title: Isoform-resolved transcriptome in patients with inflammatory bowel disease

city: New York

first: yu

last: liu

state: NY

address: 1428 Madison Ave

institute: Mount Sinai

summary: Inflammatory bowel disease (IBD) is a group of chronic diseases that cause inflammation in the intestines, including ulcerative colitis (UC) and Crohn's disease (CD). Numerous omics studies have been published to understand IBD pathogenesis and identify potential targets for therapeutics. Despite the high amount RNA-seq data from short reads sequencing platforms produced to help preclinical and clinical research in IBD, such studies fail to capture the full-length mRNAs and various isoforms from alternative splicing events. Thus, the transcriptome associated with IBD has been understudied. The IsoSeq system of PacBio Sequel platforms enables sequence large number of long transcripts, and the corresponding IsoSeq pipeline and additional bioinformatics tools can identify the full-length transcripts and various isoforms. Here, we generate isoform-resolved transcriptome in IBD patients by performing long-read RNA sequencing on samples from four IBD patients and two healthy controls and integrate the full-length transcripts data with short-read RNA sequencing data from 99 IBD patients and controls. We identify numerous unannotated isoforms transcribed from both known and unannotated genes. Our bioinformatics pipeline further identified sQTL by integrating variants predicted from RNAseq with the isoforms from IsoSeq. We also identified isoform switch associated with IBD, and genes correlated with isoforms. Further pathway analysis and gene annotation search were conducted to understand the IBD disease. Together, our findings show that the transcriptome associated with IBD is far more complex than currently known, and our results will act as a valuable resource to empower future studies for IBD.

overall_design:
Intestinal epithelial cells and lamina propria lymphocytes from IBD patient and control surgical and biopsy samples are collected, isolated and processed for RNAseq.
Tissues were prepared by following process. For surgical specimen: surgical specimens from patients undergoing bowel resection for IBD or cancer (at least 10 cm away from tumor) at the Mount Sinai Medical Center were used as a source of intestinal cells. IECs were isolated by a method described previously (PMID: 2960770). Resected surgical specimens were washed extensively with phosphate-buffered saline (PBS). The mucosa was stripped off from the submucosa, minced into small pieces, and placed in 1 mmol/L dithiothreitol (Sigma Chemical Co., St. Louis, MO) for 10 minutes at room temperature to remove mucus. The pieces were washed in PBS and incubated twice in dispase (3 mg/mL in RPMI) for 30 minutes at 37°C, vortexed every 5 minutes.  Enterocytes are located at the 0%–30% layer interface. Cells were washed with PBS and resuspended in serum-free medium (Aim V, 50 u/mL penicillin, 50 g/mL streptomycin, 2 mmol/L glutamine, all from Gibco BRL, Grand Island, NY). The purified IECs should have greater than 95% viability and contain less than 2% IELs. The remaining tissue after dispase treatment was incubated with Collagenase (1 mg/mL) and DNAse I (5 g/mL) for 1–1.5 hours at 37°C with constant shaking. Lamina propria mononuclear cells were collected from the supernatant, centrifuged through a 2-layer percoll gradient, and harvested from the 35%– 65% interface. The cell suspension was collected and centrifuged on a Percoll (Pharmacia, Piscataway, NJ) density gradient.
For biopsies: Intestinal cells were isolated according to an established protocol using Dispase II (Roche Diagnostics, Indianapolis, IN) and collagenase (Sigma Chemical Co) treatment to separate intestinal epithelial cells from lamina propria cells as cited above.  6-8 pinch biopsies were used per sample, collected from an uninflamed or inflamed region of the intestine. Pinch biopsies from the ileal or colonic mucosa of healthy individuals and IBD patients at Mount Sinai Hospital were digested at 37 °C for 1 h in 100 units ml−1 collagenase Type VIII (Sigma, St Louis, MO) and 0.150 mg ml−1 DNase (Sigma) in complete RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% fetal calf serum, 2 mM L-glutamine, 100 units ml−1 penicillin, 0.1 mg ml−1 streptomycin, and 0.05 mM 2-Mercaptoethanol. Cells were filtered through a 50-micron filter, washed with 5 ml phosphate-buffered saline (PBS), and pelleted. Next, cells were resuspended in 40% Percoll (GE Healthcare, Fairfield, CT) in complete RPMI, under-layed with 80% Percoll, and centrifuged at 2,200 r.p.m. for 20 min at room temperature. LPMCs were collected at the interface and used for subsequent flow cytometry analyses in a representative sample set. For some samples CD3 positive and negative were selected using a magnetic separation kit (Miltenyi)
We resuspended IECs and LPLs in RLT buffer (or appropriate for profiling protocol) at 10,000 cells per ul and resuspended in 500ul, counted the cells and preserved in Trizol for storage in -80C.
all six samples were processed together for full length RNA sequencing at the Icahn School of Medicine.  A randomization was employed to create batches for RNA extraction, cDNA library preparation, and full length RNA sequencing using PacBio platform.

first: Gang

last: Wang

first: Yi

last: Zhong

first: Li

last: Sun

first: Huang

first: Charles

last: Cole

first: Andrew

last: Uzilov

first: Ming

last: Xue

first: Ke

last: Hao

first: Robert

last: Sebra

first: Kristin

last: Beaumont

first: Nancy

last: Francoeur

first: Scott

last: Newman

first: Yu

last: Liu

first: Haoxiang

last: Cheng

last: Schadt

first: Lauren

last: Peters


title: The spatial architecture of pancreatic pre-invasive lesions reveal acinar metaplastic cells’ heterogeneity [scRNA-seq]

status: Public on May 01 2026

city: Jerusalem

first: Oren

last: Parnas

address: Faculty of Medicine, Ein-Kerem medical center

department: Immunology

country: Israel

institute: Hebrew University

type: Expression profiling by high throughput sequencing

summary: In this project, we utilized single-cell RNA sequencing (scRNA-seq) and MERFISH spatial transcriptomics to profile the development of pancreatic cancer in a mouse model, tracing its progression from the pre-invasive stage to full malignancy. By employing a reporter gene, we were able to dissect the heterogeneity of metaplastic acinar cells, identifying and profiling six distinct types and states of these cells. We validated their localization within pre-invasive lesions, assessed the heterogeneity across various lesions, and calculated the colocalization of different acinar metaplastic cells with stromal cells.

overall_design: Tamoxifen was injected into six-eight weeks old Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato (PRT) mice, and the pancreas was collected for single cell purification from mice seven months post-tamoxifen injection (PTI) Tamoxifen was injected into six-eight weeks old Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato (PRT) mice, and the pancreas was collected for single cell purification from mice seven months post-tamoxifen injection (PTI)

first: Juan

middle: S

last: Arcila-Barrera


title: Human regulatory B cells suppress autoimmune disease primarily via interleukin-37

city: Changchun

first: Zheng

last: Jin

address: Xinmin Street 126#

department: College of  Basic Medical Science

country: China

institute: Jilin University

summary: IL-37 is effectively induced in the majority of human Bregs upon stimulation with recombinant (r)IL-37 and/or TLR9 agonist CpG via different mechanisms.

overall_design: B cells were sorted from Human PBMC,then stimulated with or without CpG fior 24 hours. The single cells were obtained by the method of grinding through a 70 μm cell strainer.

first: Damo

last: Xu

first: Luman

last: Wang

first: Yiwei

last: Chu


title: Neurogenic Monocyte Recruitment Drives Pathogenic Enteric Nervous System Remodeling in IBD

first: yu

last: liu

country: USA

summary: The proper organization of the enteric nervous system (ENS) is critical for normal gastrointestinal (GI) physiology. Inflammatory bowel disease (IBD) dysregulates GI physiology including bowel movements (motility), but in many IBD patients, GI motility disorders persist in remission through a poorly understood pathological process. Here we uncover that post-inflammatory GI dysmotility (PI-GID) stems from structural ENS remodeling driven by a combination of neuronal loss and neurogenesis. Enteric neurons respond to mucosal inflammation by upregulating CCL2 expression and facilitating the recruitment of CCR2+ monocytes into the neural myenteric plexus of the intestinal muscle followed by the expansion of monocyte-derived macrophages, their migration into the myenteric ganglia and phagocytosis of neurons. However, excessive recruitment of monocytes promotes disproportionate ENS remodeling and PI-GID. Expansion of immune cells in the tissue also promotes tissue hypoxia. We find that enteric neurons are hypoxic upon colitis; hypoxia-induced signaling via HIF1alpha induces an adaptation program in enteric neurons to suppress CCL2 expression and limit monocyte recruitment. We demonstrate that reinforcing HIF1alpha signaling in enteric neurons prevents PI-GID by reducing colitis-associated monocyte recruitment in the myenteric plexus and mitigating ENS remodeling. In summary, our findings unveil PI-GID pathogenesis and identify a regulatory axis for its prevention.

overall_design: the longitudinal muscle/myenteric plexus (LM/MP) tissue of wild type mice GI tract (small bowel part) were isolated, processed, and analyzed by scRNAseq

first: Monalee

last: Saha

first: Qian

last: Li

first: Preston

last: White

first: Sushma

last: Nagaraj

first: Ken

first: Subhash

last: Kulkarni

first: Miliena

last: Bogunovic

first: Pankaj

middle: J

last: Pasricha


title: Single cell transcriptomes and spatial architecture of pancreatic pre-invasive lesions reveal acinar metaplastic cells' heterogeneity

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

platform_organism:
Mus musculus
synthetic construct

overall_design: Refer to individual Series


title: Identifying Potential Therapeutic Targets for Calcific Aortic Valve Disease Through Multi-Omics Approaches

city: Hangzhou

first: dilin

last: xu

state: 默认

address: 88 Jiefang Road, Shangcheng District

institute: zhejiang university

summary: Calcific aortic valve disease (CAVD) is a degenerative disease characterized by aortic valve fibrosis and calcification with no medications available for treatment. We conducted the Mendelian randomization (MR) and summary data-based Mendelian randomization (SMR) combined with transcriptomic analysis to identify potential therapeutic targets for CAVD.Protein quantitative trait loci (pQTL) from two whole plasma protein databases (deCODE and UKBppp) were utilized as the exposure. Three large CAVD cohorts from Finland (9,870 cases and 402,311 controls), the UK (3,552 cases and 425,855 controls) and France (3,163 cases and 6,531 controls) served as the outcome. Colocalization analysis was performed to determine whether CAVD and target proteins shared common causal SNPs. Single-cell and bulk RNA sequencing was further used to detect the cell-type specific and differential expression of target proteins. Drug prediction and molecular docking was applied to discover potential medications. 15 plasma proteins were identified via MR and SMR analysis with 6 strongly supported via colocalization analysis. Among them, ANGPTL4 and ITGAV was validated in the replication CAVD cohorts from the UK or France. Meanwhile, the protein levels of ANGPTL4 and ITGAV was upregulated in valvular interstitial cells (VICs). Molecular docking suggested a stable binding between the target proteins and medications.The proteome-wide MR analysis discovered 6 proteins associated with the progression of CAVD and provided novel perspective in the etiology and drug target screening strategy.

