title: Camk2a-neuron specific FLAG-Ago HITS-CLIP

status: Public on Jan 31 2023

city: Aarhus N

first: Morten

middle: T

last: Veno

state: DK

address: Aabogade 15

country: Denmark

institute: Omiics ApS

type: Non-coding RNA profiling by high throughput sequencing

summary: The HITS-CLIP procedure was used to detect miRNA mediated Argonaute (Ago) binding in Camk2a mouse neurons.

overall_design: In three independent experiments (experiment 1-3), mice that express a Flag-tagged Ago2 protein specifically in Camk2+ neurons in the forebrain (Camk2a-cre; Rosa-Stopfl/fl-Flag_x0002_Ago2) and control mice (Rosa-Stopfl/fl-Flag-Ago2) were subject to FLAG-Ago HITS-CLIP. This was done to detect potential miRNA seed targeting in Camk2a neurons.

last: Venø

first: Anne

last: Schaefer


title: Next Generation Sequencing Facilitates Quantitative Analysis of Murine Salivary Gland Transcriptomes at 8 and 32 weeks

city: Rochester

first: Catherine

middle: E.

last: Ovitt

state: NY

address: 601 Elmwood Ave, Box 611

department: Center for Oral Biology

country: USA

institute: University of Rochester Medical Center

type: Expression profiling by high throughput sequencing

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular functions. The goal of this study is to compare NGS-derived salivary gland transcriptome profilings (RNA-seq) to better understand the molecular changes in adult sublingual glands in the absence of the Nkx2.3 transcription factors.
Methods: Female sublingual salivary gland mRNA profiles were generated by deep sequencing, in 4 replicates for Nkx2.3 knockout mice, using Illumina. The sequence reads that passed quality filters were analyzed at the gene level with STAR followed by DESeq2.
Results: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the mouse genome (build mm9) and identified 496 genes that were differentially expressed between the wildtype and Nkx2.3-knockout murine female sublingual salivary glands . RNA-seq data provided valuable insights into the nature of the functional differences resulting from the Nkx2.3 disruption
Conclusions: Our study represents the first detailed analysis of sublingal salivary gland transcriptome profiling differences resulting from the Nkx2.3 disruption, with biologic replicates, generated by RNA-seq technology. Our results confirmed functions of many previously studied genes. We conclude that RNA-seq-based transcriptome characterization would offer a comprehensive and sensitive evaluation of the gene expression.

overall_design: Female sublingual salivary gland mRNA profiles of 8- and 32-week old mice were generated by deep sequencing, in 4 replicates using Illumina.

first: James

middle: E

last: Melvin


title: Gene expression in untreated and TGF-b2-treated human bone marrow-derived mesenchymal stem cells

status: Public on Jan 30 2023

city: Raleigh

first: Alix

middle: Kay

last: Berglund

state: NC

address: 1060 William Moore Dr

department: Clinical Sciences

institute: North Carolina State University

summary: To investigate the transcriptome level changes in human bone marrow-derived mesenchymal stem cells following treatment with TGF-b2, we used RNA-sequencing to compare gene expression between cells from the same donors cultured in standard media or media containing 1 ng/ml TGF-b2.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for human bone marrow-derived mesenchymal stem cells cultured in standard media or media with TGF-b2.

middle: K

first: Lauren

middle: V

last: Schnabel


title: Single-cell transcriptomics of mammalian prion diseases identifies dynamic gene signatures shared between species [mouse_scRNAseq]

city: London

first: Athanasios

last: Dimitriadis

address: 33 Cleveland Street

country: United Kingdom

institute: MRC Prion Unit at UCL

summary: Mammalian prion diseases are fatal and transmissible neurological conditions caused by the propagation of prions, self-replicating multimeric assemblies of misfolded forms of host cellular prion protein. Despite extensive studies investigating the changes in transcriptional profiles in prion diseases the mechanisms by which prion diseases induce cellular toxicity, including changes in gene expression profiles are yet to be fully characterized. Here, we took advantage of the recent developments in single-cell technologies and performed an unbiased whole-transcriptome single-nucleus transcriptomic analysis in prion disease.

