferation. Pathway enrichment analysis of RNAseq gene sets following Estradiol, ICI, or ICI plus JQ1 treatments paralleled pathway enrichment found from target genes of Megacomplex binding identified through ChIPseq. Mass-spec analysis revealed that proteins within the Megacomplex macromolecule also produced similar pathway enrichments after modulation by Estradiol or ICI. Each of these findings independently implicates Megacomplex in ESR1-driven ovarian cancer chromatin regulation. Moreover, this combined treatment strategy exhibited superior inhibition of cell proliferation and viability compared to ICI alone. Other cell lines, PEO1, OVCAR4, OVCAR8, and the normal ovarian cell line, T1074, substantiated the specificity of the response. By uncovering ESR1's resistance within the Megacomplex, the combined ICI plus JQ1 treatment elucidates a novel drug treatment vulnerability.

overall_design: Chromatin immunoprecipitation DNA-sequence (ChIP-seq) for transcription factors ESR1, PITX1, FOXA1; transcriptional coactivators Med1, BRD4 as well as histone modifications H3K27ac and H3K27me3 in PEO4 cells.

first: Sushil

last: Jaiswal

first: Kevin

last: Fedkenheuer

first: Ronak

last: Khamar

first: Hua

last: Tan

first: Valer

last: Gotea

first: Abdel

last: Elkahloun

first: Ming

last: Zhao

first: Lisa

last: Jenkins

first: Christina

last: Annunziata


title: Timecourse CAGE analysis of Madurella mycetomatis grain development in Galleria mellonella

city: Yokohama

first: Imad

last: Abugessaisa

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

institute: RIKEN

summary: Mycetoma is a neglected, chronic granulomatous infection of the subcutaneous tissue, most often caused by the fungal pathogen Madurella mycetomatis. Characteristic of the infection is the formation of grains. However, knowledge of the function and formation of the grain is limited. To map the processes leading to M. mycetomatis grain formation, we used a Galleria mellonella larvae infection model and time-course transcriptomic profiling.  In the infected G. mellonella 88.0% of the RNA sequence reads mapped to G. mellonella, only 0.01% mapped to M. mycetomatis. Differential Gene Expression analysis revealed that 3.498 G. mellonella and 137 M. mycetomatis genes were differentially expressed during infection. Most of the enriched GO terms of both host and pathogen are linked to energy pathways, nucleobase metabolic process, and cation and iron transport. Genes related to iron transport were highly expressed by both G. mellonella (transferrin and ferritin) and M. mycetomatis (SidA, SidD and SidI). A protein-protein interaction network analysis of D. melanogaster homologues genes in M. mycetomatis revealed the expression of the entire siderophore biosynthesis pathway throughout infection.  Many host and pathogen genes were differentially expressed during infection. The identification of the importance of iron acquisition during grain formation can be exploited as a potential novel diagnostic and therapeutic strategy for mycetoma.

overall_design: Galleria mellonella larvae were infected with M. mycetomatis genome strain Madurella mycetomatis. At 4h, 24h, 72h and 168h post-inoculation, RNA was extracted from larval content. LQ_ssCAGE  libraries were prepared for time-course transcriptomic profiling and analysis.

first: Mickey

last: Konings

first: Ri-Ichiroh

last: Manabe

first: Tsugumi

last: Kawashima

first: Takeya

last: Kasukawa

first: Ahmed

last: Fahal

first: Wendy

last: van de Sande


title: Regulation of MORC-1 is key to the CSR-1-mediated germline gene licensing mechanism in C. elegans

first: Colette

last: Picard

state: California

address: 610 Charles E Young Dr East, Room 4045

department: Molecular, Cell and Developmental Biology

institute: University of California - Los Angeles

summary: The Argonaute CSR-1 is essential for germline development in C. elegans. Loss of CSR-1 leads to the downregulation of thousands of germline-expressed genes, supporting a model in which CSR-1 “licenses” gene expression via a poorly understood mechanism. In contrast, a small subset of genes is upregulated in csr-1 mutants, including morc-1, which encodes a conserved GHKL-type ATPase. We show that morc-1 is overexpressed in csr-1 mutants and accumulates over CSR-1 licensed targets, coinciding with aberrant gain of H3K9me3, reduced H3K36me3, and transcriptional repression. Strikingly, loss of morc-1 fully rescues these chromatin defects and partially restores gene expression and fertility in csr-1 mutants. Conversely, ectopic overexpression of MORC-1 in the wild-type germline is sufficient to repress CSR-1 licensed targets and severely compromise fertility. These findings support a model in which CSR-1 prevents MORC-1 overexpression and consequent misregulation of CSR-1 licensed genes.