overall_design: To conducte the Mendelian randomization (MR) and summary data-based Mendelian randomization (SMR) combined with transcriptomic analysis to identify potential therapeutic targets for CAVD.

first: Dilin

first: Jin

last: Lu

first: YanFang

last: Yang


title: Primary human bronchial epithelial cells exposed to acute vs chronic woodsmoke condensate: Responses evaluated using bulk RNA sequencing

city: Chapel Hill

first: Julia

last: Rager

state: NC

address: 135 Dauer Drive, CB 7431

institute: UNC

summary: Wildfires are increasing in intensity and prevalence and are associated with respiratory health effects such as asthma, bronchitis, and respiratory infections. Recent research highlights lung cell molecular changes following acute wildfire and woodsmoke exposure. However, chronic, repeat exposure conditions that may be more typical of community-level exposures to wildfire and woodsmoke remain to be characterized in controlled, in vitro molecular toxicology studies. The goal of this project was to evaluate similarities and differences in transcriptional responses of primary human bronchial epithelial cells (HBECs) exposed to woodsmoke condensate following acute vs. chronic exposure. Woodsmoke condensate was generated from the burning of red oak at smoldering conditions, and cells were exposed for either 4 hours (acute) or 4 hours, 3 days per week, for two weeks (chronic).

overall_design: RNA-seq profiling of differentiated primary human bronchial epithelial cells (derived from n = 4 unique human donors) exposed apically to smoldering red oak condensate or PBS vehicle dissolved in media for four hours (acute exposure) or for four hours, 3 days a week, for two weeks (chronic exposure). RNA-seq signatures were acquired using bulk cellular samples.

first: Elise

last: Hickman


title: Histone post-translational modification changes upon Sfp1 depletion

city: Quebec city

first: Jacques

last: Cote

address: 9 McMahon Street

department: CHU de Québec-Université Laval Research Center

country: Canada

institute: Laval University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: In this study, we investigate the physical and functional interaction between NuA4 and Sfp1. Sfp1 was rapidly depleted from yeast nuclei, and histone modification changes were detected by ChIP-sequencing.

overall_design: ChIP-sequencing of H4K8ac, Htz1, H3K9ac and H3 upon nuclear depletion of Sfp1.


title: Functional interaction between the transcription factor Sfp1 and the NuA4 complex in response to nutrient availability [CUT&RUN]

summary: In this study, we investigate the physical and functional interaction between the transcription factor Sfp1 and the NuA4 complex in response to nutrient changes or stress. To study the functional interaction, we rapidly deplete Sfp1 or Esa1 (the catalytic subunit of NuA4) from nucleus, and performed CUT&RUN to profile all Sfp1/NuA4-regulated genes.

overall_design: CUT&RUN sequencing of Sfp1 upon Esa1 depletion, Eaf1 upon Sfp1 depletion; CUT&RUN of Sfp1 (wild-type and mutant) and Eaf1 in normal growth condition or TORC1-inhibited condition.


title: To identify the hub gene related to complex regional pain syndrome

city: Beijing

first: He

last: Zhu

address: Dongcheng street

institute: Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College

summary: To identify the hub gene related to CRPS, the human keratinocyte cell lines (China Infrastructure of Cell Line Resource, Beijing, China) were used for RNA-seq analysis after OGD/R (4 control and 4 OGD/R samples) in early stage.

overall_design: The human keratinocyte cell lines (China Infrastructure of Cell Line Resource, Beijing, China) were used for RNA-seq analysis after OGD/R (4 control and 4 OGD/R samples) in early stage. Sequencing was performed using an Illumina Novaseq 6000 instrument.


title: mTORC1-Dependent Regulation of the CCL24-CCR3 Axis Controls Granuloma Formation and Maintenance in Sarcoidosis [scRNAseq]

city: Lexington

first: Jinpeng

last: Liu

state: Kentucky

address: 800 Rose St

institute: Markey Cancer Center

summary: Sarcoidosis is a chronic inflammatory disorder characterized by the presence of tiny collections of immune cells known as granulomas. An incomplete understanding of etiology and immune-pathologic pathways have limited the development of quality biomarkers and novel targeted therapies. We found a Fsp1-Cre mediated TSC1/2 deletion mice unexpectedly led to development of a disease state, which was identified and characterized by spatial transcriptomics and single-cell RNA sequencing, similar to sarcoidosis. Notably, we identified CCL24 as a novel key chemotactic regulator in this model of sarcoidosis and found decreased CCL24 in the sarcoid group, thus translating our model and hypothesis to the human form of the disease. Intriguingly, we found that CCL24 and azithromycin significantly blocked the progression of granuloma formation in this model. Our findings not only improve an integrated understanding of the progression of sarcoidosis but also provide the basis for developing novel potential clinical interventions in sarcoidosis therapeutics.

overall_design: scRNA-seq for WT and KO lung immune cells

first: Xiongjian

middle: X

last: Rao


title: Cold stress induced dynamic chromatin accessibility facilitate histone reorganization and transcription factor recruitment in Arabidopsis.

city: Seoul

first: Nassem

last: AlBakri

address: Gwangjin-gu

department: Biomedical Science and Engineering

country: South Korea

institute: Konkuk University

summary: Plants are exposed to various environmental stresses, such as cold temperatures, that can impact growth and development. In plants, numerous genes, and pathways responsive to cold stress have been identified, suggesting dynamic changes in chromatin architecture and epigenetic modifications. Here, we used Nicking Enzyme-assisted Sequencing (NicE-seq) to investigate the dynamic changes in chromatin accessibility in Arabidopsis plants under cold stress. We show significant alterations in chromatin accessibility and histone reorganization upon cold stress without impacting gene expression. Interestingly, a subset of downregulated genes displayed highly accessible chromatin regions. This downregulation is likely mediated by CBFs/DREB1 transcription factor binding to the first exons of target genes. Specifically, our data suggests that CBF1, CBF2 and CBF3 transcription factors play a critical role in regulating the downregulation of genes in highly accessible chromatin regions during cold stress. These findings provide a novel insight into the dynamic changes in the genome-wide chromatin landscape in response to cold stress involving chromatin accessibility that facilitates position-dependent transcription factors binding for targeted gene expression regulation.

overall_design: To investigate open chromatin profling upon cold stress condition at time-point series, we performed the Nice-seq using nicking enzmye, Nt.CviPII upon cold stress  of time point series up to 24hr in Arabidopsis

first: Zein

middle: E

last: Bader

first: Hang

middle: G

last: Chin


title: mTORC1-Dependent Regulation of the CCL24-CCR3 Axis Controls Granuloma Formation and Maintenance in Sarcoidosis [RNAseq]

overall_design: RNA-seq for WT and KO MEFs


title: Spatial Transcriptomics identifies Prosaposin (PSAP) as a Novel Therapeutic Vulnerability in Pulmonary Sarcoidosis

overall_design: Spatial Transcriptomics for lungs from healthy individual and sarcoidosis patients.


title: Parkinson's disease (PD) genetic asymmetry in SNCA PFF injected mouse model

city: Grand Rapids

first: Gerry

last: Coetzee

state: MI

address: 333 Bostwick Ave NE

department: CNS

institute: Van Andel Research Institute

summary: We measured gene expression in neuronal nuclei from cortex derived from both hemispheres (injected and noninjected side) of mice (16 male, 16 female) intected with either PBS or SNCA PFFs. Mice were sacrificied at either 1 or 3 month post injection. This was a preliminary experiment done to examine a mouse model of asymmetry in a PD-like condition. RNA-sequencing was used to measure gene expression differences resulting from injecting PBS or alpha-synuclein pre-formed fibrils (PFFs), an inducer of PD like neuropathy, in the right olfactory bulb of 32 (C57BL/6J) mice, 16 male and 16 female. Cortex was then isolated for RNA extraction from both right and left hemispherical cortex in all mice at either 1 month or 3-months post-injection.   The overall conclusion was that expression changes were found but were too modest to warrant further focus using mice. Greater detail is given below. While the model has some value and the results may be useful for comparison with human data, nuisance variables were found to induce changes of similar magnitude to treatment conditions. The small magnitude in differential expression levels, coupled with strong effects by confounds, primarily cage to cage variation, resulted in less robust results than desirable. Thus, while this pilot experiment indicates that a few dozen to a few hundred genes may show hemispherical asymmetry in the mouse model across sex and timepoint, further experiments will focus on human data.   Within the experiment the features conferring the greatest difference in overall gene expression levels between the mice were sex, followed by time post injection (1-month vs. 3-month) (figure 1,2). The conditions of interest, treatment: PBS or PFF, and hemisphere: injected vs non-injected, do not confer obvious structure to overall gene expression patterns. Rather differences between individual mice as well as between cage mates outweigh overall differences for the latter 2 experimental conditions. However, some individual genes do show significant treatment or hemispherical differential expression. This is truer when treatment or hemisphere are considered while holding sex and/or timepoint constant (Table 1). Overall, across 16 statistical comparisons, 1038 statistically significant (FDR < 0.05) and high fold change (>40% change in expression) genes. Of these, excluding simple male vs. female, 342 genes are unique. These 342 genes show enrichment in processes related to neurotransmitter function, especially dopamine, cell signaling, drug binding, complement activation of the immune system, and others.   Individual comparisons are expected to be most relevant for elucidating PD asymmetry. For instance, 23 genes are significant (adj. p.value < 0.5 and logFC > 0.5) for differences between injected and non-injected hemispheres regardless of sex or timepoint. These 23 genes show some GO enrichment for axonogenesis and dopaminergic systems, among other things. It is somewhat surprising that such PD-relevant categories show differential expression in this comparison, as the PBS condition should not induce a disease like state. However, the sensitivity of the analysis may be misleading. 2/3 of the significant DE genes were also

overall_design: Frozen prefrontal cortex was prepared and sorted to isolate neuronal nuclei. RNA was preparred for bulk RNA-sequencing. Results were analyzed using generalized linear modeling after controling for unwanted variation.

first: Gerhard

middle: A

first: Steven

last: Pierce

first: Elizabeth

last: Ensink


title: Activation of Fibroblast LRP1 by AT2-secreted MDK Affects OTUB1/SLC7A11 Ubiquitination to Inhibit Fibroblast Ferroptosis in Idiopathic Pulmonary Fibrosis.

city: Wuxi

first: Hua

last: Wenjie

address: NO.1800 Lihu Avenue

institute: Jiangnan University

summary: Idiopathic pulmonary fibrosis (IPF), a chronic interstitial fibrosing pneumonia of unknown cause, is characterized by an irreversible decrease in alveolar cells and an increase in fibroblasts. In our research, the numbers of AT2 cells and fibroblasts are abnormally regulated, but AT1 cells are significantly reduced in lung tissue sections from patients with IPF and mice. By analyzing the single-cell sequence from GEO database and experimental validation both in vivo and in vitro, it is found that the abnormally regulated AT2 cells secreting a large amount of MDK to activate the CTHRC1+ fibroblasts LRP1 receptor and inhibit the ferroptosis of fibroblasts. Further, by activating the LRP1 receptor in CTHRC1+ fibroblasts, the RNA-seq results reveal that the expression level of OTUB1 increased significantly. The ubiquitination experiment results showed that OTUB1, as a deubiquitinated molecule, can directly bind to the ferroptosis inhibitory protein SLC7A11, removing the ubiquitination modification of SLC7A11 and stabilizing its expression level to inhibit ferroptosis in fibroblasts. Meanwhile, this molecular pathway was verified in mice by dropping adenovirus through the airway, and the same experimental results as in vitro were verified. In conclusion, our study verified that IPF leads to abnormal regulation of AT2 secreting MDK, which activates LRP1 receptor and increases OTUB1 in fibroblasts. Deubiquitylation modification of OTUB1 stabilizes the expression level of SLC7A11, which results in a decrease in fibroblast ferroptosis, leading to an abnormal regulation of its programmed death, ultimately the deterioration of IPF.