overall_design: FVB inbred mice were intracerebrally inoculated with infectious RML brain homogenate, uninfected CD1 brain homogenate, or PBS and culled at 5 time points (20 dpi, 40 dpi , 80 dpi, 120 dpi, and when scrapie sickness was confirmed). Half brains were flash frozen and stored at -80oC until further processing. For each sample, the brain was left to thaw and a section of the frontal lobe was cut and processed for scRNA-seq using the SPLiT-seq protocol.

first: Fuquan

last: Zhang

first: Thomas

last: Murphy

last: Trainer

first: Zane

last: Jaunmuktane

first: Christian

last: Schmidt

first: Tamsin

last: Nazari

first: Jacqueline

last: Linehan

first: Sebastian

last: Brandner

first: John

last: Collinge

first: Simon

last: Mead

first: Emmanuelle

last: Vire


title: Single-cell transcriptomics of mammalian prion diseases identifies dynamic gene signatures shared between species [human_scRNAseq]

overall_design:
We selected 10 individuals from archived tissue collected by the National Prion Clinic. For our control group, we included frontal cortex samples from individuals with low-level AD pathology or pathological ageing provided by the Queen Square Brain Bank. These samples (N = 10) were matched for sex (male = 4 sCJD and 5 controls; female = 6 sCJD and 5 controls) but not age (mean age for sCJD = 70.4 years, SD = 8.6; mean age for controls = 83.7 years, SD = 8.6).
We were also able to source 3 non-dominant frontal lobe biopsy samples from sCJD patients. The control samples for this group included frontal lobe biopsies from non-neurodegenerative disease controls with mixed clinical diagnoses and only non-specific minor histological changes (pathological non-diagnostic samples), provided by BRAIN UK. These samples (N = 3) were sampled similarly to the biopsies and individuals were matched for sex (male = 2 sCJD and 2 controls; female = 1 sCJD and 1 control) while the age was matched only partially (mean age for sCJD = 56.6, SD = 13.3; mean age for controls = 60.6, SD = 3.3).


title: Single-cell transcriptomics of mammalian prion diseases identifies dynamic gene signatures shared between species [human_bulkRNAseq]


title: RNA sequencing in wild type and GCH1-knockdown (KD) BV2 microglial cells

city: Shanghai

first: Zheyu

address: Yueyang Road

country: China

institute: CAS

summary: We conducted high-throughput sequencing and bioinformatics analysis of GCH1-KD BV2 microglial cells treated with adenovirus. Their RNA was extracted and analyzed, and the results were verified by quantitative real-time polymerase chain reaction (PCR). This study explored the lncRNAs, miRNAs, circRNAs and mRNAs regulated by GCH1 and revealed a possible mechanism of GCH1 in microglia activation.

overall_design: RNA expression profiles of wild type and GCH1-KD BV2 microglial cells were generated by high-throughput sequencing, in triplicate, using Illumina NovaSeq 6000 sequencer.

first: Chunyang

last: Meng

first: Yanhu

last: Liang


title: miR-197-3p promotes osteosarcoma stemness and chemo-resistance by inhibiting SPOPL