overall_design: Contains two sets of ChIP-seq experiments. (1) The H3K9me3 and H3K36me3 ChIPs were done identically to the ones published in Weiser et al. 2017, except adding csr-1 RNAi. These were used to check for rescue of csr-1 chromatin defects by morc-1. For these experiments, two replicates were performed for each genotype/condition/antibody, as well as two input samples per genotype/condition. (2) The MORC-1::FLAG ChIPs used a FLAG tagged into the endogenous morc-1 locus, in order to investigate the genome-wide distribution of wild-type MORC-1, as well as the distribution of MORC-1 in two different csr-1 mutants csr-1(G560R) and aid::csr-1. For the FLAG experiments, control experiments were performed using wild-type worms lacking FLAG (N2). All MORC-1::FLAG ChIPs were performed with two replicates, each with a matched input sample.

first: Jessica

last: Kirshner

first: Natasha

last: Weiser

first: Nicita

last: Mehta

first: Suhua

last: Feng

first: Victoria

last: Murphy

first: Anna

last: Vakhnovetsky

first: Connie

last: Xiao

first: Kai

last: Inoki

first: Sonia

last: El Mouridi

first: Christian

last: Frøkjær-Jensen

first: Steven

last: Jacobsen

first: John

last: Kim


title: A multisensor high-temperature signaling framework for triggering daytime thermomorphogenesis in Arabidopsis

status: Public on May 17 2025

city: Riverside

first: Meng

last: Chen

address: 2150 Batchelor Hall, 900 University Ave.

institute: University of California, Riverside

summary: The mRNA expression profiles of the albino mutant hmr-5 and hmr5pifq in Arabidopsis thaliana were analyzed under red light conditions at 50 μmol m^−2 s^−1 and warm ambient temperatures of 21°C and 27°C, with and without sucrose treatment.

overall_design: Two-day-old seedlings of the albino mutant hmr-5 and hmr-5pifq (grown at 21°C under red light at 50 μmol m^−2 s^−1) were subjected to either 21°C or 27°C for an additional two days, with or without sucrose treatment. Three replicates were performed for each condition to ensure the reliability of the results. Gene expression profiles were then analyzed to determine the impact of temperature and sucrose on the physiological responses of the hmr-5 mutant under red light conditions.

first: De

last: Fan


title: The energy-saving metabolic switch underlies survival of extremophilic red microalgae in extremely high nickel levels

status: Public on May 14 2025

city: Warszawa

first: Krzysztof

last: Goryca

address: Banacha 2c

department: CeNT

country: Poland

institute: Uniwersytet Warszawski

summary: The red microalga Cyanidioschyzon merolae inhabits extreme environments of high temperature, high acidity, and the presence of high concentrations of heavy metals and sulphites that are lethal to most other forms of life. However, information is scarce on the precise adaptation mechanisms of this extremophile to such hostile conditions. Gaining such information is important for understanding the evolution of microorganisms in the early stages of life on Earth because these extreme environments are similar. By analyzing the remodeling of the global transcriptome upon long-term exposure of C. merolae to extremely high concentrations of nickel, the key adaptive metabolic pathways and associated molecular components were identified. Our work shows that long-term Ni exposure of C. merolae cells leads to the lagged metabolic switch demonstrated by transcriptional upregulation of the metabolic pathways critical for cell survival such as DNA replication, cell cycle, and protein quality control with the concomitant downregulation of energetically costly processes including assembly of the photosynthetic apparatus and lipid biosynthesis. This study paves the way for the multi-omic studies of the molecular mechanisms of abiotic stress adaptation in phototrophs, as well as the development of the rational approaches for bioremediation of contaminated aquatic environments by modifying expression of the critical molecular components identified here.

overall_design: C. merolae was exposed to 0, 1 and 3mM and Nickel and cultivated for 5 and 10 days.

first: Sergio

last: Santaeufemia

first: Francesca

last: Marchetto

first: Patrizia

last: Romano

first: Dorota

last: Adamska

first: Jeffrey

last: Palatini

first: Ilaria

last: Corsi

first: Joanna

last: Kargul


first: Colette

last: Picard

overall_design: ATAC-seq to assess chromatin accessibility genome-wide in wild-type C. elegans. Only one replicate was performed.