overall_design: To investigate the effect of the LRP1 activated by MDK, we established the human lung primary fibroblast model stimulated with 15ug/ml MDK.

first: Wenjie

last: Hua

first: Zitao

last: Wang

first: Jingyu

last: Chen


title: Spatial Transcriptomics identifies Prosaposin (PSAP) as a Novel Therapeutic Vulnerability in Pulmonary Sarcoidosis [Mouse_Spatial]

overall_design: Spatial Transcriptomics for lungs from WT and rapamycin treated KO mice


title: Heterozygous SPTAN1 frameshift mutations cause distal myopathy with neurogenic features

city: Paris

first: Fiorella

last: Grandi

address: 105 Bld d'Hopital

department: Centre de Recherche de Myologie

country: France

institute: Sorbonne Universite

summary: Spectrinopathies represent an emerging theme with overlapping phenotypes ranging from peripheral or central nervous system involvement towards complex syndromic presentations. A total of 60 different reported SPTAN1 mutations is exemplary for this diversity with phenotypes spanning almost the entire neurospectrinopathy spectrum, except myopathic involvement.Through collaboration within the Solve-RD network we collected patients with unsolved progressive distal weakness and unreported SPTAN1 variants. We reached consensus on the interpretation of clinical picture, electrophysiology, muscle MRI and biopsy. Finally, SPTAN1 protein and RNA expression analysis was performed on available muscle biopsy on one of the patients. We provide evidence for the association of SPTAN1 frameshift variants with childhood onset distal myopathic as well as neurogenic features in 7 patients. This finding further extends the established spectrum of SPTAN1 haploinsufficiency associated with a variable penetrance of hereditary motor neuropathy and ID. Overlap between distal myopathic and neurogenic features could be present in other patients and may be overlooked due to phenotypic similarity.

overall_design: A deltoid muscle sample from one of the index families was used for RNA-sequencing.

first: Piera

last: Smeriglio

first: Tanya

last: Stojkovic


title: Transcriptome analysis of mesenchymal stem cells in eutopic endometrium and normal endometrium of patients with adenomyosis by RNA sequencing

city: Nanjing

first: Tao

last: Gui

state: Jiangsu

address: NO.100 Shizi Street, Hongshan Road

institute: Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine

type: Non-coding RNA profiling by high throughput sequencing

summary: To identify the differentially expressed miRNAs and target genes of exosomes derived from eutopic endometrial mesenchymal stem cells ( e MSCs ) in patients with adenomyosis and non-endometrial adenomyosis, and to search for miRNAs and their target genes related to macrophage polarization at the transcriptome level, so as to provide new insights into the mechanism research and treatment of adenomyosis.

overall_design: In this study, we isolated exosomes from the culture supernatant of normal eMSCs (n=3) and eutopic eMSCs (n=3). We performed miRNA Seq to identify miRNA profiles in eMSCs-Exos, and successfully obtained the results.

first: Yingying

last: Qiu


title: Single-cell RNA-sequencing of whole blood from human burn patients

city: Calgary

first: Jeff

last: Biernaskie

state: Alberta

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

institute: University of Calgary

summary: To develop effective therapeutic interventions that prevent scar formation and encourage regeneration of healthy skin, we need to better understand the cellular and molecular process that are engaged during the wound healing process in humans. However, our cellular and molecular understanding of how humans heal deep dermal wounds remains limited. Our recent work using different animal models of skin wound healing has identified the acute wound healing immune response as a key determinant of terminal fibrotic outcomes. Here, our goal is to compare the immune response during human wound healing to datasets we have generated from large animal models that are capable of scarless healing, in order to determine how to most appropriately modify the process in humans to improve outcomes (and mitigate scar).

overall_design: Our recent work using different animal models of skin wound healing has identified the acute wound healing immune response as a key determinant of terminal fibrotic outcomes. As such, in this study, we are interested in profiling the immune response during human wound healing in adults who have sustained full-thickness skin wounds. The inclusion criteria for participants was as follows: 1. <10% Total Body Surface Area; 2. Thermal or mechanical injury (irrespective of causative agent); 3. Full-thickness wound; 4. Less than 14 days post-injury; 5. Absence of re-epithelialization; 6. Consent to 6mm punches biopsies and blood collection. To examine molecular changes within specific cell compartments, a total of 15,000 single cells (containing an equal proportion of leukocytes and lymphocytes) were loaded for partitioning using 10x Genomics NextGEM Gel Bead emulsions (3′ gene expression kit, version 3.1). All samples were processed as per the manufacturer’s protocol (both PCR amplification steps were run 12×). In brief, single cells were sorted based on forward versus side scatter gating into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. Next-Generation Sequencing was performed using the Illumina NovaSeq S2 Flow cells. All raw FASTQs were aligned to the human reference genome generated using cellranger mkref pipeline. The resulting gene barcode matrix was processed using Seurat R toolkit.

first: Sarthak

last: Sinha

first: Nicole

last: Rosin

first: Keerthana

last: Chockalingam

first: Vincent

last: Gabriel


title: Dihydroartemisinin sensitizes cancer cells to ferroptosis in multiple myeloma

city: Su*zhou City

first: Wang

last: Zhiming

address: No. 199, Ren'ai Road

institute: Soochow University

summary: High-throughput transcriptome sequencing was used to identify differentially expressed protein-encoding genes after DHA treatment. Additionally, differential gene enrichment analyses using the Gene Ontology Resource (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) databases were carried out to comprehensively evaluate the effects of DHA on the signaling pathways and biological processes of U266 and LP-1 myeloma cells.

overall_design: The myeloma cells were divided into an experimental group and a solvent control group, with three replicates for each group. The experimental group was treated with Dihydroartemisinin (DHA) at its IC50 concentration, while the solvent control group was treated with an equal amount of DMSO. After 12 hours, cells were collected and subjected to transcriptome sequencing.

first: Zhiming

first: Wenzhuo

last: Zhuang

first: Bingzong

last: Li


title: Wave-forming Nanotexture-Mediated Priming of Stem Cell for Osteogenesis via Mechanotransduction Machinery and Chromatin reprogramming

city: Cheonansi

first: Ji-Young

last: Yoon

state: Chungnam

address: Dandaero 119

department: Nanbiomedical science

institute: Dankook University

overall_design: Refer to individual Series


title: Wave-forming Nanotexture-Mediated Priming of Stem Cell for Osteogenesis via Mechanotransduction Machinery and Chromatin reprogramming [ATAC-seq]

summary: Biophysical cues such as the topography of ECM can direct cell lineage specification and accelerate the regeneration of cultured tissue. Here, we show how changes in the cellular and nuclear morphologies of mesenchymal stem cells induced by clinically applicable nanotextures influence the conformation of the cells’ chromatin and their osteogenic differentiation. The wave-forming nanotextures (W-NT) were eco-friendly fabricated on medical-grade titanium by a non-thermal femto-second laser. W-NT impacted cellular elongation, nuclear architecture, and chromatin reprogramming via microtubules. The ensuing chromatin reprogramming enhanced the cells’ responsiveness to osteogenic differentiation biochemical factors, revealed by ATAC and RNA sequencing. Mechanistically, osteogenesis was enhanced by the Yap-mediated mechanotransduction machinery, involving ‘nanotexture’ - ‘microtubules’ - ‘nucleus deformation’ - ‘Yap-RUNX2 axis and histone 3 acetylation activity’. In rabbits with bone defects, implants with nanotextures enhanced bone regeneration without exogenous signaling molecules. Our findings suggest that nanotopographical modification of medical devices could be leveraged to facilitate bone regeneration through nuclear-shaped mediated chromatin priming.

overall_design: We fabricated wave-forming nanotextures (W-NT) on titanium (Ti) surfaces using a non-thermal, eco-friendly femtosecond laser (FsL) that does not require strong acids. We then compared the histone opening analysis of rMSCs cultured for 1 day on the nanotextured surfaces and flat titanium surfaces under growth conditions through ATAC-seq.

first: Jung-Hwan

last: Lee

first: Hae-Won

last: Kim


title: Chronic stress disrupts hepatic homeostasis and accelerates liver cancer progression through ADRB2 signaling

city: Shanghai

first: Lu

state: Shanghai

address: No. 650, Xinsongjiang Road, Songjiang District, Shanghai, China

department: Shanghai General Hospital

institute: Shanghai Jiao Tong University School of Medicine

summary: Chronic stress has been implicated in the dysregulation of immunological, neurochemical, and endocrinological functions, yet its impact on hepatic homeostasis and liver carcinogenesis remains elusive. In this study, by employing single-nucleus RNA sequencing and histopathological evaluation, we demonstrated that chronic stress induced profound hepatic dysfunction. Strikingly, using orthotopic murine models of liver cancer, we further found that chronic stress significantly accelerated tumor progression. Mechanistically, we identified β2-adrenergic receptor (ADRB2) signaling as the pivotal molecular pathway driving stress-accelerated cancer progression, which was in line with the poor clinical outcomes observed in cancer patients exhibiting enhanced adrenergic signaling within tumors. Collectively, our study provides compelling evidence that chronic stress compromises hepatic homeostasis and accelerates liver cancer progression via ADRB2 activation, highlighting the therapeutic potential of targeting this pathway and the clinical importance of stress management in hepatic disorders.

overall_design: Comparative gene expression profiling analysis of snRNA-seq data and bulk RNA-seq data for healthy mouse livers and stressed mouse livers (restraint stress, foot shock stress, and social defeat stress), livers from liver cancer mice and stressed liver cancer mice, livers from liver cancer mice and healthy mice, livers from Adrb2-deficient mice..

first: Huimin

last: Qin

first: Liuwei

last: Chu

first: Xiaying

last: Zheng

first: Huimei

last: Zhang

first: Yue

last: Lan

first: Ji

last: Hu


title: Early sex-related transcriptional differences in CD8+ T cells responding to chronic viral infection reveal a sex bias in exhaustion development

city: Winnipeg

first: Olivia

last: Wilkins

state: Manitoba

address: 66 Chancellors Cir

department: Biological Sciences

institute: University of Manitoba

summary: CD8+ T cell diversity is essential to control infections and chronic antigen stimulation. In acute-resolving infection, effector cells mediate acute responses and memory cells provide long-lived protection against future exposures. In chronic infection and cancer, an altered state called exhaustion occurs. Exhausted CD8+ T cells are molecularly and functionally distinct from effector and memory cells. Differences in immune responses exist between biological sexes, however, how biological sex influences the timing and transcriptional programs of CD8+ T cell responses during chronic versus acute viral infection remains unknown. Here, we show that male and female CD8+ T cells exhibit transcriptional differences in their early responses during chronic but not acute viral infection in vivo. Using single-cell RNA-sequencing analyses, we show that female CD8+ T cells adopt an early exhaustion-like program compared to males. Moreover, we found sex-related differences in single-cell gene expression dynamics and fundamental cellular processes early after chronic infection. These findings reveal new insights into sex-related differences in CD8+ T cell exhaustion development and early T cell responses that may contribute to sex differential immune responses.