city: Shenyang

first: Ming

last: He

state: Liaoning

address: No. 36 Sanhao Street

institute: Shengjing Hospital of China Medical University

summary:
First-line treatment for osteosarcoma includes chemotherapy and surgery. However, the five-year survival rate of refractory osteosarcoma remains unsatisfactory. Osteosarcoma cancer stem cells, possessing stemness and chemoresistance, are one of the critical causes for poor response to chemotherapy. Elucidating regulatory signaling pathways of osteosarcoma cancer stem cells may provide a rationale for improving regimens against chemoresistant osteosarcoma. Methotrexate (MTX)-resistant osteosarcoma cells were established. microRNA expression profiles were used for detecting differentially expressed microRNA in resistant clones and the parental cells. microRNA target databases were employed to predict potential microRNA and mRNA interaction. Flow cytometry was performed to measure stem cell marker Prominin-1 (CD133) positive cells. Im-munofluorescence staining was applied to detect CD133 expression. miR-197-3p mimic or an-ti-miR-197-3p stably transfected cells were used to generate xenograft models. In the study, we found miR-197-3p was increased in MTX-resistant cell lines. Overexpression of miR-197-3p en-hanced the expression of cancer stem cell markers CD133, Octamer-binding protein 4 (OCT4), Transcription factor SOX-2 (SOX2), and Homeobox protein NANOG (NANOG), as well as chemoresistance-associated genes ATP-dependent translocase ABCB1 (ABCB1) and Broad substrate specificity ATP-binding cassette transporter ABCG2 (ABCG2), whereas miR-197-3p knockdown inhibited stemness and recovered sensitivity to MTX. We also classified the tumor suppressor Speckle-type POZ protein-like (SPOPL) as a target of miR-197-3p. The miR-197-3p mutation that cannot combine SPOPL promoter regions was unable to sustain stemness or chemoresistance. Collectively, we discovered miR-197-3p conferred osteosarcoma stemness and chemotherapy resistance by targeting SPOPL, prompting promising therapeutic candidates for refractory oste-osarcoma treatment.
miRNA qPCR assay

overall_design: MicroRNA (miRNA) were enriched and purified by miRNeasy Micro Kit (catalog: 217084, QIAGEN, Hilden, Germany), followed by cDNA synthesis with miRCURY LNA RT Kit (catalog: 339340, QIAGEN). miRCURY LNA miRNA Custom PCR Panels (catalog: 339330, QIAGEN) were then used to investigate differentially expressed miRNA in the parental U2OS cells and MTX-resistant U2OS cells.


title: Transcriptome-wide profiling of Yeast Phosphofructokinase 1 and 2 RNA targets

city: Guildford

first: Waleed

last: Albihlal

state: Surrey

address: Stag Hill Campus

department: Microbial Sciences

institute: University of Surrey

summary: The RNA targets of the yeast Pfk1 and Pfk2 were mapped on a transcriptome scale using formaldehyde-assisted crosslinking and RNA-protein immunoprecipitation followed by high throuput sequencing (fRIP-seq). Tandem affinity purification (TAP) tagged Pfk1 and Pfk2 were usesd to capture the transcritps bound by Pfk1 and Pfk2. We also included a mock IP control i.e. wild type treated exactly the same way as the fRIP-seq samples to account for non-specific RNA interactions with the beads. Moreover, we ran total RNA-seq on wild type control for data normalization. We found that Pfk1 and Pfk2 bind mainly to mRNAs that code for proteins invovled in distinct functions . Our data suggests that, besides their well-characterized functions as glycolytic enzymes, they perfom additional functions as RNA-binding involved in posttranscriptional regulation of gene expression.

overall_design: Our aim was to map transcripts bound by the yeast Pfk1 and Pfk2 to explore their RNA-bining functions and to obtain and insight into their potential secondary function as posttranscriptional regulators of gene expression. We performed formaldehyde assisted crosslinking followed by RNA-protein immuniprecipitation (fRIP-seq) to identify RNAs bound by Pfk1 and Pfk2 under normal physiological conditon. Diploid yeast cells expressing either Pfk1 or Pfk2 with Tandem Affinity purification (TAP) tags as well as dipliod wild type cells for mock IP control were grown in rich YPD media to mid-log phase (OD600 = 0.6) then crosslinked with low concentration of formaldehyde to a final concentration of 0.3% followed by quenching of crosslinking using glycine (final concentration 125 mM). Cells were pelleted then washed three times with 1X PBS then flash frozen in liquid nitroen. Frozen cell pellets were ground under cryogenic conditions using mortar and pestel to fine powders then powders were resuspended in fRIP lysis buffer. Pfk1:TAP-RNA and Pfk2:TAP-RNA were captured using Pan mouse IgG beads. Crosslinking was reveresed followd by proteinase K digestion of proteins then RNA was extracted using standard acidic phenol method. RNA was then converted to cDNA followed by second strand synthesis then double-stranded cDNA was sheared using sonication. Sequencing libraries were constructed using Bioline JetSeq libraray preparation kit as described in product manual. We also perfomed total RNA-seq from wild type diploid cells for data normalization. Samples were sequenced on Illumina HiSeq2500 with minimum of 5 million reads per library. Our data showed that both Pfk1 and Pfk2 bind to hunderds on mRNAs coding for proteins involved in various cellular functions. Interestingly, our data suggests that Pfk1 and Pfk2 bind to mRNAs coding for proteins involved in distinct functions indicating that the two proteins are involved in different cellular functions as posttranscriptional regulators.

