title: mRNA expression data of Mes vs. Epi cells in mouse non-NE SCLC

city: Shanghai

first: yujuan

last: jin

address: Yueyang Road 320#

institute: Shanghai Institutes for Biological Sciences, CAS

summary:
Distinct mRNA populatins have been detected in Epi vs. Mes cells of mouse non-NE SCLC
The aim of the study was to globally profile the differential gene expression in Mes vs. Epi cells of mouse non-NE SCLC.

overall_design: Single clones from mouse non-NE SCLC were picked and expaned to obtain enough cells for further study. Total RNA was extracted and Labeled cRNA were purified by RNeasy mini kit (QIAGEN). Each slide was hybridized with 1.65ug Cy3-labeled cRNA using Gene Expression Kit (Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, US) following the manufacturer’s instructions. Slides were scanned by Agilent Microarray Scanner (Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution = 5 um, PMT 100%, 16 bits. Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US). GSEA was performed following the developer’s protocol (http://www.broad.mit.edu/gsea/).

first: Hongbin

last: Ji

first: Yujuan

last: Jin


title: Selection and simulated climate-warming effects on Brook Charr sperm DNA methylation

status: Public on Mar 11 2024

city: Québec

first: Kyle

last: Wellband

state: QC

address: 1030 avenue de la Médecine

department: Institut de biologie intégrative et des systèmes

country: Canada

institute: Université Laval

type: Methylation profiling by high throughput sequencing

summary: Directional selection in the domestication of fish species has resulted in rapid gains of growth, body size, and other production-relevant traits in relatively few generations. While there is clear evidence of genetic divergence contributing to selection-related phenotypic changes, emerging research suggests that intergenerational epigenetic inheritance may also be a relevant mechanism explaining rapid evolutionary change in domestic fish lines. Epigenetic changes have also been implicated in fish species’ responses to warming associated with climate change. Domestic lines of Brook Charr (Salvelinus fontinalis) are the primary source of fish used for recreational fisheries stocking in many parts of Eastern North America and there are concerns about how these fish will fare when stocked into lakes in the coming decades. We jointly investigated the effects of directional selection for performance traits (i.e., absence of early sexual maturation and increased growth) and exposure to elevated temperatures on DNA methylation in sperm cells of two experimental lines (hereafter: Selected and Control lines) of Brook Charr . We used whole-genome bisulfite sequencing to characterize DNA methylation at over 17 million methylated sites and identified 393 selection-related differentially methylated regions (DMR). The putative functions of genes in proximity to these DMRs are consistent with well-characterized phenotypic differences between the lines, including lipid metabolism and precocial maturation, and support the hypothesis that rapid evolution of traits may be partially mediated by epigenetic inheritance. We subsequently detected 85 warming-related DMRs in the Control line and 302 DMRs in the Selected line. None of these regions were shared between the two lines, indicating that the directional selection regime significantly altered the environmentally sensitive epigenetic landscape.

overall_design: Two lines of Brook Charr were maintained the ISMER for 5-6 generations. The selected line was directionally selected for low precocial maturation (immature at age 2) and then by growth (largest individuals were selected for line propagation). More details on line establishment and maintenance can be found in Bastien et al. (2011) [https://doi.org/10.1080/15222055.2011.544609]. During the period of final gamete maturation (Sept - Dec) each line was maintained in either ambient seasonal temperatures or in a simulated climate warming scenario that tracked the daily and seasonal fluctation, but with temperature consistently elevated by 2 degrees C. Gametes were collected during annual spawning activities and we used whole genome bisulfite sequencing of genomic DNA extracted from male milt (sperm). We compared effects of line (differential selection) and temperature on patterns of DNA methylation in this tissue.

first: Wellband

last: Kyle

first: Bernatchez

last: Louis


title: Epigenetic Control over IFN Type-I Response antagonizes self-renewal in acute myeloid leukemia (ChIP-seq)

city: Münster

first: Carolin

last: Walter

address: Domagkstraße 9

department: Medical Faculty of the WWU Münster

country: Germany

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Identify the genomic sites occupied and genes activated by PHF8 depending on its phosphorylation status (PHF8 AA vs. PHF8 DD).

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for PHF8 in THP1 cells.

first: Mari-Francis

last: Arteaga

first: Jan-Henrik

last: Mikesch


title: LARP4 Is an RNA-Binding Protein That Binds Nuclear-Encoded Mitochondrial mRNAs To Promote Mitochondrial Function

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

country: USA

institute: UCSD

summary: Mitochondrial associated RNA-binding proteins (RBPs) have emerged as key contributors to mitochondrial biogenesis and homeostasis. With few examples described, we set out to identify RBPs that post-transcriptionally regulate nuclear-encoded mitochondrial mRNAs (NEMmRNAs). Our analysis of RNA-target sets of RBPs made available by ENCODE identified several RBPs with a preference for binding NEMmRNAs, of which we selected LARP4, a La RBP family member, for further study. We show that LARP4’s RNA-target set is particularly enriched in mRNAs that encode for respiratory chain complex proteins (RCCPs) and mitochondrial ribosome proteins (MRPs) across multiple human cell lines. CRISPR knockout cells in conjunction with quantitative proteomics show that protein levels of RCCPs and MRPs are significantly reduced upon loss of LARP4. Furthermore, we show that LARP4 depletion reduces mitochondrial function, and this phenotype is ameliorated by LARP4 re-expression. Our findings shed light onto a novel function for LARP4 as an RBP that binds to NEMmRNAs to promote mitochondrial respiratory function.

overall_design: Identification of LARP4 targets using eCLIP-seq in HEK293 cell line

first: Benjamin

middle: M

last: Lewis

first: Chae

middle: Y

last: Cho

first: Tony

last: Hunter

middle: W


title: Identification of regulatory elements in primary sensory neurons involved in physical injury-induced neuropathic pain

city: Little Rock

first: Kimberly

middle: E

last: Stephens

state: AR

address: 4301 W. Markham St Slot 510

department: Neuroscience

institute: University of Arkansas for Medical Sciences

summary: Chronic pain is a significant public health issue that is often refractory to existing therapies. Here we combine ChIP-seq and ATAC-seq to identify cis-regulatory elements that show differential chromatin accessibility, and revealed transcription factor (TF) binding motifs with functionally regulation in the dorsal root ganglion (DRG) after nerve injury. We integrated RNA-seq to understand how differential chromatin accessibility after nerve injury may influence gene expression. We performed motif analysis of the differentially accessible H3K4me1 enriched regions to identify enrichment of TF motifs. Using TF protein arrays and ChIP-qPCR, we confirmed C/EBPg binding to a differentially accessible sequence and use RNA-seq to identify processes in which C/EBPg plays an important role. Our findings offer insights into those TF motifs that are associated with chronic pain. These data show how interactions between chromatin landscapes and TF expression patterns work together to determine gene expression programs in primary sensory neurons after nerve injury.

overall_design: chromatin accessibility in naïve and injured, female rats


title: Comparison of gene expression in the nucleus accumbens of rats expressing or not the conditioned place preference to cocaine [CPPE, nCPPE, Saline]

city: PARIS

first: Stephane

last: Le Crom

address: 46 rue Ulm

department: Genomic Core Facility

country: France

institute: IBENS

type: Expression profiling by high throughput sequencing

summary: Substance use disorder emerges in a small proportion of drug users and has the characteristics of a chronic relapsing pathology. The objective of our study was to demonstrate and characterize the variability in the expression of the reinforcing effects of cocaine in the conditioned place preference (CPP) paradigm. An unbiased cocaine-CPP paradigm in Sprague-Dawley rats with an extinction period of 12 days and reinstatement was conducted. We developed a statistical model to distinguish rats that express or do not express cocaine-induced place preference. Two groups of rats were identified: rats that did express reinforcing effects (CPP expression (CPPE), score > 102 s) and rats that did not (no CPP expression (nCPPE), score between −85 and 59 s). These two groups did not show significant differences in a battery of behavioral tests. To identify differentially expressed genes in the CPPE and nCPPE groups, we performed a whole-transcriptome RNA-sequencing analysis in the nucleus accumbens (Nac) 24 h after the CPP test. Four immediate early genes (Fos, Egr2, Nr4a1 and Zbtb37) were differentially expressed in the Nac of CPPE rats after CPP memory retrieval. Variability in cocaine-induced place preference persisted in the CPPE and nCPPE groups after the extinction and reinstatement phases. Transcriptomic differences observed after reinstatement were distinct from those observed immediately after CPP memory retrieval. These new findings provide insights into the identification of mechanisms underlying interindividual variability in the response to cocaine's reinforcing effects.

overall_design: Cocaine CPP was performed in 8-week-old Sprague-Dawley rats and the nucleus accumbens transcriptomes of CPPE, nCPPE and Saline rats were compared.