overall_design: CD8+ T cells were transferred from CD45.1 P14 donor mice to CD45.2 C57BL/6J recipient mice, including female-female, male-male, and female-male transfers. Recipients were infected with LCMV-Armstrong or LCMV-clone 13 for 4, 7, 28, or 40 days. CD8+ T cells were isolated, sorted with FACS, and analyzed using scRNA-seq.

first: Nyambura

last: Kahia

first: Sean

middle: M

last: Robertson

first: Megan

last: Rodriguez

first: Paul

last: Layug

first: Harman

last: Vats

first: Victor

last: Spicer

first: Darien

last: Yeung

first: Arzu

last: Ozturk-Aptekmann

first: Janilyn

last: Arsenio


title: The effect of NLRP3 inhibition on gene expression in intestinal fibroblasts

city: Erlangen

first: Weber

last: Simone

state: Bavaria

address: Ulmenweg 18

department: Medicine

country: Germany

summary: To test whether NLRP3 inhibition leads to alterations in the gene expression profil of intestinal fibroblasts, intestinal fibroblasts were stimulated and treated with inhibitor or left untreated.

overall_design: Fibroblasts were isolated from four IBD patients. Intestinal fibroblasts were stimulated and treated with inhibitor or left untreated.

first: Simone

last: Weber

first: Sophia

last: Sonnewald

first: Stephen

last: Reid


title: Single cell 3'UTR sequencing of neural stem cells

city: Heidelberg

first: Ana

last: Martin-Villalba

address: Im Neuenheimer Feld 280

department: Molecular Neurobiology

institute: German Cancer Research Center

summary: In cultured neural stem cells (NSCs) we report 3'UTR shortening in active neural stem cells (NSCs) versus quiescent NSCs. NSCs were treated either with BMP4 or EGF to obtain an equivalent of in vivo active and quiescent NSCs. In addition, we see differential 3'UTR usage between APLP1-/- NSCs and neuroblasts versus wildtype. (In this second experiment hash tag oligos were used to assign single cells to biological replicates.) The sequencing strategy allwos a paired-end mapping approach enabeling 3' peak calling with single cell resolution. To access CPEB4 binding to mRNAs, an RNA immunoprecipitation (RIP) assay was carried out in cultured NSCs. Here, CPEB4-bound mRNAs were pulled down with a CPEB4 antibody.

overall_design: Comparison on 3'UTR mapping position between active and quiescent NSCs as well as APLP1-/- versus wildtype NSCs. Secondly, evaluation of CPEB4 binding (position in 3'UTR and strength of binding to mRNAs).

first: Nikhil

middle: O

last: George

first: Manuel

last: Goepferich

last: Domingo-Muelas

first: Alex

last: Bizy

first: Rosa

last: Pascual

first: Isabel

last: Fariñas


title: Mapping the effects of plasma-mediated rejuvenation across brain regions

city: South San Francisco

first: Oliver

last: Hahn

state: CA

address: 1170 Veterans Blvd

institute: Calico Life Sciences, LLC

summary: Systemic factors found in plasma of young mice and humans impact cognition and can functionally rejuvenate the aging brain. However, scalable, molecular tools to profile this process are currently missing, hampering efforts to translate these findings into clinical applications. Here, we leverage the advents of brain-region-specific transcriptomics as a readout of cellular deterioration accompanying the aging process. Profiling expression shifts in cortex (Cor), hippocampus (Hip) and Cerebellum (Ceb) we identify the region-specific responses to young plasma. This atlas of brain rejuvenation supports the identification of potential mediators of plasma-derived factors, and provides the basis for finding a minimal gene expression set - a signature - to sufficiently quantify brain rejuvenation.

overall_design: mRNA-seq data of hippocampus extracted from 3 months (young), 18 months (aged) or 22 months (old) mice (C57BL/6J) were treated with one of the following paradigms: naive (N), PBS injection for 7 consecutive days (PBS), young plasma injection for 7 consecutive days (OPL), young plasma injection for 24 consecutive days (OPLTOPL) or PBS injection for 24 consecutive days (OPLTPBS). Of these mice, 3 brain regions were dissected: cerebellum (Ceb), hippocampus (Hip) and cortex (Cor), and mRNA-seq libraries were generated and sequenced using Illumina sequencing.

first: Tony

last: Wyss-Coray


title: IPA analysis of CD14-positive monocytes/macrophages from IBD patients and controls

summary: the gene expression profiles of CD14-positive cells from IBD and control patients were analyzed

overall_design: CD14-positve cells were isolated from tissue samples from four IBD and four control patients


title: Wave-forming Nanotexture-Mediated Priming of Stem Cell for Osteogenesis via Mechanotransduction Machinery and Chromatin reprogramming [RNA-seq]

first: Ji-Young

last: Yoon

overall_design: We fabricated wave-forming nanotextures (W-NT) on titanium (Ti) surfaces using a non-thermal, eco-friendly femtosecond laser (FsL) that does not require strong acids. We then compared the transcriptional profiles of rMSCs cultured on the nanotextured surfaces and flat titanium surfaces under osteogenic conditions through RNA sequencing

first: Jung-Hwan

last: Lee


title: Retinal waves are required for state-dependent visual processing

city: Cold Spring Harbor

first: Lucas

last: Cheadle

address: 1 Bungtown Rd

department: Neuroscience

institute: Cold Spring Harbor Laboratory

summary: Spontaneous activity in the form of retinal waves propagates throughout the developing visual system during the first two weeks of life in the mouse. Here, we apply a range of approaches to the visual cortex of a unique mouse model, the Beta2fl/fl; Pax6α-Cre model,  in which retinal waves are disrupted during the first week of life. We find that spontaneous activity during development is necessary for establishing neuronal transcription, synaptic connectivity, and function in the maturing brain.

overall_design: We performed single-nucleus RNA-sequencing using the inDrops approach on anterior and posterior regions of primary visual cortex in Beta2fl/fl; Pax6α-Cre mice and Cre-negative controls. We identified genes that were significantly differentially expressed between anterior and posterior regions in WT and cKO mice.


title: Up-regulation of cholesterol synthesis pathways and neurodegeneration in a knock-in Sod1 mutant mouse model of ALS.

city: Worcester

first: Alper

last: Kucukural

state: MA

address: 364 Plantation Street

department: Program in Molecular Medicine

institute: UMass Medical School

summary: Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder affecting brain and spinal cord motor neurons. Mutations in the copper/zinc superoxide dismutase gene (SOD1) are associated with ~20% of inherited and 1-2% of sporadic ALS cases. Much has been learned from mice expressing transgenic copies of mutant SOD1, which typically involve high-level transgene expression, thereby differing from ALS patients expressing one mutant gene copy. We created a knock-in point mutation in the endogenous mouse Sod1 gene leading to mutant SOD1G85R protein expression, a human ALS-causing mutation, to generate a model that more closely represents patient gene expression. Heterozygous Sod1G85R mutant mice resemble wild type, whereas homozygous mutants have reduced body weight and lifespan, a mild neurodegenerative phenotype, and express very low mutant SOD1 protein levels with no detectable SOD1 activity. Homozygous mutants exhibit partial neuromuscular junction denervation at 3-4 months of age. Spinal cord motor neuron transcriptome analyses of homozygous Sod1G85R mice revealed up-regulation of cholesterol synthesis pathway genes compared to wild type. Transcriptome and phenotypic features of these mice are similar to Sod1 knock-out mice, suggesting the Sod1G85R phenotype is largely driven by loss of SOD1 function. By contrast, cholesterol synthesis genes are down-regulated in severely affected human TgSOD1G93A transgenic mice at 4 months. Our analyses implicate dysregulation of cholesterol or related lipid pathway genes in ALS pathogenesis. The Sod1G85R knock-in mouse is a useful ALS model to examine the importance of SOD1 activity in control of cholesterol homeostasis and motor neuron survival.

overall_design: CRISPR-based mutagenesis was used to create a knock-in point mutation in the endogenous mouse copper/zinc superoxide dismutase (Sod1) gene leading to mutant SOD1-G85R protein expression, a human ALS-causing mutation. Sod1-null mice carrying a novel knock-out Sod1 mutation were also created. Laser capture microdissection was used to isolate lumbar spinal cord motor neurons from knock-in Sod1-G85R, knock-out Sod1, and WT mice at 30, 70 and 120 days of age, and from widely-used  transgenic TgSOD1-G93A mice at 120 days. Bulk RNA transcriptional profiling was performed on these populations to identify differentially expressed genes.

first: Janice

last: Dominov

first: Hesheng

last: Zhang

first: Robert

middle: H

last: Brown Jr


title: Gene expression profile at single cell level of B16 and MC38 tumor infiltrating CD45+ TILs isolated from Gpr65-deficient and WT mice

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. for Immunology

institute: University Medical Center Mainz

summary: We used single cell RNA sequencing to analyze the composition of the tumor microenvironment of B16 and MC38 tumors in Gpr65-deficient and WT mice

overall_design: B16 or MC38 tumor cells were inoculated s.c. into Gpr65-deficient and WT mice. At day 17 (B16), respectively day 16 (MC38), single cell suspensions were prepared from excised whole tumors, and CD45+ cells were enriched by magnetic bead separation and flow cytometry-based cell sort.

first: Toszka

last: Bohn

first: Jean-Fred

last: Fontaine

first: Natalia

last: Truong


title: Widespread transcriptional memory shapes heritable states and functional heterogeneity in cancer and stem cells [scRNA-seq]

status: Public on Apr 30 2026

city: Beijing

first: Yongjie

last: Lin

state: Beijing

address: No.5 Yiheyuan Road, Haidian District

institute: Peking University

summary: Recent studies show that non-genetic heterogeneity, particularly through heritable cell states, shapes cancer evolution and developmental trajectories. However, single-cell snapshots lack temporal information to identify these states. We employ lineage-resolved single-cell transcriptomics to map heritable cell states that persist across divisions, distinguishing them from transient fluctuations. We uncover that heritable states are underpinned by widespread transcriptional memory, whereby heritable gene expression defines two classes of states: clustered states, characterized by clustered gene expression, and latent states, marked by non-clustered gene expression. This memory shows conservation across cell types and conditions and appears to be maintained by robust epigenetic mechanisms resistant to environmental perturbations. Functionally, memory genes predict critical behaviors including metastatic potential and lineage commitment, with latent-state genes often outperforming clustered-state genes. Our findings establish transcriptional memory as a potential basis of heritable cellular heterogeneity, providing a framework for understanding functional cellular variations across biological systems. A record of this paper’s transparent peer review process is included in the supplemental information.