first: W

middle: S

first: A

middle: P

last: Gerber


title: Throughout in vitro first spermatogenic wave: next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants

city: Paris

first: Hélène

last: Lopez-Maestre

address: 25-28 rue du Dr Roux

country: France

institute: Institut Pasteur

summary: The in vitro spermatogenesis procedure, once optimized, should be proposed to survival prepubertal patients at high risk for malignant contamination in their testis, for example in case of acute leukaemia. Differentially expressed genes between the in vitro cultured testes and the aged-matched in vivo controls will be explored by a transcriptomic analysis using the RNA-Seq technology (single-read 50 bp, PolyA-enriched) at D0, D4, D16 and D30 of culture using our reference culture medium (one-step culture), and at the same time points after an initial culture step with growth factors (two-step culture). This approach will allow us to identify the signalling pathways or molecular mechanisms that are deregulated in vitro. Moreover, a comparison of transcriptomic analyses to cryopreserved testicular fragments will allow us to identify the potential impact of cryopreservation procedures for fragments after thawing (D0) and explants at the end of culture with our reference culture medium (D30).

overall_design: Testicular fragments from 6.5 days postpartum (dpp) mice were cultured from fresh or controlled slow frozen (CSF) testicular tissue. Cultures were stopped at key time points of the first spermatogenic wave: day 4, D16, and D30 for fresh tissue in order to evaluate the impact of in vitro culture and stopped at D30 for frozen-thawed tissue to evaluate the impact of cryopreservation. Testes from mice of 6.5, 10.5, 22.5, and 36.5 dpp correspond to respective age-matched in vivo controls. RNAs from testicular explants (n=3×6 fragments per condition) as well as their respective in vivo controls (n=3×6 fragments per condition) were extracted and analysed by RNA-seq. Biological replicates samples: in vivo 6.5 (fresh: n=3; CSF: n=2), 10.5 (n=3), 22.5 (n=3), 36.5 (n=2) dpp; in vitro D4 (n=3), D16 (n=2), and D30 (fresh tissue: n=3; CSF: n=1).

first: Ludovic

last: Dumont

middle: L

last: Maestre

first: Frédéric

last: Chalmel


title: Targeting METTL3 reprograms tumor microenvironment to improve cancer immunotherapy [RNA-seq]

city: Wuhan

first: Haisheng

last: Yu

state: Hubei

address: 115, Donghu road, Wuchang district

department: Medical Research Institute

institute: Wuhan University

summary: Methyltransferase-like 3 (METTL3), a core catalytic subunit for RNA N6-methyladenosine (m6A) modification, plays a crucial role in regulating gene expression to govern various physiological and pathological processes. However, whether and how METTL3 regulates TME and anti-tumor immunity in non-small-cell lung cancer (NSCLC) remains poorly understood. To understand the function and mechanism of METTL3 in NSCLC TME, we compare the differential expression genes (DEGs) in STM2457 (METTL3 specific inhibitor) versus DMSO treated lewis lung carcinoma cells (LLC).

overall_design: RNA-seq in LLC cells treated with 5μM STM2457 versus DMSO for 46 hours.

first: Jing

last: Liu

first: Xia

last: Bu

first: Zhiqiang

last: Ma

first: Yingmeng

last: Yao

first: Jinfeng

last: Li

first: Tiantian

last: Zhang

first: Wenjing

last: Song

first: Xiangling

last: Xiao

first: Yishuang

last: Sun

first: Wenjun

last: Xiong

first: Jie

last: Shi

first: Panpan

last: Dai

first: Bolin

last: Xiang

first: Hongtao

last: Duan

first: Xiaolong

last: Yan

first: Fei

last: Wu

first: Wen

middle: C

first: Haojian

first: Frank

middle: J

last: Slack

first: Housheng

middle: H

first: Gordon

last: Freeman

first: Wenyi

last: Wei

first: Jinfang


title: The role of a mammalian network in regulating ischemic stroke outcome III

city: Kuopio

first: Flavia

last: Scoyni

address: Neulaniementie 2

department: A.I. Virtanen Institute for Molecular Sciences

country: Finland

institute: University of Eastern Finland

summary:
To detect circRNAs, lncRNAs and mRNAs in oxygen and glucose deprivation (OGD) conditions we cultured cortical neurons Embryonic day 15 and subjected them to OGD
We performed differentially expression analysis of RNA-seq of wild-type and ciRS-7 KO neurons