first: Luisa

middle: A

last: Atehortua Martinez

first: Nawel

last: Mekdad

first: Emmanuel

last: Curis

first: Claire

last: Larrieu

first: Cindie

last: Courtin

first: Laurent

last: Jourdren

first: Corinne

last: Blugeon

first: Jean

middle: L

last: Laplanche

first: Bruno

last: Megarbane

first: Cynthia

last: Marie-Claire

first: Nadia

last: Benturquia


title: Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

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

overall_design: Refer to individual Series


title: Splicing Factor RBM22 Broadly Affects RNA Polymerase II 5' Pausing, Elongation Rate and Termination and Prevents Genome Instability [C3'-seq]

status: Public on Mar 10 2024

city: Wuhan

first: Rui

last: Xiao

state: Hubei

address: Medical Research Institute, Wuhan University

department: Medical Research Institute

country: China

institute: Wuhan University

summary: Splicing factors are vital for the regulation of RNA splicing, but the underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood. Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity and provokes transcriptional readthrough genome-wide, coupled with downstream-of-gene (DoG) transcript production and genome instability. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level. Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII, and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

overall_design: We studied the impact of RBM22 on mRNA 3' end processing using c3'-seq with 3 replicated experiments in HepG2 cells

first: Xian

last: Du

first: Wenying

last: Qin


title: C-terminal binding protein (CTBP2) is a novel tumor suppressor targeting the MYC-IRF4 axis in multiple myeloma

city: Shatin

first: Margaret HL

last: Ng

address: 30-32 Ngan Shing Street

country: Hong Kong

institute: The Chinese University of Hong Kong

summary: Multiple myeloma (MM) cells are addicted to MYC and its direct transactivation target IRF4 for proliferation and survival. MYC and IRF4 are still considered “undruggable” as the majority of small molecule inhibitors suffers from low potency, suboptimal pharmacokinetic properties and undesirable off-target effects. Indirect inhibition of MYC/IRF4 emerges as a therapeutic vulnerability in MM. Here, we uncover an unappreciated tumor suppressive role of C-terminal Binding Protein 2 (CTBP2) in MM via strong inhibition of the MYC-IRF4 axis. In contrast to epithelial cancers, CTBP2 is frequently downregulated in MM, in association with shortened survival, hyperproliferative features and adverse clinical outcomes. Restoration of CTBP2 exhibited potent anti-tumor effects against MM in vitro and in vivo, with marked repression of MYC-IRF4 network genes. Mechanistically, CTBP2 impeded transcription of MYC and IRF4 by histone H3 lysine 27 deacetylation (H3K27ac), and indirectly via activation of MYC repressor IFIT3. In addition, activation of interferon gene signature by CTBP2 suggested its concomitant immunomodulatory role in MM. Epigenetic studies revealed contribution of polycomb-mediated silencing and DNA methylation to CTBP2 inactivation in MM. Notably, inhibitors of Enhance of zeste homolog 2 (EZH2), histone deacetylase (HDACs) and DNA methyltransferase (DNMTs) currently under evaluation in clinical trials were effective in restoring CTBP2 expression in MM. Our findings indicated that loss of CTBP2 plays an essential role in myelomagenesis and decipher an additional mechanistic link on MYC-IRF4 dysregulation in MM. We envision that identification of novel critical regulators would facilitate the development of selective and effective approaches for treating this MYC/IRF4-addicted malignancy.

overall_design: To elucidate the global transcriptome changes by CTBP2 in MM, we ectopically overexpressed CTBP2 or empty-vector control in human myeloma cell lines MM.1S and NCI-H929. Biological duplicates of each transduction were subjected to RNA-sequencing.

first: CHY

last: Cheung

first: MHL


title: Splicing Factor RBM22 Broadly Affects RNA Polymerase II 5' Pausing, Elongation Rate and Termination and Prevents Genome Instability [ChIP-seq]

overall_design: Investigation of the role of splicing factor RBM22 in trancriptional control using ChIP-seq with 2 replicated experiments in HepG2 cells


title: Next Generation Sequencing Facilitates Quantitative Analysis of mRNA expression prolifes in placentas from wild Type,early-onset and later-onset PE mouse models

first: Wen-Long

last: Zhao

address: Guangyuan road 145#

institute: The International Peace Maternity and Child Health Hospital

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling (RNA-seq) of E17.5 placentas from control, early-onset (EO) and later-onset (LO) preeclampsia (PE) mice.
Methods: E17.5 placental mRNA profiles of offspring from control, EO and LO PE mice were generated by deep sequencing, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks.
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the E17.5 placentas from control EO and LO PE mice with BWA workflow and 34,115 transcripts with TopHat workflow. R
Conclusions: Our study represents the first detailed analysis of E17.5 placentas transcriptomes, with biologic replicates, generated by mRNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within tissue. We conclude that mRNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: E17.5 placental mRNA profiles  from  Control (Con), EO and LO PE mice were generated by deep sequencing, in quadruplication, using Illumina GAIIx

first: Xue-Yuan

last: Liu


title: FTO-mediated autophagy inhibition promotes non-small cell lung cancer progression by reducing SESN2 mRNA stability [MeRIP-Seq]

status: Public on Mar 09 2024

city: Luzhou

first: Kai

last: Wang

address: No. 1, Section 1, Xianglin Road, Longmatan District

institute: Southwest Medical University

summary: N6-methyladenosine (m6A) serves as the most abundant mRNA modification within eukaryotes that is involved in autophagy and progression of non-small cell lung cancer (NSCLC). The role of the m6A demethylase FTO in NSCLC progression has recently received widespread attention. However, the mechanism of FTO-mediated autophagy regulation in NSCLC progression is unclear. In this study, we found that FTO was significantly up-regulated in NSCLC, and down-regulation of FTO blocked the growth, invasion and migration of NSCLC cells via autophagy induction. Importantly, SESN2 was involved in m6A regulation during autophagy in NSCLC cells. FTO deficiency prompted IGF2BP1 to interact with SESN2 mRNA, which led to an increase in SESN2 mRNA stability as well as enhanced SESN2 protein levels. However, FTO inhibited autophagic flux via down-regulation of SESN2, thereby promoting the growth, invasion and migration of NSCLC cells. Our findings uncovered that FTO-mediated autophagy inhibition promotes malignant progression of NSCLC via reduction of SESN2 mRNA stability, thus providing novel notions for the prevention and treatment of NSCLC.

overall_design: To investigate autophagy-mediated alterations in m6A modification of specific genes, we performed MeRIP-seq and RNA-seq analyses with autophagy-activated H460 cells treated with rapamycin for 24h. Then, the data obtained from RNA-seq or MeRIP-seq was used to perform differentially gene or differentially methylated gene expression profiling analysis. Thereafter, the two datasets were overlaid with pre-existing autophagy-associated genes to obtain key common genes for subsequent experiments.


title: RNA sequencing of BM myeloid cells treated with AAV-IL-27/ctrl in vivo

first: Jianmin

last: Zhu

state: Shanghai

address: Dongfang road 1678

institute: Shanghai Children's Medical Center

summary: Interleukin-27 (IL-27) is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells and has a potential to be used as a therapeutic for cancer. We have recently demonstrated that systemic delivery of IL-27 using adeno-associated virus (AAV-IL-27) exhibits potent inhibition of tumor growth in mouse models. In this work, we demonstrate that AAV-IL-27 treatment leads to significant expansion of CD11b+Gr1+ myeloid cells (MCs). AAV-IL-27-induced expansion of CD11b+Gr1+ cells is IL-27R-dependent, requires Stat3 signaling but is inhibited by Stat1 signaling. AAV-IL-27 treatment does not increase the self-renewal capacity of CD11b+Gr1+ cells but induces significant expansion of hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitor (GMP) cells. IL-27-induced Ly6G+ MCs upregulate MHC class I/II molecules but are less immune suppressive in vitro, and their expansion does not promote, but rather inhibit tumor growth in vivo. In the tumor microenvironment, IL-27 gene therapy appears to promote Ly6G+ MCs to differentiate into MHC class I/IIhigh and F4/80high macrophages. Thus, IL-27 gene therapy induces expansion of CD11b+Gr1+ myeloid cells and promotes their differentiation into anti-tumor M1 macrophages in tumor microenvironment.Bone marrow myeloid cells of tumor-bearing C57BL/6 mice treated with AAV-IL-27/ctrl for 2 weeks were sepreated by MACS. Transcriptome profiling (RNA-seq) of these purified Ly6G+ cells (>95%) was completed by BGI. Changing in gene express profiling resulted by IL-27 was analyzed in this study.

overall_design: C57BL/6 mice were subcutaneously injected with B16.F10 1 week before AAV-ctrl/IL27 were intramuscularly injected. Ly6G+ myeloid cells were derived from 4 mice. 2 mice treated with AAV-ctrl and 1 mouse treated with AAV-IL-27.

first: Xue-Feng

last: Bai


title: Patient-derived organoids for precise medicine in gastric cancer

city: Beijing

first: Ye-guang

last: Chen

address: Tsinghua Road, Medical Building

department: School of life science

institute: Tsinghua University

summary: Gastric cancer (GC) is a challenging malignant disease among gastrointestinal tumors. The clinical treatments for GC include surgery, neoadjuvant therapy and adjuvant chemotherapy. As patients’ response to chemotherapeutic drugs varies, an approach to predict the efficacy of chemotherapeutic treatment is urgently needed. Here, a biorepository of 17 human gastric tumor organoids (GTOs) and 9 normal organoids (GNOs) derived from GC patients was established, which contains the intestinal, diffuse and mixed-type of GC. GTOs retained morphologic, histopathological and genetic features of the tumors from which they were derived, and patient-derived organoid xenograft model indicated tumorigenicity of organoids. GTOs responses to clinically respective chemotherapy treatment positively correlated with the individual patient’s clinical response. Meanwhile, GNOs, which maintained original characteristics of corresponding tissues, could be separated from GTOs. The use of GNOs predicted the cytotoxic effect of chemotherapy on the organism. Our study implies that patient-derived organoids predict GC patient responses to chemotherapy and provide a solution for personalized treatment.

overall_design: In order to investigate the molecular characteristics of GTOs, we performed whole exome sequencing on tumor tissues and GTOs derived from 8 patients to explore the genomic landscape and identify genetic alterations in GTOs compared to their corresponding tumor tissues. By comparing these findings to the corresponding drug screening outcomes using organoids, we aim to elucidate the molecular evolution of GTOs and shed light on their potential as models for precision medicine applications in cancer research and therapy.

first: Ting

first: Wanlu

last: Song

first: Qingyu

last: Meng


title: The role of quiescent thymic progenitors in TAL/LMO2-induced T-ALL chemotolerance

city: Worcester

first: Michelle

last: Kelliher

state: Massachusetts

address: 364 Plantation Street, Lazare Research Building, 470H

institute: UMass Chan Medical School

summary: Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4−CD8−CD25+CD44−) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle–restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a− thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

overall_design: Thymocytes from Wild type FVB/N, Tal1/Lmo2 transgenic FVB/N preleukemic, or Tal1/Lmo2 transgenic FVB/N leukemic mice were sorted for viability (7-AAD negative) using fluorescence-activated cell sorting (FACS) and analyzed by scRNA-seq

first: Kevin

last: O'Connor

first: Kensei

last: Kishimoto

first: Lihua

middle: J


title: Viral integration promotes SV40T-induced immortalization by disturbing the expression of DNA/chromosome- and ECM-associated functional genes

city: Xi’an

first: zihang

last: li

address: 277 West Yanta Road

institute: the First Affiliated Hospital of Xi’an Jiaotong University

summary: Lentivirus containing simian virus 40 large T antigen (SV40T) is routinely used to induce cell immortalization. However, the roles of viral integration itself in this progress is still controversial. Here, we transformed primary mouse embryonic fibroblasts (MEFs) with SV40T lentivirus and studied the roles of viral integration in the immortalization using RNA-seq and whole genome sequencing (WGS). During the immortalization, differentially expressed genes (DGEs) are enriched in viral infection and several diverse activities. However, DEGs between immortalized and aging cells are significantly enriched in DNA/chromosome- and extracellular matrix (ECM)-associated activities. Gene regulatory network (GRN) analysis shows that although p53 is a key regulatory factor, many other transcription factors also play critical roles in the process, like STAT1. Of these DEGs, 32 genes have viral integration in their coding and/or regulatory regions. Our findings suggest that viral integration may promote SV40T-mediated immortalization by disturbing the expression of DNA/chromosome- and ECM-associated genes.