overall_design: This study used lineage-resolved single-cell RNA-seq with lentivirus barcodes, bulk RNA-seq data and bulk RRBS data and NGS of lineage barcode in cell populations to study transcriptional memory. This dataset demonstrates single-cell lineage tracing data.

first: Xiangru

last: Chen

first: Long

last: Wu

first: Yutong

last: Zhou

first: Yihan


title: Widespread transcriptional memory shapes heritable states and functional heterogeneity in cancer and stem cells [RRBS]

type: Methylation profiling by high throughput sequencing

overall_design: This study used lineage-resolved single-cell RNA-seq with lentivirus barcodes, bulk RNA-seq data and bulk RRBS data and NGS of lineage barcode in cell populations to study transcriptional memory. This dataset demonstrates RRBS data.


title: Effects of anti-CD40 agonist and Cabozantinib on the immune microenvironment of mouse hepatocellular carcinoma

city: Taipei

first: Chia-Lang

last: Hsu

address: No. 7, Zhongshan S. Rd.

department: Department of Medical Research

country: Taiwan

institute: National Taiwan University Hospital

summary: CD45+ cells were extracted from mouse HCC model with or without treatment of anti-CD40 and cabozantinib for the scRNA-seq processing.

overall_design: scRNA-Seq

first: D

middle: L

last: Ou

first: C


title: Single-cell transcriptomic and immunoglobulin profiling in WT and IkB-deficient mouse early B cell progenitor and precursor populations

city: Los Angeles

first: Yu-Sheng

state: California

address: 611 Charles E. Young Drive East

department: MIMG

institute: University of California, Los Angeles

summary: Aging and inflammation have been shown to reduce the supply of functional B-cells; the prevailing explanation is that hematopoietic differentiation decisions are skewed towards the myeloid lineage. Here, we have addressed how inflammaging may affect B-lymphopoiesis itself. Using a new NFkB reporter mouse, we uncovered dramatic dynamics in RelA control during early B lymphopoiesis in young but not in all aged mice. When genetically perturbing NFkB RelA dynamics with specific IkB mutants, we found severe, cell-intrinsic defects in B lymphopoiesis, that could not be attributed to a differentiation block or diminished cell survival. Careful quantification of B-lymphoid progenitors allowed us to fit a mathematical model of the differentiation pathway, which led to the seemingly paradoxical prediction – confirmed in vitro – that mutant B-cell progenitors ‘rush through’ the differentiation pathway. Further analysis revealed that the differentiation hypermorph leads to diminished mature cell populations given that differentiation pauses are essential for population expansion. Transcriptomic profiling at single-cell resolution revealed that RelA dynamic dysregulation resulted in an accelerated progression of transcriptomic cell states that are not in synch with the classically defined progenitors and – importantly – override the preBCR checkpoint. As this is a major quality control step to ensure proper immunoglobulin heavy chain recombination, the mutant triggers premature κ-light chain recombination, resulting in B-cells with nonfunctional immunoglobulin. Our findings establish a new paradigm for how aging and inflammation impact humoral immunity via dysregulation of RelA dynamics: accelerated differentiation diminishes the size and the quality of the B-cell pool available for adaptive immune responses.

overall_design: Primary B cell progenitor and precursor populations, including Hardy Fraction A, Fraction B, Fraction C, Fraction C', and Fraction D, were sorted by flow cytometry from pooled mouse bone marrow cells of each experimental group.

first: Y

first: A

last: Hoffmann


title: PROS1 expression is regulated by BAP1 in uveal melanocytes and melanomas.

city: Miami

first: Christopher

middle: Joseph

last: Kaler

state: FL

address: Biomedical Research Building 1501 NW 10th AVE

department: Ophthalmology

institute: University of Miami

summary: To investigate if knockdown of BAP1 result in an accumulation of the activation mark H3K27ac at the PROS1 locus. Uveal melanoma is a highly metastatic cancer of the eye which is notoriously resistant to therapy. Elucidating the mechanisms of metastasis in order to devise effective therapies has been a major challenge. The strongest genetic risk factor for metastasis in uveal melanoma is the mutational inactivation of the BAP1 tumor-suppressor gene. However, it remains unknown how BAP1 loss promotes tumor progression. Here, we show that BAP1 loss leads to increased expression of PROS1 in uveal melanocytes and melanoma cells, which in turn leads to phosphorylation and activation of the receptor tyrosine kinase MERTK on adjacent macrophages, driving them into a suppressive M2-polarized state. This mechanism could help explain the suppressive tumor immune microenvironment that is characteristic of BAP1-mutant uveal melanomas, and it suggests that BAP1 loss may lead to metastasis at least in part by facilitating immune escape. These findings provide new insights into the role of BAP1 in uveal melanoma, and they nominate new strategies for increasing the efficacy of immunotherapy in this cancer.

overall_design: Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) was performed using 20 million cells per experiment, which were crosslinked for 7 minutes with 1% formaldehyde. Chromatin was sonicated to an average fragment size of 200-500 base pairs with a Covaris M220 sonicator. 10 μg of antibody was used for each ChIP experiment. For experimental uveal melanoma cell line Mel202, shRNA vectors targeting CGTCCGTGATTGATGATGATA, CCCTGTATATGGATTTATCTT, and CCACAACTACGATGAGTTCAT of human BAP1 cDNA (shBAP1) were individually cloned into a pLKO-TET-puro vector (Addgene #21915) and packaged into lentiviral particles by transient transfection into H293T cells.  Mel202cells were then transduced with shBAP1-containing lentiviral particles and selected with puromycin (2 μg/mL) 48 hours after initial transduction. Cells were clonally selected for optimal doxycycline-inducible BAP1 knockdown (>85% knockdown) and expanded for use in this study. Cells were grown to <80% confluency and induced with 1 μg/mL of doxycycline for 5 days.

first: Kurtenbach

last: Stefan


title: miR-375 Regulates Intestinal Crypt Cell Landscape, Proliferation, and Regenerative Capacity

city: Ithica

first: Praveen

last: Sethupathy

address: Cornell University College of Veterinary Medicine, Box 17

institute: Cornell University

summary: The role of miRNAs in small intestinal epithelial homeostasis is under-explored.  In this study, we discovered that miR-375 is among the most significantly enriched miRNAs in crypts, and in both slowly- and actively-cycling intestinal stem cells (ISCs). We then showed by multiple manipulations, including CRISPR/Cas9 editing, of mouse enteroids that miR-375 is strongly suppressed by Wnt signaling.  Single cell RNA-seq analysis of jejunal crypt cells from miR-375 knockout (375-KO) mice revealed elevated numbers of tuft cells and increased expression of Wnt-pathway genes in ISCs.  In accordance with these findings, 375-KO mice displayed significant improvement in the clearance of helminth infection and in the regenerative response to whole-body irradiation.  The effects of miR-375 on gut epithelial survival and proliferation were further confirmed by ex vivo perturbations in murine enteroids and in vivo over-expression experiments in Drosophila stem/progenitor cells. Functional studies in enteroids revealed that suppression of miR-375 partially compensates for the effects of Yap1 inhibition on cell survival.  Consistent with these results, we also find that miR-375 is significantly reduced in mouse and human intestinal tumors.  Taken together, this study demonstrates that miR-375 is a master regulator of intestinal epithelial function, proliferation, and regenerative capacity.

overall_design: single-cell RNA-seq of WT and 375-KO intestinal cells, miRNA-seq and mRNA-seq from sorted or dissected intestinal cell types including from mice on specific diets or conventional or germ free mice.

first: Matt

last: Kanke


title: Widespread transcriptional memory shapes heritable states and functional heterogeneity in cancer and stem cells

overall_design: This study used lineage-resolved single-cell RNA-seq with lentivirus barcodes, bulk RNA-seq data and bulk RRBS data and NGS of lineage barcode in cell populations to study transcriptional memory.


title: Heterogenous microglial reactivity contrasts with stable vascular transcriptional programs in mouse models of Alzheimer’s, CADASIL, and Traumatic Brain Injury (Spatial data)

city: Stockholm

first: Michael

last: Vanlandewijck

address: Blickagången 6

department: Department of Medicine

country: Sweden

institute: Karolinska Institute

summary: The extent to which the cerebrovasculature is affected in various brain disorders is still not well understood. To address this, we established a transcriptomic repository of major vascular cell types and microglia to compare the global transcriptomic response in mouse models of three human brain disorders linked to neuroinflammation and associated vascular reactivity: Alzheimer’s disease (AD), traumatic brain injury (TBI), and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Single-cell analysis of >250,000 cells at different disease stages led to identification of two previously unknown vascular cell subtypes, expanded the endothelial zonation spectrum and allowed for a detailed analysis of the cellular and molecular responses. Surprisingly, most vascular cell types lacked major transcriptomic changes across the three conditions, while microglia exhibited significant, disease-specific transcriptional changes. Notably, microglial responses converged between late-stage TBI and AD, offering new insights into the predisposition for neurodegeneration following TBI.

overall_design: Traumatic Brain Injury (TBI) was performed by controlled cortical injury (CCI). In brief, the mice were anesthetized with intraperitoneal anesthesia ketamine and medetomidine 10mL/g (solution of 10mg/mL (Ketador Vet, Orion Pharma, Espoo, Finland) + 0.05mg/mL (Domitor, Orion Pharma, Espoo, Finland)) and buprenorphine (Temgesic, Schering-Plough Kenilworth, NJ, USA) (0.05 mg/kg) subcutaneously for analgesia, and attached to a stereotaxic frame. The heads were clipped, and local anesthesia (0.10 mL of bupivacaine ((Marcaine, Pfizer, New York, NY, USA) 0.25%) was provided prior to a midline incision and microsurgical dissection to expose the skull. A 3x4 mm bone flap was removed above the right parietal lobe and the animals were exposed to a 2 mm deep impact by a 3 mm in diameter piston. The skin was sutured, and the mice were placed on a heating pad until fully awake. As controls, sham surgery was performed which included all steps except the impact. Tissue was collected at 3 days or 28 days after CCI.