overall_design: Comparative gene expression analysis of RNA-seq data for wild-type and ciRS-7 KO cortical neurons treated for 12h with OGD in respect of the normoxic conditions (n= 5 biological replicates)

first: Luca

last: Giudice


title: Effect of overexpression of PHF19 in multiple myeloma cells [RNA-seq]

city: Boston

first: TENGTENG

last: YU

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

summary:
To investigate the transcriptomic effect of PHF19 overexpression in MM cells.
We then performed gene expression profiling analysis using data obtained from RNA-seq of AMO1 cells with differential PHF19 expression.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for empty vector MM cells and its PHF19  overexpression cells (PHF19 OE  and EV cells)

first: Tengteng

last: Yu

first: Mengyu

last: Zhao


title: The role of a mammalian network in regulating ischemic stroke outcome II

summary:
To detect mRNAs in ischemic stroke animals we dissected contralateral (CL) and peri-ischemic (PI) cortex of transient middle cerebral artery occlusion (tMCAo) mice wild-type and ciRS-7 KO
We performed differentially expression analysis of RNA-seq of wild-type and ciRS-7 KO tMCAo ischemic mice

overall_design: Comparative gene expression analysis of RNA-seq data for wild-type and ciRS-7 KO animals subjected to tMCAo 1 day post ischemia (1dpi) (n= 6 biological replicates)


title: Effect of PFKFB3 depletion on gene expression of primary murine vascular smooth muscle cells isolated from c57bl/6

city: Guangzhou

first: Dongxing

last: Zhu

state: Guangdong

address: Xinzao, Panyu District

department: School of Basic Medical Sciences

institute: Guangzhou Medical University

summary: To investigate the functional role of PFKFB3 in VSMCs osteogenic differentiation, we knockdowned PFKFB3 expression in VSMCs using siRNAs

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different cells transefected with siScrambled and 3 different cells transfected with siPFKFB3.Comparative gene expression profiling analysis of RNA-seq data for control VSMCs and VSMCs with PFKFB3 depletion


title: Proteolytic Regulation of a Galectin-3/Lrp1 Axis Controls Osteoclast-Mediated Bone Resorption

first: Lingxin

address: 237 Luoyu Road, Hongshan District, School and Hospital of Stomatology, Wuhan University

summary: To begin screening global effects on osteoclast activity in an unbiased fashion, we conducted a transcriptome analysis comparing the expression profiles of wild-type, Mmp9/Mmp14 DKO osteoclasts, and Mmp9/Mmp14 DKO osteoclasts treated with GCS-100

overall_design: Comparative gene expression profiling analysis of RNA-seq data for wild-type, Mmp9/Mmp14 DKO osteoclasts, and Mmp9/Mmp14 DKO osteoclasts treated with GCS-100.

first: Stephen

last: Weiss


city: Zhanjiang

first: Ji-Xin

last: Tang

address: No.57, Renmin Avenue

institute: Affiliated Hospital of Guangdong Medical University

summary: Transcription factors can affect autophagy activity by promoting or inhibiting the expression of autophagy and lysosomal related genes. As a zinc finger family DNA-binding protein, ZKSCAN3 has been reported to function as a transcriptional repressor of autophagy, silencing of which can induced autophagy and promoted lysosome biogenesis in cancer cells. However, the studies in Zkscan 3 knockout mice showed that the deficiency of Zkscan3 did not induce autophagy and in-crease lysosome biogenesis. In order to further explore the role of ZKSCAN3 in the transcriptional regulation of autophagy in human cells, we generated ZKSCAN3 knockout HK-2 and Hela cells by CRISPR/Cas9 system and analyzed the differences in gene expression between ZKSCAN3 deleted cells and non-deleted cells through fluorescence quantitative PCR, Western blot and transcriptome sequencing, with special attention to the differences in gene expression of autophagy and lysosomal biogenesis. We found that ZKSCAN3 is not an essential regulator of autophagic or lysosomal gene expression, as the absence ZKSCAN3 had no significantly effect on the expression of autophagy or lysosomal genes.