overall_design: Isolated MEFs at the primary stage were divided into three equal portions for no infection or infection with SV40T lentivirus or empty lentivirus, which were assigned as control, SV40T and vehicle group, respectively. This is the time point 1. Once the cells in either control or vehicle group stopped proliferating, all cell cultures were terminated and this was the time point 2.

first: Zihang

last: Li

first: Peng

last: Gao


title: Enzyme-mediated proximity labeling identifies small RNAs in the endoplasmic reticulum lumen

first: Ziqi

last: Ren

address: Chengfu Road No.292

department: College of Chemistry and Molecular Engineering

institute: Peking University

summary: In eukaryotic cells, the subcellular targeting of RNA controls many fundamental aspects of cellular physiology. RNA molecules are broadly distributed throughout the nucleoplasm and cytoplasm, but are conventionally believed to be excluded from the secretory pathway compartments, including the endoplasmic reticulum (ER). Recent discovery of RNA N-glycan modification (glycoRNAs) has challenged this view, but direct evidence of RNA localization in the ER lumen has been lacking. In this study, we applied enzyme-mediated proximity labeling to profile the ER lumen-localized RNAs in human embryonic kidney 293T cells and rat cortical neurons. Our dataset revealed the presence of small non-coding RNAs in the ER lumen, including U RNAs, sco/sca RNAs and Y RNAs, which partially overlap with glycoRNAs. These findings shed light on novel RNA targeting mechanism and raise interesting questions regarding the biological functions of ER lumenal RNAs.

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: Comparative gene expression profiling analysis of RNA-seq data from HEK293T and rat cortical neuron under different treatment captured by MERR HRP-seq

first: Ran

first: Xinyue

last: Zhou

first: Yu

first: Yuxin

last: Fang

last: Zou


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Extended donors]

city: Boston

first: Chen

last: Weng

state: OH

address: 415 Main St, Cambridge

department: Genetics

institute: Broad Institute and Whitehead Institute

summary: The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived hematopoietic stem cells (HSCs). Perturbations to this process underlie a diverse set of diseases, but the clonal contributions to human hematopoiesis and how this changes with age remain incompletely understood. While recent insights have emerged from barcoding studies in model systems, simultaneous detection of cell states and phylogenies from natural barcodes in humans has been challenging, which has limited the ability to explore functional differences between HSC clones. Here, we introduce an improved single-cell lineage tracing system based on deep detection of naturally-occurring mitochondrial DNA (mtDNA) mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs, and map the physiological state and output of these clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as differences in total HSC output as well as biases toward the production of different mature blood and immune lineages. We also find that the diversity of HSC clones decreases dramatically with age leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides the first clonally-resolved and cell-state aware atlas of human hematopoiesis at single-cell resolution revealing an unappreciated functional diversity of human HSC clones both in young and aged individuals and more broadly paves the way for refined studies of clonal dynamics across a range of tissues in human health and disease.

overall_design: We developed single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling (ReDeeM), which is a modified massive parallel single cell protocol to simultaneously profile multi-omics with deep mtDNA sequencing based on the 10X Genomics platform. With ReDeeM, three separate libraries are generated, including enriched mtDNA library for deep sequencing and mutation profiling, RNA library for gene expression, as well as ATAC library for chromatin accessibility profiling, all of which are linked via matchable single cell barcodes. Following the principle of our previous work, we firstly modified the droplet-based 10X genomics multi-omics protocol (Catalog #100283) by processing the whole cell instead of nuclei with fixation and mild permeabilization to maximally retain mitochondrial DNA. Next, we designed mtDNA specific probe sets to enrich the mitochondrial fragments using DNA hybridization. The RNA and ATAC library preps follow the standard 10X protocol with some modifications.  More method details are described in the method section and an open ReDeeM Protocol is available. ReDeeM is further computationally supported by consensus variant calling pipeline redeemV , and an inhouse R package redeemR for the downstream mutation quality control, as well as single cell phylogenetic and integrative analysis.  This SubSeries contains data of CRISPR mouse lineage tracing with ReDeeM

first: Vijay

middle: G

last: Sankaran

first: Jonathan

middle: S

last: Weissman


title: Integrated transcriptomic analysis identifies coordinated responses to nitrogen and phosphate deficiency in rice

city: 杭州

first: Fei

last: Wang

state: 浙江

address: 紫金港校区

institute: 浙江大学

summary: Nitrogen (N) and phosphorus (P) are two primary components of fertilizers for crop production. Coordinated acquisition and utilization of N and P are crucial for plants to achieve nutrient balance and optimal growth in a changing rhizospheric nutrient environment. However, little is known about how N and P signaling pathways are integrated. We performed transcriptomic analyses and physiological experiments to explore gene expression profiles and physiological homeostasis in the response of rice (Oryza sativa) to N and P deficiency. We determined that the transcript levels of 763 core genes changed under both N or P starvation conditions. Among these core genes, we focused on the transcription factor gene NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) and show that its encoded protein is a positive regulator of P homeostasis and a negative regulator of N acquisition in rice. These results provide new clues about the mechanisms underlying the interaction between plant N and P starvation responses.

overall_design: To obtain molecular insight into transcriptional network changes and identify genes associated with both N and P homeostasis, we carried out RNA sequencing of rice root in response to control nutrient and N or P deficiency treatment. Each treatment contained three biological replicates.

first: Yan

first: Luying

last: Ying

first: Hong

last: Lu


title: Ex vivo cultured mouse LT-HSCs RNA-seq

city: Hangzhou

first: lu

last: huan

address: 866 Yuhangtang Road

institute: Zhejiang University medical center

summary: Mouse LT-HSCs were co-cultured with polymer nanospheres for ex vivo expansion

overall_design: Mouse total bone marrow cells were isolated and  co-cultured with different polymer nanospheres for 3 days, LT-HSCs were sorted by FACS to perform bulk RNA-seq

first: Qiwei

first: Huan

first: Pengxu

last: Qian


title: Transcriptome Investigation of HFD-fed mice treated or not with Glycoursodeoxycholic Acid

city: Xi'an

first: Lele

last: Cheng

institute: The First Affiliated Hospital of Xi’an Jiaotong University

summary: Recent studies reveal that bile acid metabolite composition and its metabolism are changed in metabolic disorders, such as obesity, type 2 diabetes and metabolic associated fatty liver disease (MAFLD), yet its role and the mechanism remain largely unknown. Hepatic whole transcriptome analysis identified glycoursodeoxycholic acid (GUDCA), glycine-conjugated bile acid produced from intestinal bacteria, differentially regulated 189 genes involved in biological processes including sterol, cholesterol and steroid biosynthesis, lipid metabolism, circadian rhythm and regulation, and the cellular component in cytosol, cytoplasm and endoplasmic reticulum, and molecular function including catalytic activity and platelet-derived growth factor binding.

overall_design: Hepatic mRNA profiles of Vehicle-treated and GUDCA-treated mice receiving HFD diet (60% fat) (n=3/group).

first: Manyun

last: Guo

first: Junhui

last: Liu


title: Extensive and dynamic changes in the human blood transcriptome and its circadian organisation when lying in bed, head down, for 60 days

city: Guildford

first: Simon

last: Archer

address: Stag Hill

country: United Kingdom

institute: University of Surrey

summary: We investigated the human 24-h time series blood transcriptome in 20 individuals during a 90-day constant bed rest protocol.

overall_design: A total of 637 samples, involving 20 human subjects. Each subject contributed up to 36 samples, gathered across 24 hours over 6 different days.

middle: N

first: Carla

last: Moller-Levet

first: Maria-Angeles

last: Bonmati-Carrion

first: Emma

last: Laing

first: Derk-Jan

last: Dijk


title: LIN28A inhibited DUSP family phosphatases and activated MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells

city: yangling

first: Wu

last: Xiaolong

state: Shannxi

address: College of Veterinary MedicineShaanxi Centre of Stem Cells Engineering and Techn

department: College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology

institute: Northwest A&F University

summary: LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism of LIN28A maintaining pluripotency in piPSCs remains unclear. The aim of this study was to explore the functions of LIN28A in piPSCs. Here we explored the functions of LIN28A in piPSCs by overexpressing and knocking down LIN28A. We performed total RNA sequencing (RNA-seq) on piPSCs and the expression levels of relevant-genes were detected by qRT-PCR, western blot and immunofluorescence staining. The proliferation ability of piPSCs was related to the expression level of LIN28A and the proliferation ability decreased when LIN28A was knocked down. When the expression level of LIN28A was lower than 20% compared with the negative control (shNC) group, the pluripotency of piPSCs would disappear and differentiate to the neuroectoderm. We also found that LIN28A inhibited the expression levels of DUSP (dual-specificity phosphatases) family phosphatases and then activated mitogen-activated protein kinases (MAPK) signaling pathway. This work revealed that LIN28A activated MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our results provided a new resource for exploring the functions of LIN28A in piPSCs.