first: Katrine

last: Dahl Bjørnholm

first: Hao

last: Li

first: Francesca

last: Del Gaudio

first: Giuseppe

last: Mocci

first: Wenchao

last: Shao

first: Elena

last: Baldisseri

first: Shreyas

last: Rao

first: Caroline

last: Lindblad

first: Alexander

last: Fletcher-Sandersjöö

first: Elisa

last: Vázquez-Liébanas

first: Riikka

last: Pietilä

first: Lars

last: Muhl

first: Richeng

last: Jiang

first: Chrysoula

last: Kalantzi

first: Julie

last: Cheung

first: Chao-bo

last: Jin

first: Mikael

last: Svensson

first: Syvänen

last: Stina

first: Saskia

last: Lesnik Oberstein

first: Maarja

last: Andaloussi Mäe

first: Reidun

last: Torp

first: Urban

last: Lendahl

first: Helena

last: Karlström

first: Eric

middle: P

last: Thelin

first: Per

last: Nilsson


title: MURV-seq reveals the genome-wide diversity of 5’ termini and leader sequences across poxvirus transcripts

city: Chicago

first: Chorong

last: Park

address: 303 E Chicago Ave

institute: Northwestern Univeristy

summary: Viral RNA polymerases (vRNAPs) can slip on homopolymeric sequences to expand their coding potential in diverse ways. This includes poxviruses, whose vRNAP slips at thymidine-rich motifs to generate non-templated polyadenosine (polyA) leaders on certain transcripts. However, we lack a comprehensive genome-wide understanding of poxviral 5’ leaders and their capping status. Here, we developed Mapping 5′ Untranslated Regions of Viral transcripts (MURV-seq), a customized transcript capture, sequencing and data analysis strategy that distinguishes 5′ triphosphate (5′ppp) versus 7-methylguanosine (m7G) termini and provides high-resolution sequences for the leaders of all annotated poxvirus open reading frames (ORFs). MURV-seq reveals extensive diversity and temporal shifts in the use of templated and non-templated leaders by individual genes. We further define three distinct polyA leader types and refine our understanding of variance in their lengths both between kinetic classes and across individual genes. Contrary to recent suggestions that long polyA leaders generally lack canonical caps, we find that this only occurs on a small fraction of transcripts while all viral transcript types predominantly retain m7G caps. Moreover, while many early genes utilize templated leaders, as expected, transcription upstream of canonical vRNAP slippage sites produces alternative templated leaders for many intermediate and late ORFs. Computational analysis suggests that some of these harbor short upstream ORFs that likely limit their use as 5’ untranslated regions (UTRs), while others are predicted to be functional for the primary ORF or in producing N-terminally extended isoforms. Combined, MURV-seq provides a genome-wide framework for understanding poxvirus transcription start sites and leader diversity.

overall_design: Twelve 5′ end–enriched RNA-seq libraries were generated from HAP1 cells under mock or Vaccinia virus (VacV) infection conditions, including three biological replicates of mock-treated cells at 24 h (RppH), four replicates of VacV-infected cells at 24 h (RppH), three replicates at 5 h post-infection (RppH), and two replicates of VacV-infected cells at 24 h enriched for 5′ triphosphorylated transcripts (5′ppp).  Libraries were prepared following rRNA depletion, spike-in normalization, selective enrichment of capped or triphosphorylated RNAs via enzymatic treatments (RppH or polyphosphatase), and sequential adapter ligation to capture and distinguish 5′ end structures.

first: Hannah

last: Serio

first: Daniel

last: Arango

first: Derek

last: Walsh


title: Disordered protein LAT encodes relative levels of signaling pathways in T cell activation [Perturb-CITE-seq]

city: Cambridge

first: Adam

last: Rubin

state: Massachusetts

address: 415 Main St.

institute: Broad Institute

summary: Immune cells transduce environmental stimuli into responses essential for host health via complex signaling cascades. T cells, in particular, leverage their unique T cell receptors (TCRs) to detect specific Human Leukocyte Antigen (HLA)-presented peptides. TCR activation is then relayed via linker for activation of T cells (LAT), a TCR-proximal disordered adapter protein, which organizes protein partners and mediates the propagation of signals down diverse pathways including NFAT and AP-1. Here, we studied how balanced downstream pathway activation is encoded in the amino acid sequence of LAT. To comprehensively profile the sequence-function relationship of LAT, we developed a pooled, single-cell, high-content screening approach in which a large series of mutants in the LAT protein were analyzed to characterize their effects on T cell activation. Measuring epigenetic, transcriptomic, and cell surface protein dynamics of single cells harboring distinct LAT mutants, we found functional regions spanning over 40% of the LAT amino acid sequence. Conserved sequence motifs for protein interactions, along with charge distribution, are critical sequence features, and contribute to interpretation of human genetic variation in LAT. While mutant defect severity spans from moderate to complete loss of function, nearly all defective mutants, irrespective of their position in LAT, confer balanced defects across the NFAT and AP-1 pathways. To understand the molecular basis for this observation, we performed proximal protein labeling, which demonstrated that disruption of LAT interaction with a single partner protein indirectly disrupts other partner interactions, likely through the dual roles of these proteins as effectors of downstream pathways and bridging factors between LAT molecules. Overall, we report widely distributed functional regions throughout a disordered adapter and a precise physical organization of LAT and interacting molecules which constrains signaling outputs. More broadly, we describe an approach for interrogating sequence-function relationships for proteins with complex activities across regulatory layers of the cell.

overall_design: Jurkat or human primary CD4 T cells were stimulated through their T cell receptor and subjected to single-cell ATAC-seq or CITE-seq. These protocols were modified for the detection genetic barcodes identifying the open reading frame (ORF) delivered to each cell by lentivirus.

first: Alex

middle: K

last: Shalek


title: Disordered protein LAT encodes relative levels of signaling pathways in T cell activation [Perturb-ATAC-seq]


title: Transciptional effects of coronary vessel loss on epicaridal cells in juvenile zebrafish

city: New York

first: Michael

last: Harrison

state: NY

institute: Weill Cornell Medicine

summary: During cardiac development, the myocardium expands in response to physiological demands to achieve proper cardiac morphology and functional contractility, while simultaneously integrating with the developing coronary vasculature. However, the mechanisms governing this ordered expansion and the role of coronary vasculature formation in this process remain poorly understood. Here, we found that regional hypoxia drives local tissue thickening, which in turn exacerbates a hypoxic microenvironment. We demonstrate that epicardial hypoxia serves as a central regulatory mechanism, coordinating both coronary angiogenesis and myocardial expansion during juvenile zebrafish heart development. This mechanism activates discrete spatial patterns of epicardial gene expression, including for vegfaa, loxl2a, and col12a1b. Through live and fixed imaging, we find that cardiomyocytes and endothelial cells exhibit coordinated migration patterns through third-party epicardial signals. We further demonstrate that epicardial subpopulations are required for both coronary development and myocardial expansion. Using cxcr4aum20 mutants lacking functional coronary vessels, we show that coronary vessels function as a negative feedback mechanism to epicardial hypoxia, while positively responding to the same hypoxic cues that drive myocardial expansion. Importantly, disruption of this negative feedback on epicardial hypoxia also leads to increased myocardial stiffness through dysregulated extracellular matrix crosslinking. Thus, we provide mechanistic insights into pathological conditions characterized by myocardial stiffness such as cardiomyopathies. These findings establish the role of regional epicardial hypoxia within a fundamental regulatory network that drives appropriate regional tissue growth with integrated vascular supply during cardiac morphogenesis.

overall_design: Ventricles were isolated from Tg(tcf21:nEGFP) zebrafish at 70 days post-fertilization (dpf), with similar body size. Cell dissociation was performed. For isolation of epicardial cells, tcf21:nEGFP-positive cells were sorted using a MA900 cell sorter (Sony). For total RNA extraction, sorted cells were immediately lysed in RLT buffer from the RNeasy® Micro Kit (Qiagen), and RNA was purified according to the manufacturer’s protocol. Purified RNA was submitted to the Genomics Core Facility of Weill Cornell Medicine for bulk RNA-seq library preparation using SMART-Seq v4 Ultra Low Input RNA plus Nextera XT DNA Sample Preparation. Libraries were sequenced on an Illumina NovaSeq X Plus using a paired-end flow cell with 2 × 100 cycles.


title: Overlapping roles of Irx3 and Irx4 in the compaction of the ventricular myocardium

city: Ottawa

first: Kyoung-Han

last: Kim

state: Ontario

address: 40 Ruskin Street

institute: University of Ottawa Heart Institute

summary: To determine Irx3 and Irx4 regulatory pathways during heart compaction, RNA-seq analyses on WT, Irx3KO, Irx4KO, and Irx3;Irx4DKO mutants hearts were performed to determine misregulated pathways upon the loss of these transcription factors. Our analyses revealed that there are overlapping functions between Irx3 and Irx4, and that both Irx3 and Irx4 are required for the regulation of the BMP/TGFβ signalling pathway known to be involved in endocardial cushion formation.

overall_design: RNA-Seq of E14.5 WT, Irx3KO, Irx4KO, Irx3+/-;Irx4+/-; and Irx3;Irx4DKO ventricles.

first: Weifan

last: Liu

first: Wen-Chi

last: Yin


title: Disordered protein LAT encodes relative levels of signaling pathways in T cell activation [ATAC-seq]

overall_design: Jurkat T cells were stimulated through their T cell receptor for various durations and subjected to bulk ATAC-seq to monitor dynamics of chromatin accessibility genome-wide.


title: Sustained MYB activity drives emergent enhancer activation and precise enhancer-promoter interactions [MCC]

city: Oxford

first: I-Jun

last: Lau

address: Old Rd

department: Botnar Research Centre

country: United Kingdom

institute: University of Oxford

summary: Transcription factors (TFs) are key effectors of enhancer activity. MYB is a critical hematopoietic TF that is frequently dysregulated in cancer. Despite its well-established role, the exact mechanisms by which MYB influences enhancer function—and the specific stages of enhancer activation at which it operates—remain poorly understood. Using high resolution Micro-Capture-C, we show that upon MYB degradation, highly defined enhancer-promoter interactions at specific MYB binding sites are lost. Loss of these interactions, together with other hallmarks of enhancer activity—reduced H3 lysine-27 acetylation and enhancer RNA transcription—correlates with significant downregulation of target gene expression in leukemia, indicating that MYB mediates transcription activation via maintenance of enhancer function. When anchored to DNA within a gene desert region that is devoid of histone marks and active transcription, the MYB transactivation domain is sufficient and necessary for the nucleation of an enhancer-like region. This results in the activation of transcription from distal cryptic elements and the establishment of long-range chromatin interactions up to 400 kb away from the anchor point. Together, these results indicate that MYB activity alone is sufficient to induce long-range interactions and transcription, achieving this through highly precise enhancer-promoter crosstalk.

overall_design: Micrococal-Capture-C (MCC) in SEM cells with MYB endogenously tagged with FKBP12(F36V) ±dTAG-13 treatment.

first: Nicholas

middle: T

last: Crump

first: Thomas

last: Milne


title: Dynamics of glioma-associated microglia and macrophages reveals their divergent roles in the immune response of brain

city: Shanghai

first: Jiawen

last: Qian

address: 130 Dong'an Road

department: Immunology

institute: Fudan University

summary: Glioma microenvironment contains numerous myeloid cells, including brain-resident microglia and recruited monocytes and macrophages (Mo/Mφ). Previous researches studied these cells collectively and revealed their pro-tumor effects. Yet, little is known about the differences among these myeloid populations. Here we revealed the phenotypic characteristics, spatial variances, and dynamic changes of these relatively heterogeneous cell populations by single-cell sequencing analysis. Microglia were pro-inflammatory transiently, and soon skewed to restore the homeostasis of the central nervous system (CNS). Notably, a subset of glioma-associated microglia shared similar trait in a diverse spectrum of neuropathogenesis. Mo/Mφ, on the other hand, were responsible for various tumor-promoting effects, such as angiogenesis, tumor invasion, and immune evasion. Moreover, the Mo/Mφ subsets presented prognostic and classificatory value in clinical application. Conclusively, these results eliminate the long-existing ambiguity about the role of microglia and Mo/Mφ in glioma pathogenesis, and reveal their prognostic and therapeutic value for glioma patients.