overall_design: To further and comprehensively characterize the transcriptome-wide transcriptional alterations caused by ZKSCAN3 deficiency, we performed whole transcriptome sequencing in WT and ZKSCAN3-KO cells. In HK-2 cells, we found that deletion of ZKSCAN3 resulted in 1408 genes significantly down regulation and 1270 genes significantly up regulation. Whereas in Hela cells, ZKSCAN3 deficiency led to 894 significantly genes down regulation and 617 genes significantly up regulation. Of these significantly differentially expressed genes, 161 were present in both HK-2 and Hela ZKSCAN3-KO cells.

first: Xiao-Min

last: Li

first: Jun-Hao

last: Wen

first: Yun-Shan

first: Hua-Feng

last: Liu


title: HDAC Inhibitors Impair GATA-3 DNA Binding and Target Gene Expression in T-cell Neoplasms

city: Ann Arbor

first: Chenguang

last: Wang

state: Michigan

address: 1500E Medical Center Dr, Room 4411

department: Department of Internal Medicine

institute: University of Michigan

summary: To investigate early transcriptional changes upon HDACi treatment, a well characterized and p53-deficient CTCL cell line (H9) and primary malignant T cells isolated from the peripheral blood of Sezary Syndrome (SS) patients (n=3) were cultured for 6 hours in the presence of vehicle control, romidepsin (romi) or belinostat (beli) and transcriptionally profiled by RNA-seq

overall_design: Comparative gene expression profiling analysis of RNA-seq data for cell lines and patients in the presence of belinostat romidepsin.

first: Ryan

last: Wilcox


title: CircRNA in Frailty

city: San Sebastián

first: David

last: Otaegui

state: Guipuzcoa

address: Paseo Dr begiristain s/n

department: Neurosciences

country: Spain

institute: Biodonostia Research institute

summary: This work describes for the first time a different circular RNA (circRNAs) expression pattern between frail and robust individuals. Moreover, the level of some circRNAs is modulated after a physical intervention. These results suggest that they could be used as minimally invasive biomarkers of frailty. Frailty is an intermediate and reversible geriatric syndrome that often precedes dependency. Therefore, its identification is essential to prevent dependency. Diverse molecules have been proposed as biomarkers of frailty, but none of them has reached clinical practice. Recently, circular RNAs have emerged as new non-coding RNAs. Their regulatory role together with their high stability in biofluids makes them good candidates as biomarkers for several processes, but, to date, no studies have characterized circRNA expression in frailty. We studied RNA from leukocytes of 35 frail and 35 robust individuals. After RNA-Sequencing, circRNA detection was performed by CIRI2 and Circexplorer2 and differential expression analysis by DESeq2. Validation was performed by Quantitative-PCR. A Linear Discriminant Analysis was performed to determine the best circRNA combination to discriminate frails from robusts. Besides, CircRNA candidates were studied in 13 additional elder donors before and after a 3-month physical intervention. We found 89 differentially expressed circRNAs (p-value<0.05, FC>|1.5|) with frailty. The upregulation of hsa_circ_0007817, hsa_circ_0101802 and hsa_circ_0060527 in frail individuals was validated. The combination of hsa_circ_0079284, hsa_circ_0007817 and hsa_circ_0075737 levels showed a great biomarker value with a 95.9% probability of correctly classifying frail and robust individuals. Moreover, hsa_circ_0079284 levels decreased after physical intervention in concordance with an improvement in frailty scores.