overall_design: mRNA profiles of Negative Control and shLin28a

first: Xiaolong

last: Wu

first: Zhenshuo

last: Zhu

first: Xia

last: Xiao

first: Zhe

last: Zhou


title: Integrated transcriptome analysis of rats exposed to bisphenol mixtures from fetus to developmental stage

status: Public on Mar 08 2024

city: Daejeon

first: Soontag

last: Jung

address: 141 Gajeong-ro, Yuseong-gu

department: Developmental and reproductive toxicology

country: South Korea

institute: Korea Institute of Toxicology

summary: Bisphenol A (BPA), widely used in plastics and resins, raised health concerns for its endocrine-disrupting effects. BPA analogues like bisphenol S (BPS) and bisphenol F (BPF) emerged as alternatives but were found to exhibit similar risks. Despite many countries have implemented BPA regulations, alternatives remain insufficiently regulated Although the safety of BPS and BPF has not been sufficiently verified, they have already been detected in various surrounding environments and human urine, raising serious concerns Bisphenols are expected to have various adverse effects, but research on this is lacking. This study explores the adverse effects of bisphenol mixtures on rats from fetus to young adulthood, analyzing transcriptomes by tissue and gender to identify key genes impacted by bisphenol exposure. Dams were orally administered test substances from gestational day 6 to lactation day 6. F1 pups received the same substances at half the concentration from postnatal day 7 to day 63. The tissues collected from the pups were subjected to transcriptome analysis, and core genes were identified through integrated analysis. The study identifies core genes associated with high-density lipoprotein and hormone secretion. These genes provide insights into the mechanisms through which BPA may cause hormonal imbalances. Furthermore, the study suggests that a complex exposure of BPA, BPS, and BPF can exerts different effects than BPA alone, pronounced effects on the thyroid and reproductive organs, even though individual concentrations were below the no-observed-adverse-effect-level. It highlights the potential cumulative impact of endocrine disrupting chemicals in the body.

overall_design: The dose of a single bisphenol (Single) was selected as BPA 500 mg/kg/day and bisphenol mixture (Mix) was made at 500 mg/kg/day proportionally combined each of the three chemicals (BPA, 160 mg/kg/day; BPF, 160 mg/kg/day; BPS, 180 mg/kg/day). The pregnant SD rat assigned to 3 groups (VC, Single and Mix) were orally administered each test substance from gestational day 6 to lactation day 6. On postnatal day (PND) 4, F1 pups were culled to three male and female per F0 dam. F1 pups were orally administered the same test substance as F0 dam at half the concentration (250 mg/kg/day) from PND 7 to PND 63. For transcriptome analysis, pups were humanely sacrificed, and liver, kidney, thyroid, and reproductive organs including testis and ovary were collected.

first: Jung

last: Soontag

first: Quah

last: Yixian

first: Park

last: Dong-Ju

first: Ham

last: Onju

first: Kim

last: Sang-Yun

first: Jeong

last: Ji-Seong

first: Lee

last: Seung-Jin

first: Yu

last: Wook-Joon


title: The covariance environment defines cellular niches for spatial inference

city: NY

first: Doron

last: Haviv

state: NY

address: 1275 York Avenue

institute: Memorial Sloan Kettering Cancer Center

summary: The tsunami of new multiplexed spatial profiling technologies has opened a range of computational challenges focused on leveraging these powerful data for biological discovery. A key challenge underlying computation is a suitable representation for features of cellular niches. Here, we develop the covariance environment (COVET), a representation that can capture the rich, continuous multivariate nature of cellular niches by capturing the gene-gene covariate structure across cells in the niche, which can reflect the cell-cell communication between them. We define a principled optimal transport-based distance metric between COVET niches and develop a computationally efficient approximation to this metric that can scale to millions of cells. Using COVET to encode spatial context, we develop environmental variational inference (ENVI), a conditional variational autoencoder that jointly embeds spatial and single-cell RNA-seq data into a latent space. Two distinct decoders either impute gene expression across spatial modality, or project spatial information onto dissociated single-cell data. We show that ENVI is not only superior in the imputation of gene expression but is also able to infer spatial context to disassociated single-cell genomics data.

overall_design: Brain Single Nuclei RNA sequencing of mouse with melanoma metastatis to the leptomeninges

first: Jan

last: Remsik

first: Adrienne

last: Boire

first: Dana

last: Pe'er


title: Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance

city: Bethesda

first: Michael

middle: C

last: Kelly

state: MD

address: 41 Center Drive

department: Center for Cancer Research

institute: NCI

summary: Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations; namely, Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21 that attracts developing thymocytes to the medullary region. Here we report that CCL21+ mTECs and Aire+ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detected a cooperation for self-tolerance between Aire and Fezf2, which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance.

overall_design: For SCAF1213_S_1119 and SCAF1315_S_1224, cells from the third pharyngeal region were collected. For SCAF695_190313_P5, SCAF621_P5, SCAF622_P6, SCAF696_190313_P6, SCAF804_P5 and SCAF805_P6, thymic epithelial cells from B5T-Venus embryos were isolated using FACS cell sorting according to the presence or absence of Venus signals. For SCAF2439_IRF6flox, thymic stromal cells were obtained through CD45 depletion using CD45 MicroBeads. All cells underwent analysis via Single Cell 3' end-counting gene expression (scRNA-Seq) using the 10X Genomics platform.

first: Yousuke

last: Takahama

last: Tanaka

first: Alison

last: Jacques

first: Meiting

last: Yang


title: KRASG12D Transforms Murine B-Cell Progenitors and Causes Aggressive ALL in Vivo

city: Houston

first: Xiaoping

last: Sun

state: Texas

address: 2130 Westholcombe Blvd, LSP12.3014

department: Laboratory Medicine

institute: UT MD Anderson Cancer Center

summary: Expression profile of KRAS transformed murine B cell progenitors and empty vector transduced counterparts by DNA microarray analysis

overall_design: We did triplicate for both KRAS transformed cells and control cells by using Affymetrix Mouse Genechip 2.0 ST arrays.

first: Sun

last: Xiaoping

first: June

last: Li

first: Kevin

last: Lin

first: Yue

last: Lu

first: Yun

last: Gong

first: Haifa

last: Shen

first: Bingliang

last: Fang

first: Xin

last: Han

first: Laura

last: Baseler

first: Lawrence

last: Kwong

first: Yingli

last: Wu

first: Peng

last: Huang

first: Saradhi

last: Mallampati

first: Jianfang

last: Zeng


title: Hepatocyte CCAAT/enhancer-binding protein alpha restricts liver fibrosis  by inhibiting secreted phosphoprotein 1 expression

city: Bethesda, MD

first: Frank

last: Gonzalez

address: Bldg. 37 National Institutes of Health

institute: National Institutes of Health, National Cancer Institute

summary: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Persistent NAFLD can progress to nonalcoholic steatohepatitis (NASH) with inflammation and fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor that modulates glycolipid homeostasis, cell differentiation and tumor progression. MTL-CEBPA, a first-in-human small activating RNA therapeutic, has been used to overexpress CEBPA and treat hepatocellular carcinoma by targeting myeloid cells in clinical trials. However, whether and how hepatocyte-specific CEBPA modulates NASH are unknown. Here we found that CEBPA expression was inhibited in the livers of high-fat, high-cholesterol, and high-fructose diet (HFCFD)-fed mice, NASH patients and palmitic/oleic acid-treated primary hepatocytes. Liver-specific Cebpa knockout mice (Cebpa-dLiv) and liver-specific tamoxifen-inducible ERT2-Cre mice (Cebpa-dLiv-ERT2) were then generated. Both Cebpa-dLiv and Cebpa-dLiv-ERT2 mice (treated with tamoxifen) developed enhanced NASH and fibrosis compared to control mice. Global transcriptome analyses demonstrated hepatic secreted phosphoprotein 1 (Spp1) encoding the fibrosis-promoting factor SPP1, was markedly induced by hepatocyte CEBPA knockout in HFCFD-fed mice. Consistently, hepatic SPP1 was upregulated by NASH and negatively correlated with CEBPA expression in humans. Hepatocyte loss of CEBPA enhanced hepatic SPP1 expression and serum SPP1 levels both at the early and late stage of NASH in mice. CEBPA-knockout primary hepatocytes released more SPP1 to the culture medium than wild-type mouse hepatocytes, resulting in enhanced fibrogenesis of hepatic stellate cells, a phenotype that was rescued by the Spp1 shRNA. Hepatocyte-directed AAV8-deliverey of Spp1 shRNA rescued the fibrosis in HFCFD-fed Cebpa-dLiv mice. Mechanistically, CEBPA overexpression reduced, while CEBPA knockout enhanced, SPP1 expression in primary hepatocytes in vitro. The Spp1 promoter had functional CEBPA response elements, while CEBPA modulated histone acetylation to restrict SPP1 expression in a novel feedback loop. Furthermore, overexpression of CEBPA in hepatocytes with AAV8-TBG-CEBPA, inhibited hepatic SPP1 expression and reduced fibrosis in HFCFD-fed wild-type mice. This study demonstrates a novel hepatocyte CEBPA-SPP1 axis involved in modulating NASH fibrosis that supports hepatocyte CEBPA as an anti-NASH target.

overall_design: Examination of gene expression change in cell populations in liver of hepatocyte-specific CEBPA knockout mice fed a NASH-promoting 40% HFCFD using scRNASeq based on10x Chromium Single Cell 3' library preparation.

first: Yan

last: Tingting

first: Gonzalez

last: Frank

first: Sawaswong

last: Vorthon


title: One-pot method for preparing DNA, RNA, and protein for multiomics analysis I

city: Little Rock

first: Stephanie

middle: D

last: Byrum

state: Arkansas

address: 501 Jack Stephens Dr, Jackson T. Stephens Bldg 4th floor

institute: University of Arkansas for Medical Sciences

summary: Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

overall_design: To assess the quality and reproducibility of our novel multiomics sample preparation workflow, we prepared DNA, RNA, and peptides from four triple-negative breast cancer cell lines that exhibit different degrees of malignancy: MCF10A (non-tumorigenic), MCFNeoT (benign hyperplasia), MCFT1 (atypical hyperplasia), MCFCA1 (invasive cancer). Three biological replicates were carried out with each cell line.