first: Jessica

last: Kirshner

first: Natasha

last: Weiser

first: Nicita

last: Mehta

first: Suhua

last: Feng

first: Victoria

last: Murphy

first: Anna

last: Vakhnovetsky


overall_design: RNA-seq data for two sets of experiments: (1) comparing gene expression in our two csr-1 mutants and in csr-1(G560R); morc-1 vs. matched controls to see csr-1 expression defects & their rescue by morc-1, and (2) comparing expression in various samples/treatments of our morcOE transgenic line (prefix "rna_morcOE") to see if germline MORC-1 overexpression causes expression defects. Experiment (1) samples were done in 4 replicates, and for csr-1(G560R) samples the control is wild-type (N2), for aid::csr-1 treated w/ 1mM auxin, the control is aid::csr-1 treated with 0mM auxin. In (2), morcOE worms were taken through three generations (P0, F1, F2), with F1 worms split into two separate batches processed in parallel, which were treated as biological replicates in all analyses. For each sample within each batch, most were done in triplicate, with a few in duplicate when worms were too sick. Some samples were maintained on neomycin to force transgene expression, whlie others were left on non-selective media and manually separated into transgene expressing (mCherry ON) vs. transgene off (mCherry OFF) samples in the F2 generation. All samples in (2) were compared to a control grown in parallel that lacks the transgene and just has a flag tag on the endogenous MORC-1 copy ("rna_morcOE_ctrl" samples).

first: Connie

last: Xiao

first: Kai

last: Inoki

first: Sonia

last: El Mouridi

first: Christian

last: Frøkjær-Jensen

first: Steven

last: Jacobsen

first: John

last: Kim


title: The role of the 3D genome organization on platinum drug induced DNA damage susceptibility

first: Ye

last: Wang

address: 611 Charles E Young Dr E

department: Bioinformatics

summary: Platinum drugs (Pt drugs) are one of the most widely used chemotherapy drugs in cancer treatment. Although the cytotoxic and resistant mechanisms of Pt drugs have been thoroughly explored, it remains elusive what factors affect the receptiveness of DNA to Pt drug-induced damage. A large fraction of the genome shows significantly altered Pt drug-induced DNA damage susceptibility in the in vivo nuclear environment in comparison to isolated DNA, which cannot be explained only by the transcriptional status and DNA accessibility of the chromatin regions in vivo. Here, we demonstrate that, besides local chromatin structure, the global nuclear locations of genomic regions play a key role in modulating Pt drug-induced DNA damage susceptibility in vivo. By integrating data from damage-seq experiments with 3D genome structure information we show that the preferential nuclear locations of genomic regions relative to specific nuclear bodies and nuclear compartments can explain patterns of Pt drug DNA damage susceptibility. Thus, our observations are consistent with recent in vitro observations of an enrichment of Pt drugs in biomolecular condensates linked to certain nuclear bodies. Finally, when mapping observed DNA damage-seq signals onto 3D genome structures, we found that the 3D nuclear distribution of Pt-drug induced DNA damage differs in drug resistant cells in comparison to drug sensitve cells. In particular DNA damage increases in gene poor chromatin preferentially located in the lamina compartment, while DNA damage is decreased in gene rich regions preferentially located at nuclear speckles. This change may deter Pt-drug induced DNA damage from more viable gene dense chromatin regions that are crucial for short term cell survival. This observation suggests a selective spatial redistribution of Pt drug action in the nucleus during the emergence of chemoresistance.  These observations are relevant for a better understanding of Pt drug action and the development of cancer resistant cells.

overall_design:
HCT116 cell lines, categorized as either sensitive or resistant to oxaliplatin treatment, are treated with oxaliplatin to induce DNA damage. In-situ Hi-C is performed at oxaliplaint-resistant cell to determine the chromatin structure. Subsequently, genome-wide damage map is generated by damage-seq. Following this, the joint analysis of damage map and chromatin structure is performed to investigate how the nuclear architecture influence the damage patterns during the development of chemoresistance.
Invitro.oxa.damage.15min = oxaliplaint-sensitive HCT116 treated with oxaliplatin for 15 min
RES.oxa.damage.3hrs = oxaliplaint-resistant HCT116 treated with oxaliplatin for 3hrs
SEN.oxa.damage.3hrs = oxaliplaint-sensitive HCT116 treated with oxaliplatin for 3hrs
RES.oxa.damage.24hrs = oxaliplaint-resistant HCT116 treated with oxaliplatin for 24hrs
SEN.oxa.damage.24hrs = oxaliplaint-sensitive HCT116 treated with oxaliplatin for 24hrs
RES.control.HiC = Hi-C of oxaliplatin-resistant HCT116 (without any treatment)

first: Frank

last: Alber

first: Xianghong

last: Zhou