overall_design: Frail vs Robust pools analysis

first: Leire

last: Iparraguirre

first: Ainhoa

last: Alberro

first: Itziar

last: Vergara

first: Ander

last: Matheu


title: Single-cell transcriptomics of mammalian prion diseases identifies dynamic gene signatures shared between species

first: Athanasios

last: Dimitriadis

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: A next generation three-dimensional hydrogel-based culture system for studying host-pathogen interaction and drug efficacy in tuberculosis

city: Bengaluru

first: Rachit

last: Agarwal

state: Karnataka

address: CV Raman road

department: Biosystems Science and Engineering

country: India

institute: Indian Institute of Science, Bangalore

summary: An accessible, simple, reliable, next-gen in-vitro platform to further the screening for Mtb drugs and understand TB host-pathogen interaction.

sample_organism:
Homo sapiens
Mycobacterium tuberculosis H37Rv

overall_design:
Dual RNA sequencing to dissect the host-pathogen interaction mechanisms by identifying their transcriptomic differences in the collagen gels 14 days post-infection.
Please note that the 'Dual_Mtb3' sample was not considered for analysis.

first: Vishal

last: Gupta


title: The role of a mammalian network in regulating ischemic stroke outcome I

first: Flavia

last: Scoyni

summary:
To detect miRNAs in oxygen and glucose deprivation (OGD) conditions we cultured cortical neurons Embryonic day 15 and subjected them to OGD
We performed differentially expression analysis of miRNA-seq of wild-type and ciRS-7 KO neurons

overall_design: Comparative gene expression analysis of miRNA-seq data for wild-type and ciRS-7 KO cortical neurons treated for 12h with OGD in respect of the normoxic conditions (n= 5 biological replicates per group)

first: Scoyni

last: Flavia

first: Giudice

last: Luca


title: Characterisation of VDR signaling in prostate cancer health disparities (small RNA-Seq)

city: WEST HOLLYWOOD

first: Moray

last: Campbell

state: CA

address: 8687 Melrose Avenue, Office B227.5G

institute: Cedars-Sinai Medical Center

summary: To investigate the effect of 1,25(OH)2D3 activation on small RNA expression in prostate cell lines derived from European Americans and African Americans

overall_design:
RNA was extracted from cells in the presence of 1α,25(OH)2D3 (100nM, 8hr) or EtOH in biological triplicate samples and analyzed by RNA-Seq. Cell lines were treated as RNA-Seq and library preparation included ligation of 5’ and 3’ RNA adapters to the mature miRNAs 5ʹ‐phosphate and 3ʹ‐hydroxyl groups and 11-13 PCR cycles using a universal primer and a primer containing one of 48 index sequences, which allowed pooling of libraries and multiplex sequencing.  Prior to pooling, each individual sample’s amplified cDNA construct was visualized on a DNA-HS Bioanalyzer DNA chip (Agilent Technologies) for mature miRNA and other small RNA products (140-150bp).  Successful constructs were purified using a Pippen prep (Sage Inc.), using 125 – 160 bp product size settings with separation on a 3% agarose gel. The purified samples were validated for size, purity and concentration using a DNA-HS Bioanalyzer chip. Validated libraries were pooled at equal molar to a final concentration of 10nM in Tris‐HCI 10 mM, pH 8.5, before 50 cycle sequencing on a MiSeq (Illumina, Inc.).
Comparative small RNA gene expression profiling analysis of RNA-seq data for prostate cell lines treated with 1α,25(OH)2D3 (100 nM, 8h)

first: Clayton

last: Yates


title: Fully accessible fitness landscape of oncogene negative lung adenocarcinoma

city: Stanford

first: Gabor

last: Boross

address: 450 Serra Mall

institute: Stanford University

summary: Multiple genetic mutations are needed for cancer to develop. This is even more pronounced in the case of oncogene negative cancer, where a single dominant oncogenic mutation that can start tumor growth is lacking and substituted by a combination of driver mutations. To shed light on how tumors can subsequently acquire these independent mutations and to test the predictability of cancer evolution, we have characterized the fitness landscapes of a triple-mutant oncogene negative genotype leading to potent lung adenocarcinoma formation. We found that, irrespective of the genetic background, landscapes are surprisingly accessible, where each additional mutation leads to growth advantage and hence prediction of the order of mutations in oncogene negative tumors cancer evolution is expected to be challenging.