last: Biedka

first: Duah

last: Alkam

first: Charity

last: Washam

first: Svitlana

last: Yablonska

first: Aaron

last: Storey

first: Jonathan

last: Minden


title: SWI/SNF-dependent genes are defined by their chromatin landscape [RNA-seq]

city: Seville

first: Jose

middle: Antonio

last: Guerrero-Martinez

state: Seville

address: Av. Americo Vespucio, N 24

department: Genome Biology

country: Spain

institute: CABIMER

summary: The SWI/SNF complex was the first chromatin remodeling machinery discovered. Although it has been extensively investigated, numerous aspects of its regulation and activity are still poorly understood, especially in higher eukaryotes. In mammals, there is not a single SWI/SNF complex (also called BRM or BRG1 associated factors, BAF, complex) but rather a polymorphic family of complexes, with three main subtypes called canonical BAF (cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF), with relatively different specificities. The enzymatic motors of the complexes are two mutually exclusive ATPases of the SNF2 family called BRAHMA (BRM, also called SMARCA2) and BRAHMA RELATED GENE 1 (BRG1, also called SMARCA4). Recently, an specific inhibitor of BRM and BRG1 ATPase activity, called BRM014, has been developed. We have extensively investigated the effect of BRM014 on the transcriptome and the chromatin landscape of non-tumoral normal murine mammary (NMuMG) epithelial cells.

overall_design: RNA sequencing of NMuMG mouse glandular epithelial cells treated with DMSO or BRM014 at 6h and 30h were generated by deep sequencing, in duplicates, using Illumina NovaSeq 6000.

last: Basurto-Cayuela

last: Guerrero-Martínez

last: Reyes


title: Identification of RBM5 and RBM10-dependent splicing events in 293Flp-In cells.

city: LOS ANGELES

first: Chia-ho

last: Lin

address: 6577 MRL. 675 CHARLES E YOUNG DR. SOUTH

institute: UCLA

summary: RBM5 and RBM10 are RNA-binding proteins and splicing regulators. These two proteins are putative paralogs in mammalian cells, sharing common domain organization and extensive protein sequence similarity, but their RNA-binding preferences differ. We developed a sensitive system to identify splicing events regulated by RBM5 and/or RBM10, deleting all RBM5 alleles in 293Flp-In cells, and reducing the expression of RBM10, which is partially rescued by activation of a cryptic exon (CE) in one of the RBM10 alleles. RBM5 or RBM10 transgenes were then introduced in these RBM5-null, RBM10 mutant cells (RBM5-/-;RBM10-/CE), allowing controlled protein expression, induced with Doxycycline. Using this system we identify thousands of RBM5 and RBM10-regulated cassette exons, and note a substantial overlap between the two sets.

overall_design: CRISPR-Cas9 gene deletion, followed by isogenic insertion of tetracycline-inducible transgenes.

first: Andrey

last: Damianov

first: Chia-Ho

first: Jeffrey

last: Huang

first: Douglas

last: Black


title: SWI/SNF-dependent genes are defined by their chromatin landscape [ChIP-seq_2]

summary: The SWI/SNF complex was the first chromatin remodeling machinery discovered. Although it has been extensively investigated, numerous aspects of its regulation and activity are still poorly understood, especially in higher eukaryotes. In mammals, there is not a single SWI/SNF complex (also called BRM or BRG1 associated factors, BAF, complex) but rather a polymorphic family of complexes, with three main subtypes called canonical BAF (cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF), with relatively different specificities. The enzymatic motors of the complexes are two mutually exclusive ATPases of the SNF2 family called BRAHMA (BRM, also called SMARCA2) and BRAHMA RELATED GENE 1 (BRG1, also called SMARCA4). Recently, an specific inhibitor of BRM and BRG1 ATPase activity, called BRM014, has been developed. We have extensively investigated the effect of BRM014 on the transcriptome and the chromatin landscape of non-tumoral normal murine mammary (NMuMG) epithelial cells.

overall_design: Binding profiles of H3K27ac, BRD9, DPF2 and PBRM1 from NMuMG mouse glandular epithelial were generated by deep sequencing using Illumina NovaSeq 6000.

first: Laura


title: SWI/SNF-dependent genes are defined by their chromatin landscape [MNase-seq]

overall_design: Mnase-seq analysis of NMuMG mouse glandular epithelial cells treated with DMSO or BRM014 at 6h and 30h were generated by deep sequencing, in duplicates, using Illumina NovaSeq 6000. Include samples with crosslinking and without crosslinking.


title: SWI/SNF-dependent genes are defined by their chromatin landscape [Cut&Tag]

overall_design: Binding profiles of H3K27me3 using Cut&Tag technology from NMuMG mouse glandular epithelial cells treated with DMSO or BRM014 at 30 hours were generated by deep sequencing, in duplicates, using Illumina NovaSeq 6000.


title: Resident Kupffer cell maladaptation in Liver Fibrosis is Rescued by Monocyte-Derived Syncytia of Macrophages

city: Calgary

first: Paul

last: Gordon

state: AB

address: 3330 Hospital Drive NW

institute: University of Calgary

summary: To investigate the effects of liver fobrosis and alteration in liver architecture in the regulation of embryonic Kupffer cells identity and funtion, we used a well stablished carbon tetrachloride model to induce liver fibrosis.

overall_design: Comparative gene expression profiling analysis of data obtained from RNA-seq of naïve KCs and KCs isolated from fibrotic livers.


title: SWI/SNF-dependent genes are defined by their chromatin landscape [ChRNA-seq]

overall_design: Chromatin-enriched RNA sequencing of NMuMG mouse glandular epithelial cells treated with DMSO or BRM014 at 6h and 30h were generated by deep sequencing, in duplicates, using Illumina NovaSeq 6000.


title: SWI/SNF-dependent genes are defined by their chromatin landscape [ATAC-seq]

overall_design: ATAC-seq analysis of NMuMG mouse glandular epithelial cells treated with DMSO or BRM014 at 6h and 30h were generated by deep sequencing, in duplicates, using Illumina NovaSeq 6000. Please note that each processed data file was generated from both replicate samples and is linked to the corresponding rep1 sample records.


title: SWI/SNF-dependent genes are defined by their chromatin landscape

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

overall_design: Refer to individual Series


title: Evaluating the toxicity of two endocrine disrupting chemicals, Raloxifene and Resorcinol using a larval zebrafish transcriptomic approach

city: Saskatoon

first: Morgan

last: Kirzinger

state: SK

address: 110 Gymnasium Place

institute: National Research Council

summary: We use a larval zebrafish model to assess the transcriptomic alterations caused by sub-phenotypic concentrations of two endocrine disrupting drugs: Raloxifene and Resorcinol.

overall_design: We use two approaches to evaluate potential toxicological mechanisms; we compare serial dilutions of each drug to the vehicle control and we determine the Point of Departure (POD) for each drug by modelling gene expression changes as continuous systems.

first: Michael

last: Morash

first: Lee

last: Ellis


title: ORE15, a PLATZ transcription factor, determines root meristem size by modulating auxin and cytokinin signaling

city: Hyeonpung-eup

first: Hyunsu

last: Park

state: Dalseong-gun, Daegu

address: E4-620, 333, Techno jungang-daero

department: New biology

institute: DGIST

summary: An optimal size of postembryonic root apical meristem (RAM) is achieved by a balance between cell division and differentiation. Despite extensive research, molecular mechanisms underlying the spatiotemporal coordination of cell division and differentiation remain largely unknown. Here, we report that ORESARA 15 (ORE15), an Arabidopsis PLATZ transcription factor preferentially expressed in the RAM, determines RAM size. The primary root length, RAM size, cell division rate, and stem cell niche activity were reduced in an ore15 loss-of-function mutant but enhanced in an activation-tagged line overexpressing ORE15 compared with the wild type. We further demonstrate that ORE15 regulates auxin transport and distribution, at least partly, by modulating the expression and localization of the auxin importer AUXIN RESISTANT 1 (AUX1). ORE15 forms mutually positive and negative feedback loops with auxin and cytokinin signaling, respectively. Collectively, our findings imply that ORE15 controls the RAM size by mediating the antagonistic interaction between auxin and cytokinin.

overall_design: mRNA profiling from ORE15 in root tips in Arabidopsis

first: Rupak

last: Timilsina

first: Yongmin

last: Kim

first: Sanghoon

first: Hyunsoo

first: Sung-Jin

first: Jin

middle: H

first: Ji-Hwan

first: Doa

first: Youn-Il

first: Daehee

last: Hwang

first: Jong-Chan

last: Lee

first: Hong

last: Nam

first: Hye

middle: R

last: Woo


title: SWI/SNF-dependent genes are defined by their chromatin landscape [ChIP-seq]

first: Jose

last: Guerrero-Martinez

overall_design: Binding profiles of H3K27me3 from NMuMG mouse glandular epithelial were generated by deep sequencing using Illumina NovaSeq 6000.

first: Laura

last: Basurto-Cayuela

last: Guerrero-Martínez


title: One-pot method for preparing DNA, RNA, and protein for multiomics analysis

first: Stephanie

last: Byrum


title: Single cell TCR sequencing of allogeneic HSC-derived CAR-engineered NKT cells

status: Public on Mar 07 2024

city: Los Angeles

first: Lili

last: Yang

state: California

address: 610 Charles E. Young Dr. East

department: Microbiology Immunology & Molecular Genetics

institute: University of California, Los Angeles

summary: CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control.

overall_design: Sngle cell TCR sequencing of allogeneic HSC-derived CAR-engineered NKT cells, compared with those of PBMC-derived conventional T and NKT cells

first: Yan-Ruide

last: Li

first: Yang

last: Zhou

first: Jiaji

last: Yu

first: Jeong

first: Miao


title: TDP-43, a novel rhythmic protein, regulates the ubiquitination degradation of BMAL1, cellular glucose utilization and energy synthesis

city: jiangsunantong

first: hu

middle: han

last: jing

address: Chongchuan District Xincheng Bridge street

institute: nantonguniversity

summary: In this study, we discovered that TDP-43 expression followed a rhythmic pattern, which was influenced by sleep deprivation. Knockdown of TDP-43 altered the mRNA and protein expression of multiple circadian genes, including BMAL1, CLOCK, CRY1, and PER2. Furthermore, TDP-43 knockdown affected the autonomous circadian wheel behavior, as well as the cognitive and balance abilities of mice. We also revealed that TDP-43 regulated the deubiquitination of BMAL1 by influencing the alternative splicing of USP13, thereby affecting the expression of USP13 and the ubiquitin-mediated degradation of BMAL1. Additionally, TDP-43 modulated the AMPK signaling pathway, leading to changes in cellular glucose uptake and ATP production.

overall_design: M17 cells were infected with LentiCRISPR v2/TDP-43KO or control viruses, and synchronized for 36 hrs, and then collected with TRIzol reagent at CT12.