overall_design: scRNA-seq profiles for the CD45hi/loCD11b+ cells isolated from the tumor-bearing hemispheres of the GL261 glioma model at day 10, day 20, and terminal stage.

first: Chen

last: Wang


title: Heterogenous microglial reactivity contrasts with stable vascular transcriptional programs in mouse models of Alzheimer’s, CADASIL, and Traumatic Brain Injury (ArcSwe data)

last: Vanlandewijck

summary: The extent to which the cerebrovasculature is affected in various brain disorders is still not well understood. To address this, we established a transcriptomic repository of major vascular cell types and microglia to compare the transcriptomic response in  mouse models of three human brain disorders linked to neuroinflammation and associated vascular reactivity: Alzheimer’s disease (AD), traumatic brain injury (TBI), and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Single-cell analysis of >250,000 cells at different disease stages led to identification of two previously unknown vascular cell subtypes, expanded the endothelial zonation spectrum and allowed for a detailed analysis of the molecular responses in the vascular cells and microglia. Surprisingly, all vascular cell types remained transcriptomically normal across the three conditions, while microglia exhibited significant, disease-specific transcriptional changes. Notably, microglial responses converged between late-stage TBI and AD, offering new insights into the predisposition for neurodegeneration following TBI.

overall_design: Using a microvascular isolation protocol (PMID 37056377), single cell suspension of adult murine brain vasculature were extracted. The following mouse models were used: An Alzheimer mouse model (tg App ArcSwe) and C57Bl6 control mice

first: Katrine

last: Dahl Bjørnholm

first: Hao

last: Li

first: Francesca

last: Del Gaudio

first: Giuseppe

last: Mocci

first: Wenchao

last: Shao

first: Elena

last: Baldisseri

first: Shreyas

last: Rao

first: Caroline

last: Lindblad

first: Alexander

last: Fletcher-Sandersjöö

first: Elisa

last: Vázquez-Liébanas

first: Riikka

last: Pietilä

first: Lars

last: Muhl

first: Richeng

last: Jiang

first: Chrysoula

last: Kalantzi

first: Julie

last: Cheung

first: Chao-bo

last: Jin

first: Mikael

last: Svensson

first: Syvänen

last: Stina

first: Saskia

last: Lesnik Oberstein

first: Maarja

last: Andaloussi Mäe

first: Reidun

last: Torp

first: Urban

last: Lendahl

first: Helena

last: Karlström

first: Eric

last: Thelin


title: Inhibition of METTL3 by STC-15 induces RNA misprocessing that results in dsRNA formation and activates innate immunity

first: Harry

last: Fischl

address: Babraham Research Campus

institute: Storm Therapeutics

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Proteomic screening identifies HNRNPA2B1 as a novel factor that controls Epstein-Barr virus reactivation through chromatin regulation

city: Pittsburgh

first: Febri

last: Sugiokto

state: Pennsylvania

address: 5117 Centre Avenue

institute: University of Pittsburgh

summary: Epstein-Barr virus (EBV) establishes lifelong persistent infection in most human populations. The virus persists as an episome in the host cells during latency and periodically reactivates through transcriptional activation of the immediate-early genes. While epigenetic regulation is central to maintaining viral latency, the host factors that enforce repression at these promoters remain incompletely defined. Here, we employed a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation coupled with mass spectrometry (enChIP-MS) approach to identify proteins associated with the promoter of EBV immediate-early (IE) gene ZTA. This approach revealed an enrichment of multiple heterogeneous nuclear ribonucleoproteins and identified HNRNPA2B1 as a potential regulator of EBV ZTA gene expression. Functional analyses across multiple EBV+ cell models demonstrated that HNRNPA2B1 acts as a restriction factor for EBV lytic reactivation. Depletion of HNRNPA2B1 led to increased expression of IE and downstream lytic genes, enhanced RNA polymerase II recruitment to the ZTA and RTA promoters, and elevated the proportion of cells entering the lytic cycle. Conversely, enforced expression of HNRNPA2B1 suppressed EBV lytic reactivation. Mechanistically, HNRNPA2B1 enhances repressive viral chromatin states by facilitating recruitment of the histone demethylase KDM1A to EBV IE gene promoters, thereby limiting the activating histone H3K4 trimethylation. Together, these findings identify HNRNPA2B1 as a key epigenetic regulator of EBV latency and link RNA-binding proteins to chromatin control of viral reactivation.

overall_design: RNA-seq profiling of wiltype HNRNPA2B1 and depleted HNRNPA2B1 in Akata (EBV+) cell line

first: Sugiokto

last: Febri Gunawan

first: Li

last: Renfeng


title: Prenatal cell-free DNA methylome detects association with autism and maternal obesity

city: Davis

first: Logan

middle: Allan

last: Williams

address: 1275 Med Sci Dr

department: Medical Microbiology and Immunology

institute: University of California, Davis

summary: Early identification of autism spectrum disorder (ASD) remains a critical challenge, particularly in utero when non-genetic factors such as maternal obesity (MO) are implicated. Here, we report results of whole-genome bisulfite sequencing of cell-free DNA (cfDNA) from third-trimester maternal plasma in a high-likelihood ASD pregnancy cohort associated with child (3 y) ASD diagnosis and/or MO. Differentially methylated regions (DMRs) between ASD and control cfDNA were strongly enriched for synaptic functions and  genes previously implicated in ASD. These cfDNA ASD DMRs recapitulated those observed in ASD placenta and postmortem cortex and significantly overlapped with MO DMRs. Our findings establish cfDNA methylation derived from maternal blood as a minimally invasive window into fetal brain ASD etiology, providing a framework for future mechanistic and early intervention studies. Future studies could investigate additional prenatal environmental exposures interacting with genetics during neurodevelopment.

overall_design: CfDNA from 51 pregnant mothers (48 reported here) in a prospective ASD cohort was isolated and subjected to whole-genome bisulfite sequencing. Variable child autism diagnosis outcomes and variable maternal body-mass indexes allowed for the identification of differentially methylated regions in cfDNA at late third trimester associated with ASD and/or MO.

first: Ensieh

last: Habibi

first: Meghan

last: Miller

first: Irva

last: Hertz-Picciotto

first: Cheryl

last: Walker

first: Rebecca

last: Schmidt

first: Janine

last: LaSalle


title: Single-phage profiling illuminates viral individuality during cell fate determination

city: Urbana

first: Ido

last: Golding

state: IL

address: 1110 W Green St.

department: Physics

institute: University of Illinois Urbana-Champaign

summary: The choice between cell death (lysis) and viral dormancy (lysogeny) following bacteriophage infection serves as a founding paradigm for the emergence of cellular heterogeneity in a genetically uniform population. The determination of host fate arises through the stochastic transcription from multiple viral genomes present within each cell, but this activity remains hidden from empirical interrogation, which typically stops at the whole-cell level. Here we use parallel sequential fluorescence in situ hybridization (par-seqFISH), followed by spatial clustering of phage-encoded transcripts within each cell, to profile the transcriptional activity of individual phages during synchronized infection of Escherichia coli (E. coli) by bacteriophage lambda. At the whole-cell level, transcription kinetics capture the developmental choice between lysis and lysogeny, and further demonstrate that viral replication is required for the emergence of diverging fate decisions. Zooming in to the single-phage level illuminates an individuality of viral activity during infection. We find that, while cells pursuing lysogeny display consensus activity of all in-habiting phages, lytic cells may contain phages that exhibit lysogenic activity. These findings support an earlier suggestion that consensus among coinfecting phages is required for cell dormancy. More broadly, our results highlight the need to identify how whole-cell behavior emerges from the activity of physically distinct copies of the same genetic circuit.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Total RNA-seq was performed on E. coli MG1655 wild-type cells infected with phage lambda at high or low multiplicity of infection (MOI), or mock-infected with buffer, at multiple time points post-infection. As controls, libraries were similarly generated from uninfected MG1655 host cells and lambda lysogens. Two biological replicates were done for each condition.

first: Tianyou

last: Yao

first: Yuncong

last: Geng

first: Ehsan

last: Homaee


title: Lodiculeless Spikelet is a Mutation of MicroRNA172b Controlling Cleistogamy in Rice

city: Chuncheon

first: Dong-Hoon

last: Jeong

state: Gangwon-do

address: Room #8306, Department of Life Science, 1 Hallymdaehak-gil

department: Dept. of Life Science

institute: Hallym University

summary: Cleistogamy is a useful agronomic trait to prevent gene flow in transgenic crops and to maintain genetic purity in autogamous crops including rice. Previously, we isolated a natural cleistogamous rice mutant, lodiculeless spikelet (ld). Map-based cloning approach identified about 4 kb deletion spanning two microRNAs, the entire MIR806a precursor and upstream of MIR172b precursor, in the ld locus. CRISPR/Cas9-mediated transgenic approach validated that mutations of MIR172b, not MIR806a, was responsible for the cleistogamy phenotype. Small RNA sequencing revealed that the expression of miR172b was almost completely reduced in the ld mutants. Quantitative RT-PCR analysis indicated that expression of AP2 target genes of miR172b was not changed in the ld mutants. Finally, we discussed possible regulatory mechanisms on how the defect of MIR172b causes cleistogamy in rice. This is the first report that mutation of a MIR gene causes cleistogamy in rice.

overall_design: Examinination of miRNA expression patterns in rice ld mutants

first: Jong-Seong

last: Jeon

first: Hee-Jong

last: Koh

first: Rihua

last: Piao

first: Sang-Kyu

last: Lee

first: So

middle: Y

last: Park


title: Heterogenous microglial reactivity contrasts with stable vascular transcriptional programs in mouse models of Alzheimer’s, CADASIL, and Traumatic Brain Injury (Main dataset)

first: Michael

last: Vanlandewijck

overall_design: Using a microvascular isolation protocol (PMID 37056377), single cell suspension of adult murine brain vasculature were extracted. The following mouse models were used: An Alzheimer mouse model (App-NLGF knock-in), a CADASIL mouse model (Tg-N3-R182C) and a control cortical impact model for traumatic brain injury

first: Katrine

last: Dahl Bjørnholm

first: Hao

last: Li

first: Francesca

last: Del Gaudio

first: Giuseppe

last: Mocci

first: Wenchao

last: Shao

first: Elena

last: Baldisseri

first: Shreyas

last: Rao

first: Caroline

last: Lindblad

first: Alexander

last: Fletcher-Sandersjöö

first: Elisa

last: Vázquez-Liébanas

first: Riikka

last: Pietilä

first: Lars

last: Muhl

first: Richeng

last: Jiang

first: Chrysoula

last: Kalantzi

first: Julie

last: Cheung

first: Chao-bo

last: Jin

first: Mikael

last: Svensson

first: Saskia

last: Lesnik Oberstein

first: Maarja

last: Andaloussi Mäe

first: Reidun

last: Torp

first: Urban

last: Lendahl

first: Helena

last: Karlström

first: Eric

last: Thelin

first: Per

last: Nilsson


title: Widespread transcriptional memory shapes heritable states and functional heterogeneity in cancer and stem cells [RNA-Seq]

first: Yongjie

last: Lin

institute: Peking University

summary: Recent studies show that non-genetic heterogeneity, particularly through heritable cell states, shapes cancer evolution and developmental trajectories. However, single-cell snapshots lack temporal information to identify these states. We employ lineage-resolved single-cell transcriptomics to map heritable cell states that persist across divisions, distinguishing them from transient fluctuations. We uncover that heritable states are underpinned by widespread transcriptional memory, whereby heritable gene expression defines two classes of states: clustered states, characterized by clustered gene expression, and latent states, marked by non-clustered gene expression. This memory shows conservation across cell types and conditions and appears to be maintained by robust epigenetic mechanisms resistant to environmental perturbations. Functionally, memory genes predict critical behaviors including metastatic potential and lineage commitment, with latent-state genes often outperforming clustered-state genes. Our findings establish transcriptional memory as a potential basis of heritable cellular heterogeneity, providing a framework for understanding functional cellular variations across biological systems. A record of this paper’s transparent peer review process is included in the supplemental information.