title: Transcriptomic analysis of bovine endometrial epithelial cells in response to interferon tau and hormone stimulation

city: Wuhan

first: Jie

address: Zhijinchangshan Road No.2, Jinkou, Jiangxia District

department: Institute of Animal Science and Veterinary Medicine

institute: Wuhan Academy of Agricultural Sciences

summary: The embryonic loss during early stage of gestation is one of the major causes of infertility for domestic ruminants, causing huge economic losses to pasture. Maternal recognition of pregnancy and implantation are the crucial process for determining the successful establishment and development of pregnancy in cattle. The research on molecular mechanisms of pregnancy recognition will facilitate illustrating the complex process of pregnancy establishment and help to improve pregnancy outcomes. In this study, we performed transcriptomic analysis of primary bovine endometrial epithelial cells (BEND) with or without IFNT and hormones intervention through RNA sequencing. We eventually identified 608 differentially expressed genes (DEGs) including 409 up-regulated genes and 199 down-regulated genes in IFNT and hormones-treated group compared with control group. Gene Ontology (GO) enrichment analysis demonstrated that the majority of DEGs were implicated in immune system process, response to external stimulus, response to cytokine, regulation of response to stress. Results from KEGG analysis showed a significant enrichment of NOD-like receptor signaling pathway, antigen processing and presentation, necroptosis, oxidative phosphorylation, RIG-I-like receptor signaling pathway. Additionally, a set of promising candidate genes, including (USP18, STAT1, PSMB8, IFIH1, MX2, IFI44, DHX58, CASP8, DRAM1, CXCR4), were characterized by constructing an integrated interaction network.

overall_design: To investigate the gene-interaction network and underlying molecular mechanisms regulating maternal recognition of pregnancy in ruminants, we established primary bovine endometrial epithelial cells (BEND), and performed RNA-seq to dissect the changes of gene expression profile in BEND under the combinatory treatment of IFNT, P4 and E2. The BEND were cultured with DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin, and  divided into three groups of three replicates, which includes cells of control group (CON group), cells treated with E2 and P4 (GA group), cells treated with E2, P4 and IFNT (GB group) for sequencing. Comparative gene expression profiling analysis of RNA-seq data for BEND were performed.

first: Lei

last: Cheng


title: Transcriptomic profiling of human SHARPIN deficiency

city: Cologne

first: Hirotsugu

last: Oda

state: NRW

address: Joseph-Stelzmann-Str. 26

department: CECAD

institute: University of Cologne

summary: The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN, and is essential for proper immune responses. Patients with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage. In mice, the loss of Sharpin leads to severe dermatitis due to excessive cell death in keratinocytes. Here we report two patients with SHARPIN deficiency manifesting autoinflammatory symptoms but unexpectedly, no dermatologic manifestations. Patient fibroblasts and B cells showed attenuated canonical NF-κB response and propensity to cell death mediated by TNF superfamily members. Both SHARPIN- and HOIP-deficient patients showed substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient patient with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical role of LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.

overall_design: RNAseq using the pre- and post-treatment whole blood RNA from a patient with a biallelic loss-of-function mutation in SHARPIN

first: Daniel

last: Kastner


title: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow

city: Shenzhen

first: Tongling

last: Huang

address: Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences  1068 Xueyuan Avenue,Shenzhen University Town,  Nanshan District, Shenzhen, China

summary: Extensive bone marrow adipocytes (BMAds) accumulation occurring under diverse pathophysiological conditions leads to bone deterioration.Estrogen-related receptor α (ESRRA) is a key regulator responding to metabolic stress.To explore the transcriptional role of ESRRA in regulating BMAds autonomous genes，bulk RNA-seq transcriptome analysis was performed in adipocyte-specific ESRRA deficiency BMAds lineage cells at 7 days upon adipogenic induction.

overall_design: Bone marrow stromal/stem cells (BMSCs) were isolated from Esrrafl/fl and Adipoq-Cre;Esrrafl/fl mice and were subjected to adipogenic induction for 7 days. Total RNA was extracted and RNA-seq transcriptome analysis was performed in BMSCs-derived BMAds lineage cells.

first: Zhaocheng

last: Lu

first: Zihui

last: Wang

first: Lixin

first: Lu

last: Gao

first: Jun

first: Ning

last: Zhang

first: Chang-An

last: Geng

first: Xiaoli

last: Zhao

first: Huaiyu

first: Chi-Wai

last: Wong

first: Kelvin

last: Yeung

first: Haobo

last: Pan

first: William

first: Min

last: Guan


title: Obesity-disrupted H3K4me3 in hematopoietic stem progentior cells in mice

city: Denver

first: Pluto

last: Bio

state: CO

address: 3575 Ringsby Ct Suite 414

institute: Pluto Bio

summary: Obesity alters long-term functions of hematopoietic stem cells (HSPCs), which are associated with myelopoitic bias and maladapted pro-inflammatory phenotype. H3K4me3 histone modification is generally considered an activating histone marks and is also liked with epigenetic programming for trained immunity. We aim to identify how obesity in mice alter H3K4me3 marks in HSPCs and study its impact on HSPC functions in vitro and in vivo.

overall_design: Comparative H3K4me3 marked peak analysis of ChIP-seq data for hematopoietic stem progenitor cells (Lin-Sca1+cKit+) obtained from the bone marrow of C57B6/J mice fed a high-fat diet or a normal-fat diet.

first: Norifumi

last: Urao

first: Kentaro

last: Takahashi


title: Single-cell RNA-Seq of porcine peripheral blood monocytes

first: Pingping

last: Han

state: Hubei

address: Shizishan Street, Hongshan District

institute: Huazhong Agricultural University

summary: We performed 10× Genomics single-cell RNA sequencing on a mixed population of CD3+ PBMCs isolated from three healthy Large White pigs. Considering that cell resolution of scRNA-seq analysis can be improved based on large datasets, we also downloaded 7 pbulic PBMC scRNA-seq datasets using the same technique (10× Genomics). After rigorous quality control and the removal of batch effects, a total of 14,595 transcriptomes were obtained from 34,220 cells in 8 PBMC samples for subsequent analyses. Using the graph-based uniform manifold approximation and projection (UMAP) clustering algorithm in Seurat package, we identified 14 major cell types (32 clusters) from peripheral blood circulating cells of pigs, and observed remarkable heterogeneity among CD8 T cell types. Upon re-clustering of CD8+ T cells, we defined 4 CD8 T cell subtypes and revealed their potential differentiation trajectories and transcriptomic differences among them. Through single-cell regulatory network inference, we identified key regulatory factors during CD8 T cell differentiation. Moreover, cell-cell communication analysis underscored extensive intercellular interactions among different cell types. Lastly, our cross-species analysis highlighted both similarities and potential differences between species.

overall_design: Peripheral blood mononuclear cells (PBMCs) were collected from 3 healthy Large White pigs at the experimental pig farm of Huazhong Agricultural University. Peripheral blood samples (2 mL) were collected into EDTA anticoagulant tube and gently inverted 2-3 times to ensure thorough mixing. Subsequently, the mixed blood samples were transferred to 15 mL centrifuge tube and treated with 6 mL of red blood cell lysis solution for 15 min, followed by centrifugation at 1600 rpm for 10 min at 4℃ to collect cell pellets. The cell pellets were then re-suspended in cold phosphate-buffered saline (PBS), passed through a 100 μm cell strainer, and incubated with 3 μL CD3ε antibody (clone BB23-8E6-8C8, BD Pharmingen) for 30 min at 4℃ in the dark, followed by FACS to obtain living CD3+ PBMCs. The cell viability assay using trypan blue staining. After cell counting, the samples from the three healthy pigs were mixed in equal volumes to obtain 9×105 cells. The mixed cells were used for constructing 10× genomics sequencing library and subsequent sequencing by Genergy Biotechnology (Shanghai, China)

first: Wei

first: Daoyuan

first: Yalan

last: Wu

first: Xinyun

last: Li

first: Shuhong

first: Mengjin

last: Zhu


title: The plant POLYMERASE-ASSOCIATED FACTOR1 complex links transcription and H2B monoubiquitination genome wide [ChIP-seq]

city: Valencia

first: Jaime

last: Pérez

address: Calle del Ingeniero Fausto Elio, s/n

institute: IBMCP

summary: We have determined the distribution of two subunits of Paf1C, namely ELF7 and VIP3. We have investigated the contribution of the Paf1 complex to the monoubiquitination of H2B genome-wide by ChIP-seq. Finally, we have investigated the effect of defective Paf1C on the distribution of total RNAPII.

sample_organism:
Arabidopsis thaliana
Drosophila melanogaster

overall_design:
FLAG-ELF7 and GFP-VIP3 ChIP-seq of Arabidopsis transgenic lines.
H2Bub and H2B ChIP-seq of wild type and elf7-3 plants. Drosophila spike-ins were added to account for global changes in H2Bub levels.
RNAPII ChIP-seq of wild type and elf7-3 plants.

first: Noel

last: Blanco-Touriñán

last: Pérez-Alemany

first: David

last: Alabadí


title: The plant POLYMERASE ASSOCIATED FACTOR1 complex links transcription and H2B monoubiquitination genome-wide [RNA-seq]

summary: The transcriptome of WT, elf7-3 and hub1-4 seedlings was obtained by RNA-seq in order to determine the effect of mutations on transcript levels and to correlate them with chromatin features determined by ChIP-seq.

overall_design: RNA-seq of wild type, elf7-3 and hub1-4 plants.


title: Single cell RNA sequencing of in vivo OVCAR8 tumor cells

first: Lili

last: Yang

address: 610 Charles E. Young Dr. East, TLSB 3045

institute: UCLA Lili Yang Lab

overall_design: Gene expression profiling of tumor cells in an in vivo study using an MM-FG human multiple myeloma xenograft NSG mouse model

first: Yan-Ruide

first: Yang

last: Zhou

first: Jiaji

last: Yu

first: Jeong

last: Kim

first: Miao


title: Multi-omics analysis of green lineage osmotic stress pathways unveils crucial roles of different cellular compartments

city: Mountain View

first: Josep

middle: Vilarrasa

last: Blasi

state: CA

address: 787 corto street

institute: Aetna

summary: Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allowed us to infer their evolutionary conservation and divergence across these lineages.  76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling.  We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

overall_design: Investigate the gene expression upon osmotic treatment using sodium chloride and mannitol of Chlamydomonas reihardtii (CC-4533)

last: Vilarrasa Blasi

first: José

last: Dinneny


title: One-pot method for preparing DNA, RNA, and protein for multiomics analysis II

first: Stephanie

last: Byrum

summary: Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe a method for preparing high-quality, sequencing-ready DNA and RNA, and either intact proteins or mass-spectrometry-ready peptides for whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver, and lung tissue.

overall_design: To assess the quality and reproducibility of our novel multiomics sample preparation workflow, we prepared DNA, RNA, and peptides from four triple-negative breast cancer cell lines that exhibit different degrees of malignancy: MCF10A (non-tumorigenic), MCFNeoT (benign hyperplasia), MCFT1 (atypical hyperplasia), MCFCA1 (invasive cancer). Three biological replicates were carried out with each cell line.

last: Biedka

first: Duah

last: Alkam

first: Charity

last: Washam

first: Svitlana

last: Yablonska

first: Aaron

last: Storey

first: Jonathan

last: Minden


title: Single cell RNA sequencing to study the in vivo behaviour of allogeneic HSC-derived CAR-engineered NKT cells

overall_design: Gene expression profiling of therapeutic cells in an in vivo study using an MM-FG human multiple myeloma xenograft NSG mouse model

last: Li


title: Single cell RNA sequencing of the in vivo mouse TME

overall_design: Gene expression profiling of mouse immune cells in an in vivo study using an MM-FG human multiple myeloma xenograft NSG mouse model


title: Single cell RNA sequencing monitoring the development of allogeneic HSC-derived CAR-engineered NKT cells


title: Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNAmethylation profiling [EPIC v1]

city: Darlinghurst

first: Timothy

middle: John

last: Peters

state: NSW

address: 384 Victoria St

department: Immunology

country: Australia

institute: Garvan Institute of Medical Research

summary: Genome wide DNA methylation profiling of a suite of biological samples for technical evaluation of the HumanMethylationEPIC v2.0 BeadChip. Samples include prostate (LNCaP, PrEC) and breast cancer (MCF7, TAMR) cell lines, as well as primary tumour samples from prostate (SYN*) and breast (HCI*|Gar*).

overall_design: To assess the performance of the EPICv2 array we profiled and ran a series of technical analyses on 40 DNA samples from a range of sample types, including: a transformed prostate cancer cell line (LNCaP), primary cell cultures of prostate epithelial cells (PrEC), fresh-frozen human prostate tumour samples (PrCa), transformed breast cancer cell lines (MCF7 and TAMR), and human breast cancer patient derived xenografts (PDX). LNCaP and PrEC were assayed each in triplicate at three different dilutions (500ng, 250ng and 125ng). A subset of the TAMR cells and PDXs had undergone treatment with the hypomethylating drug decitabine.

first: T

first: R

last: Pidsley

first: B

last: Meyer

first: J

last: Achinger-Kawecka

first: S

last: Clark


title: Characterisation and reproducibility of theHumanMethylationEPIC v2.0 BeadChip for DNAmethylation profiling [EPIC v2]


title: Epigenetic sequencing of allogeneic HSC-derived CAR-engineered NKT cells

first: Shuo

address: 630 Charles E Young Dr S

overall_design: Genomic DNA from samples was isolated with QIAGEN DNeasy Blood & Tissue kit following the corresponding manufacturer’s protocols. The isolated genomic DNA was sonicated by Covaris system. Size selection by Ampure XP beads (Beckman-Coulter) was performed to enrich DNA fragments of around 250 bp in average sizes before library construction. In brief, 0.7 volume beads were added to the fragmented genomic DNA. After incubation, the supernatant was transferred to a new tube and an additional 1.3 volume beads were added. After washing two times with 80% ethanol, genomic DNA was eluted with 40 µl EB buffer. Subsequently, 200 ng genomic DNA was used for DNA library preparation with NEBNext Ultra II DNA library prep Kit (Cat#E7645). DNA library preparation has the following steps: DNA fragments end prep, adaptor ligation, size selection or purification of Adaptor-ligated DNA with Ampure XP beads, bisulfite conversion by Epitect Fast DNA Bisulfite Kits (QIAGEN, Cat#59826), PCR enrichment of Adaptor-ligated DNA, and purification of PCR product with 0.9 x Ampure XP beads. The average size of resulting DNA fragments was around 480 bp, validated by Tapestation. The libraries were then subjected to sequencing on Illumina NovaSeq sequencer with 2 x 150 bp configuration (Azenta). The raw bisulfite sequencing reads were trimmed by cutadapt to remove sequencing adapters. The trimmed reads were then aligned to hg19 reference genome by Bismark. Then duplicated reads from PCR amplification were identified and removed by Bismark. The deduplicated reads were then sorted and indexed using Samtools. After that, the methylated and unmethylated cytosines were counted at every CpG site by Bismark. The methylation at a gene promoter region was quantified as the beta value, i.e., the ratio between the number of methylated cytosines and the total number of cytosines mapped to the region. The promoter region of a gene was identified by GeneHancer with the highest confidence score. The beta values were then used for heatmap visualization.

first: Yan

first: Yang

last: Zhou

first: Jiaji

last: Yu

first: Lili

last: Yang


title: Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNAmethylation profiling

overall_design: Refer to individual Series


title: Expression analysis of hCMEC/D3 PATJ KD cells.

city: Palma

first: Cristofol

last: Vives-Bauza

address: Crta Valldemossa km.7.5

institute: University of Balearic Islands

summary:
Stroke is a leading cause of long-term disability worldwide. Patients with good recovery after ischemic stroke present a higher downregulation of PATJ comparing with patients with marked disability. To understand the effects of PATJ downregulation we generated PATJ-knockdown human microvascular ECs.
We used microarrays to detail the gene expression to identify the modulated genes due to the depletion of the PATJ gene.

overall_design: To deeper study the molecular events underlying PATJ downregulation in response to ischemic stroke, we generated hCMEC/D3 cell clones stably depleted of PATJ by using lentiviral particles harboring shRNA against PATJ. Three PATJ KD clones were selected for further analysis due to a progressive depletion of the PATJ species

first: Vives-Bauzá

last: Cristòfol

first: Fleischer

last: Aarne


title: Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling [WGBS]

summary: Genome wide DNA methylation profiling of a suite of biological samples for technical evaluation of the HumanMethylationEPIC v2.0 BeadChip. Samples include prostate (LNCaP, PrEC) and breast cancer (MCF7, TAMR) cell lines, as well as primary tumour samples from prostate (SYN*) and breast (FD*).

overall_design: To assess the performance of the EPICv2 array we profiled and ran a series of technical analyses on 40 DNA samples from a range of sample types, including: a transformed prostate cancer cell line (LNCaP), primary cell cultures of prostate epithelial cells (PrEC), fresh-frozen human prostate tumour samples (PrCa), transformed breast cancer cell lines (MCF7 and TAMR), and human breast cancer patient derived xenografts (PDX). LNCaP and PrEC were assayed each in triplicate at three different dilutions (500ng, 250ng and 125ng). A subset of the TAMR cells and PDXs had undergone treatment with the hypomethylating drug decitabine. Matched WGBS of prostate tumour samples was used for technical comparisons.


title: The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

city: Cleveland

first: Ann

last: Harris

state: OH

address: 2109 Adelbert Rd

department: Genetics and Genome Sciences

institute: Case Western Reserve University

summary: We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line.  The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

overall_design: Analysis of the 3D chromatin architecture with 4C-seq at the CFTR locus to determine interaction frequencies between elements in 16HBE14o- or Caco2 cells with non-coding deletions between enhancers and the CFTR promoter

first: Jenny

last: Kerschner

first: Fredrick

last: Meckler

first: Giuliana

last: Coatti

first: Nirbhayaditya

last: Vaghela

first: Alekh

last: Paranjapye


title: Gene expression by BRAF overexpression in DU145 cells

city: Jinan

first: Jing

last: Hu

address: Wenhua xi road

department: Department of Pathology

institute: Shandong University

summary: The aim of this research was to confirm the regulatory effect of wild-type and mutant BRAF on gene expression in the development of prostate cancer.

overall_design: In this research, there were three samples examed including sample of negtive control cells (DU145-P-Enter), sample of cells with wild-type BRAF overexpressed (DU145-WT) and sample of cells with BRAFL597R mutant plasmids transfected(DU145-L597R).

first: Bo

last: Han


title: Neonatal CD8+ T cells resist terminal exhaustion during chronic infection [RNAseq_day30]

city: Ithaca

first: Andrew

last: Grimson

address: 445 Biotechnology Building

department: Molecular Biology & Genetics

institute: Cornell University

summary: Chronic viral infections represent a major public health problem.  Although it is well understood that neonates and adults respond differently to chronic viral infections (HIV, HCV), the underlying mechanisms remain poorly understood.  In this study, we transferred neonatal and adult CD8+ T cells into a mouse model of chronic infection (LCMV clone 13) and dissected out the key cell-intrinsic differences that alter their ability to protect the host.  Interestingly, we found that neonatal CD8+ T cells preferentially become effector cells early in chronic infection when compared to adult CD8+ T cells, and resist commitment to the exhausted differentiation trajectory. Further, neonatal CD8+ T cells are preferentially maintained as stem-like exhausted progenitors rather than terminally exhausted cells during the chronic phase of infection. The altered differentiation trajectories of neonatal and adult CD8+ T cells is functionally significant, for the neonatal cells protect from viral replication. Together, our work demonstrates how cell-intrinsic differences between neonatal and adult CD8+ T cells influences key cell fate decisions during chronic infection.

overall_design: Adult (8-12 weeks old) and neonatal (5-7 days old) P14 transgenic CD8+ T cells were magnetically enriched, then co-adoptively transferred to congenically-distinct wildtype recipient mice (2,000 of each cell type). 1 day after adoptive transfer, recipient mice were infected with 2e6 PFU LCMV clone 13 systemically. 30 days post-infection, adult and neonatal donor cells were FACS sorted to at least 95% purity and subjected to RNA sequencing.