first: Xiangru

last: Chen

first: Long

last: Wu

first: Yutong

last: Zhou

first: Yihan


title: Microglial TDP-43 mediates myelin refinement and represses Tyrobp cryptic exon inclusion in mice [RNA-seq]

city: Berlin

first: Andranik

last: Ivanov

address: Chariteplatz 1

department: Core Unit Bioinformatics

institute: Berlin Institute of Health

summary: TDP-43 proteinopathy is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis and frontotemporal dementia where mislocalization of TDP-43 has been observed in neurons and glial cells. However, the role of TDP-43 in microglia and the consequences of its loss-of-function remain unexplored. Combining magnetic resonance imaging, confocal, and electron microscopy we uncovered structural changes and myelin abnormalities in the early postnatal brain of mice lacking microglial TDP-43. Spatial transcriptomics further revealed an enriched interferon-response signature associated with oligodendrocyte dysfunction. Early microglial TDP-43 depletion resulted in motor deficits in adult mice. Mechanistically, knocking out TDP-43 impaired microglial ability to engulf and degrade myelin. It also led to cryptic exon inclusion in the Tyrobp mRNA, resulting in truncated DAP12 protein, thus causing defective TREM2 signaling. Our findings reveal a novel role for TDP-43 in regulating the TREM2-DAP12 axis in mice, highlighting a previously unrecognized mechanism by which TDP-43 controls microglial function.

overall_design: To investigate the role of microglial TDP-43 in brain development, we generated an inducible cKO mouse line by crossing Cx3cr1-CreERT2 and Tardbp-floxed strains, which also express Rosa26-LSL-TdTomato. We perfomed Bulk RNA sequencing from FACS isolation of microglial cell from P15 mice Control (WT 3 mice) or KO (5 mice) for TDP-43. Comparative gene expression of RNA-seq data for Control and cKO microglia was analysed.

first: Anne-Claire

last: Compagnion

first: Anil

last: Rana

first: Felipe

last: Espinoza

first: Thomas

last: Sandmann

first: Fanny

last: Martineau

first: Katia

last: Monsorno

first: Robera

last: Facchineti

first: Alessandro

last: Matera

first: Lionel

last: Rougé

first: Fernando

last: Ibáñez

first: Clarissa

last: Catale

first: Mateo

last: Bizzotto

first: Sonia

last: Garel

first: Michela

last: Matteoli

first: Yutaro

last: Kashiwagi

first: Ryuta

last: Koyama

first: Christian

last: Haass

first: Marie-Eve

last: Tremblay

first: Dieter

last: Beule

first: Ileana

last: Jelescu

first: Valerio

last: Zerbi

first: Gilbert

last: Di Paolo

first: Rosa

middle: C

last: Paoliceli


title: Single-cell RNA sequencing of differentiated human iPSC organoid-derived kidney tubuloids (iPSCod tubuloids)

city: Utrecht

first: Carla

last: Pou Casellas

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting ‘iPSC organoid-derived (iPSCod)’ tubuloids can be exponentially expanded for at least 2.5 months, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.

overall_design: scRNA-seq of FAF-differentiated human iPSCod kidney tubuloids (1 sample from a bulk tubuloid culture)

first: Fjodor

last: Yousef Yengej

first: Carola

last: Ammerlaan

first: Maarten

middle: B

last: Rookmaaker

first: Marianne

last: Verhaar

first: Hans

last: Clevers


title: Metabolically and epigenetically reprogrammed splenic TRNP1hiCD8+ T cells exacerbate liver fibrosis

first: Liyuan

last: Zhang

state: China

address: Xiangyin road

department: National Key Laboratory of Medical Immunology & Institute of Immunology

institute: Naval medical university

summary: MASLD, especially its severe form MASH, progresses to liver fibrosis, leading to cirrhosis and liver cancer. The cross-talk between spleen and liver in MASLD/MASH progression remains poorly understood. Here, we find the enlarged spleens in MASLD patients, and the induced TRNP1hiCD8+ T cells in the spleens of MASLD/MASH mouse models and confirmed in MASLD patients, with pro-fibrotic property by secreting INSR-α. Mechanistically, demethylated DNA and H3K27me3, increased H3K27ac, together with bolstered enhancer-promoter contact, synergistically reorganized chromatin topologically associating domains spatially to initiate the expression of transcription factor TRNP1 in splenic CD8+ T cells. TRNP1 then transcriptionally activates the expression of FURIN and CTSD, promoting the maturation and ectodomain shedding of INSR-α thereby facilitating its secretion to activate hepatic stellate cells. In vivo blockade of INSR-α using neutralizing antibodies amelioratesalleviates MASLD/MASH-induced liver fibrosis. Therefore, this study reveals splenic TRNP1hiCD8+ T cells with pro-fibrotic property and suggests a potential anti-fibrotic strategy.

overall_design: ATAC-seq profiling of splenic CD8+ T cells from ND or CDAHFD (7 weeks) induced MASH mice models.

first: Yahui

last: Wang

first: Kai

last: Wei

first: Wen

last: Nie

first: Yayuan

last: Feng

first: Zhiwen

last: Shi

first: Jin

last: Hou


title: Loss of Hepatocyte PI3Ka Protects Mice from Hepatocellular Carcinoma in association with altered lipid metabolism gene expression

city: Gothenburg

first: Bart

last: Edelbroek

address: Medicinaregatan 3B

department: Bioinformatics and Data Centre, Core Facilities

institute: University of Gothenburg

summary: Hepatocellular Carcinoma (HCC) is a disease with an increasing incidence and a high mortality rate; therefore, targeted therapies for HCC are urgently needed. PI3K-AKT-mTORC1 signaling is frequently induced in solid tumors and is associated with tumor progression and with the most aggressive type of HCC. However, complete inhibition of all PI3K isoforms is unlikely to achieve a favorable therapeutic index for HCC treatment due to on-target side effects. Here, we investigate the role of hepatocyte PI3Ka in HCC using conditional knockout mice. We found that PI3Ka in the hepatocyte is dispensable for AKT phosphorylation in HCC and in normal liver, and hepatocytes during compensatory proliferation following administration of the hepatocarcinogen. We have also found that AKT phosphorylation induced by hepatocyte growth factor (HGF) and epidermal growth factor (EGF) is mediated by redundant PI3Ka and PI3Kb activities. Nonetheless, mice lacking hepatocyte PI3Ka showed reduced HCC proliferation and reduced hepatocyte proliferation induced by DEN. This phenotype was associated with a gene expression signature indicating altered lipid metabolism and reduced formation of lipid droplets. Together, these results indicate that PI3Ka is a promising target for the treatment of HCC.

overall_design: mRNA-seq of PI3Ka LoxP floxed mice (PI3Ka F/F) and mice specifically lacking PI3Ka in the hepatocyte (PI3Ka HEP). The mice were injected with 25 mg/kg of N-nitrosodiethylamine (DEN) at 2 weeks old, fed either standard chow diet (Chow) or high fat diet (HFD; 60% of calories from fat) from week 6, and sacrificed at 36 weeks of age. Sequencing was performed either of the non-tumor liver tissue or of Hepatocellular carcinoma tissue (HCC).

first: Barbara

last: Becattini

first: Claudia

last: Sardi

first: Amit

last: Chand Gupta

first: Toshima

last: Parris

first: Khalil

last: Helou

first: Giovanni

last: Solinas


title: Microglial TDP-43 mediates myelin refinement and represses Tyrobp cryptic exon inclusion in mice [Spatial Transcriptomics]

first: Andranik

last: Ivanov

first: Anne-Claire

last: Compagnion

first: Anil

last: Rana

first: Felipe

last: Espinoza

first: Thomas

last: Sandmann

first: Fanny

middle: S

last: Martineau

first: Katia

last: Monsorno

first: Robera

last: Facchineti

first: Alessandro

last: Matera

first: Lionel

last: Rougé

first: Fernando

last: Ibáñez

first: Clarissa

last: Catale

first: Mateo

last: Bizzotto

first: Sonia

last: Garel

first: Michela

last: Matteoli

first: Yutaro

last: Kashiwagi

first: Ryuta

last: Koyama

first: Christian

last: Haass

first: Marie-Eve

last: Tremblay

first: Dieter

last: Beule

first: Ileana

last: Jelescu

first: Valerio

last: Zerbi

first: Gilbert

last: Di Paolo

first: Rosa

last: Paoliceli


title: Targeting XPO1/DDX1-mediated nuclear export of R-loops in inflammaging [CUT&Run]

city: Houston

first: Yuan

last: Qi

state: TX

address: 1400 Pressler St

department: Bioinformatics & Computational Biology

institute: University of Texas M.D. Anderson Cancer Center

summary: R-loops are three-stranded nucleic acids containing DNA-RNA hybrid and a third displaced ssDNA and are widely distributed across the genome. Dysregulation in R-loop dynamics cause DNA damage and genome instability. Although its role has been extensively investigated in various pathological processes, whether R-loops are perturbed during senescence and tissue aging remains unclear. Here we observe a global decline in R-loop stalling across the genome during senescence. While R-loops in nucleus were dramatically decreased, we detected their significant accumulation in the cytoplasm in senescent cells. We identify their source as nuclear R-loops with a specific enrichment in alpha-satellites, a subset of repetitive elements. Notably, these cytoplasmic R-loops localize into cytoplasmic chromatin fragments (CCF) to induce cGAS-STING innate immune response, and this nucleocytoplasmic transport is dependent on exportin 1 (XPO1)/DEAD-Box helicase 1 (DDX1) complex. Targeting R-loop nuclear export by XPO1 inhibitor Selinexor (KPT-330) suppresses the inflammatory response in senescent cells and relieves inflammaging in aged mice. In summary, our findings linked aberrant accumulation of cytoplasmic R-loops to inflammaging, rendering their nucleocytoplasmic transport process an ideal target for suppressing inflammaging.

overall_design: We conducted CUT&RUN method-based MapR-seq in proliferating and senescent IMR90 cells to chracterize R-loop dynamics during senescence.

first: Xue

last: Hao

first: Qingqing

last: Yan

first: Andrew

last: Kossenkov

first: Rugang

last: Zhang