first: Viviana

middle: I

last: Maymí

first: Hongya

last: Zhu

first: Mason

last: Jager

first: Shawn

last: Johnson

first: Rodman

last: Getchell

first: James

last: Casey

first: Jennifer

middle: K

last: Grenier

first: John

last: Wherry

first: Norah

last: Smith

first: Brian

last: Rudd


title: Human fallopian tube single-cell analysis reveals monocytic transcriptional and interaction changes in high-grade serous ovarian cancers

city: Madison

first: Huy

last: Dinh

state: WI

address: 1111 Highland Avenue

institute: University of Wisconsin-Madison

summary: Tumorigenesis for most high-grade serous ovarian cancers (HGSCs) likely initiates from fallopian tube (FT) epithelia. While epithelial subtypes have been characterized using single-cell RNA-sequencing (scRNA-Seq), heterogeneity of other cellular compartments and their involvement in tumor progression are poorly defined. Integrated analysis of human FT scRNA-Seq data and other relevant tissues, including HGSC tumors, revealed greater transcriptional diversity of immune and stromal cells. We identify an unprecedented abundance of monocytes in human FT myeloid cells across two independent donor cohorts. The ratio of macrophages to monocytes are relatively similar between benign FTs, ovaries, and adjacent normal tissues, but is significantly greater in tumor. FT-defined monocyte and macrophage signatures, cell-cell communication, and gene set enrichment analysis identified monocyte- and macrophage-specific ligand-receptor interactions and functional pathways in tumors and adjacent normal tissue. Further reanalysis of tumor scRNA-Seq from HGSC patients suggested different monocyte and macrophage subsets associated with neoadjuvant chemotherapy treatment. Taken together, our work provides evidence that an altered FT immune composition could inform early detection markers in HGSCs.

overall_design: Single-cell RNA sequencing of human ovarian cancers.

first: Joshua

last: Brand

first: Marcela

last: Haro

first: Xianzhi

last: Lin

first: Stephanie

last: McGregor

first: Kate

last: Lawrenson

middle: Q


title: Repression of Cebpa-L1-AD-En2 enhancer decreases Cebpa expression in inguinal white adipose tissue and affects adipose tissue development

city: Chengdu

first: Xiaokai

last: Li

state: Sichuan

address: Huiming road #211

institute: Sichuan Agricultural University

summary: To investigate whether Cebpa-L1-AD-En2 produces a marked effect on adipogenesis in mouse adipose tissue, we injected the dCas9-KARB-En2 lentiviral system (iWAT_Cebpa-En2 group) or dCas9-KRAB-GFP (Control group) directly into mouse inguinal white adipose tissue (iWAT). We conducted RNA-seq analysis on the iWAT of iWAT-En2 group mice and control group mice at 13 weeks of age

overall_design: Gene expression profiling analysis of mRNA-seq data for  iWAT-En2 group and control group in  mouse iWAT at 13 weeks of age.

first: Sha

last: Zeng

first: Yu

last: Zhang


title: Transcriptomic and metabolic effect of immune checkpoint inhibitors on cellular components in renal cell tumor microenvironment and in Sunitinib resistant renal cell carcinoma [II]

city: Silver Spring

first: Carole

last: Sourbier

address: 10903 New Hampshire Ave

department: CBER

institute: FDA

summary: Tumor microenvironmental cellular components like bone marrow cells stromal cells macrophages changes due to sunitinib resistance may affect their efficacy in advanced stage renal cell carcinomas. Immune checkpoint inhibitors (ICIs) have been approved as 2nd line therapy over acquired resistance. Each ICI uniquely affected different cellular components. Better understanding of transcriptomic, metabolomic or proteomic changes due to ICI treatment is necessary to development proper immunotherapy.

overall_design: Stromal cells were treated with two PD1 inhibitors ICIs (P: Pembrolizumab, Niv: Nivolumab) for 24hr and RNA was extracted after each treatment. Differential gene expression analysis of each of the treated samples was performed using RNA-seq.

first: Susmita

last: Ghosh

first: Chao-Kai

last: Chou

first: Wells

last: Wu


title: Transcriptomic and metabolic effect of immune checkpoint inhibitors on cellular components in renal cell tumor microenvironment and in Sunitinib resistant renal cell carcinoma [III]

overall_design: Stromal cells were treated with three ICIs (At: Atezolizumab, Av: Avelumab and D: Durvalumab) for 24hr and RNA was extracted after each treatment. Differential gene expression analysis of each of the treated samples was performed using RNA-seq.


title: CD74 deficiency impairs Treg accumulation and function in the tumor

city: Paris

first: Eliane

last: Piaggio

address: 26 rue d'Ulm

institute: Institut Curie

summary: Regulatory T cells (Tregs) are known to actively adapt to the microenvironment in which they reside. However, how Tregs are reshaped in the tumor tissue remains under defined. We observed that in human tumors, Tregs selectively overexpress at the surface CD74, the MHC class II-associated invariant chain. Beyond its role in antigen presentation, CD74 has been described to impact gene transcription and cell migration, however, its role in Treg biology remains unknown. Here, we found that CD74KO Tregs exhibited major defects in the organization of their actin cytoskeleton and intracellular organelles and, consistently, they failed to accumulate in tumors and to suppress the anti-tumoral T-cell response. Strikingly, this phenotype did not result from a generic defect of CD74-deficient Tregs as their phenotype, proliferation, and suppressive function in vitro and in vivo during Graft-versus-Host disease were not affected. Our results reveal a new role for CD74 in Tregs and uncover CD74 potential as novel target to interfere with Treg anti-tumor activity.

overall_design: Regulatory T cells isolated from healthy-donor blood are expanded for bulk-RNA sequencing analysis

first: Elisa

last: Bonnin

first: Jimena

last: Tosello Boari


title: Transcriptomic and metabolic effect of immune checkpoint inhibitors on cellular components in renal cell tumor microenvironment and in Sunitinib resistant renal cell carcinoma

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


title: Transcriptomic and metabolic effect of immune checkpoint inhibitors on cellular components in renal cell tumor microenvironment and in Sunitinib resistant renal cell carcinoma [1]

overall_design: Bone marrow cells were treated with two PD1 inhibitors ICIs (P: Pembrolizumab, Niv: Nivolumab) for 24hr and RNA was extracted after each treatment. Differential gene expression analysis of each of the treated samples was performed using RNA-seq.


title: Neonatal CD8+ T cells resist terminal exhaustion during chronic infection [RNAseq_day8]

first: Andrew

last: Grimson

state: NY

summary: Chronic viral infections represent a major public health problem.  Although it is well understood that neonates and adults respond differently to chronic viral infections (HIV, HCV), the underlying mechanisms remain poorly understood.  In this study, we transferred neonatal and adult CD8+ T cells into a mouse model of chronic infection (LCMV clone 13) and dissected out the key cell-intrinsic differences that alter their ability to protect the host.  Interestingly, we found that neonatal CD8+ T cells preferentially become effector cells early in chronic infection when compared to adult CD8+ T cells, and resist commitment to the exhausted differentiation trajectory. Further, neonatal CD8+ T cells are preferentially maintained as stem-like exhausted progenitors rather than terminally exhausted cells during the chronic phase of infection. The altered differentiation trajectories of neonatal and adult CD8+ T cells is functionally significant, for the neonatal cells protect from viral replication at the cost of early-onset immunopathology. Together, our work demonstrates how cell-intrinsic differences between neonatal and adult CD8+ T cells influences key cell fate decisions during chronic infection.

overall_design: Adult (8-12 weeks old) or neonatal (5-7 days old) P14 transgenic CD8+ T cells were magnetically enriched, then adoptively transferred to congenically-distinct wildtype recipient mice (4,000 of each cell type). 1 day after adoptive transfer, recipient mice were infected with 2e6 PFU LCMV clone 13 systemically. 8 days post-infection, adult or neonatal donor cells were FACS sorted to at least 95% purity and subjected to RNA sequencing.

first: Viviana

last: Maymí

first: Hongya

last: Zhu

first: Mason

last: Jager

first: Shawn

last: Johnson

first: Rodman

last: Getchell

first: James

middle: W

last: Casey

first: Jennifer

last: Grenier

first: John

middle: E

last: Wherry

first: Norah

last: Smith

first: Brian

last: Rudd


title: Resistance to Naïve and Formative Pluripotency Conversion in RSeT Human Embryonic Stem Cells

city: Bethesda

first: Kory

state: MD

address: 10/3B01, 9000 Rockville Pike

department: DIR IT & Bioinformatics

institute: NINDS/NIH

summary: One of the most important properties of human embryonic stem cells (hESCs) is related to their primed and naïve pluripotent states. Our previous meta-analysis indicates the existence of heterogeneous pluripotent states derived from diverse naïve protocols. In this study, we have characterized a commercial medium (RSeT)-based pluripotent state under various growth conditions. Notably, RSeT hESCs can circumvent hypoxic growth conditions as required by naïve hESCs, in which some RSeT cells (e.g., H1 cells) exhibit much lower single cell plating efficiency, having altered or much retarded cell growth under both normoxia and hypoxia. Evidently, hPSCs lack many transcriptomic hallmarks of naïve and formative pluripotency (a phase between naive and primed states). Integrative transcriptome analysis suggests our primed and RSeT hESCs are close to the early stage of post-implantation embryos, similar to the previously reported primary hESCs and early hESC cultures. Moreover, RSeT hESCs did not express naïve surface markers such as CD75, SUSD2, and CD130 at a significant level. Biochemically, RSeT hESCs exhibit a differential dependency of FGF2 and co-independency of both Janus kinase (JAK) and TGFβ signaling in a cell-line-specific manner. Thus, RSeT hESCs represent a previously unrecognized pluripotent state downstream of formative pluripotency. Our data suggest that human naïve pluripotent potentials may be restricted in RSeT medium. Hence, this study provides new insights into pluripotent state transitions in vitro.

overall_design: There were 24 hESC samples, representing three different hESC lines (H1, H7, H9), cultured in hESC medium (Primed) and RSeT medium under normoxia or hypoxic growth condition.

first: Kevin

last: Chen

first: Kyeyoon

last: Park

first: Forest

last: Yang

first: Yang

last: Fann

first: Pamela

last: Robey

first: Barbara

last: Mallon


title: Cebpa in adipocyte differentiation

first: Xiaokai

last: Li