overall_design: Ultra-deep sequencing of DNA barcode amplicon

first: Maryam

last: Yousefi

first: Laura

last: Andrejka

first: Monte

middle: M

last: Winslow

first: Dmitri

middle: A

last: Petrov


title: RNA samples of human peripheral blood mononuclear cells for Methylated RNA immunoprecipitation-seq

city: hefei

first: Jun

last: Wu

state: Anhui

address: Meishan Road 81

institute: Anhui medical university

summary: We established m6A modification profiles using MeRIP-seq  in peripheral blood mononuclear cells (PBMCs) from  systemic lupus erythematosus (SLE) patients and controls (HC), and investigated m6A-related lncRNAs in  SLE for novel  potential roles in SLE. Compared with controls,  m6A level was lower in SLE patients,426 lncRNAs and 2,331 mRNAs were differentially expressed in SLE patients.

overall_design: RNA samples of PBMCs from three new-onset SLE patients and three matched controls (HC) were utilized for Methylated RNA immunoprecipitation-seq (MeRIP-seq)

first: Dong-qing

last: Ye


title: Insights into the evolution of dermal armour: osteoderms in a mammal, the spiny mouse

city: Gainesville

first: William

middle: Bradley

last: Barbazuk

state: Florida

address: UF-CGRC Room 407 2033 Mowry Rd

department: Biology

institute: University of Florida

summary: The goals of this study are to compare NGS-derived transcriptomes (RNA-Seq) derived from skin sampled from the distal and the proximal regions. Using Acomys cahirinus we describe the structure and development of osteoderms in the tail which develop in a proximal to distal and dorsal to ventral sequence over a 6-week period postnatally. We use RNA sequencing to identify which gene regulatory networks are involved in their development which together suggests a deep molecular homology exists in the mammalian dermis which can be activated at any time in evolution.

overall_design: RNA was isolated from the distal and proximal regions of 6 newborn Acomys pups. The RNA was used in the construction of Illumina paired-end RNA-SEQ libraries, which were sequenced on the Illumina NovaSeq6000 platform.

first: Barbazuk

middle: W

last: Bradley

first: Polvadore

last: Trey

first: Malcolm

last: Maden


title: Photostimulation of retinal organoids reveals light-regulated microRNA molecules in human retina.

city: Brno

first: Tomas

last: Barta

address: Kamenice 3

department: Faculty of Medicine

country: Czech Republic

institute: Masaryk University

summary: To investigate light-regulated miRNAs in the human retina we photostimulated retinal organoids and found 51 light-regulated miRNAs that are significantly up- or down-regulated.

overall_design: Retinal organoids were unstimulated (dark), or photostimulated using 100 lx white light for 1 hour and 3 hours, respectively.

first: Canan

last: Celiker

first: Kamila

last: Weissová

first: Kateřina

last: Černá

first: Jan

last: Oppelt

first: Tomáš

last: Bárta


title: Conserved reduction of m6A marks during aging and neurodegeneration is linked to altered translation of synaptic transcripts

city: Gottingen

first: Andre

last: Fischer

state: NS

address: Von Sieboldstr 3a

department: Epigenetic Mechanisms in Dementia

country: Germany

institute: German Center for Neurodegenerative Diseases (DZNE)

summary: N6-methyladenosine (m6A) plays diverse roles in the regulation of mRNA metabolism. In the mammalian brain it has been linked to developmental processes, memory function and plasticity. However, the precise role of m6A in the context synaptic plasticity and especially during impaired cognition are not fully understood. Here, we describe the mouse and human brain m6A epi-transcriptome in a tissue-specific manner. We furthermore show that m6A levels undergo massive decreases across mouse brain regions as a consequence of aging. Equally, Alzheimer’s disease in humans correlates with decreased N6-methylation in a similar population of transcripts that are linked to synaptic function and localized to synapses, such as the calcium/calmodulin-dependent kinase II (CaMKII). In line with this we show that reduced m6A levels impair synaptic protein-synthesis of CAMKII. Our results suggest that N6-RNA-methylation is an important mechanism to control synaptic protein synthesis which is affected early in cognitive diseases. This data submission just includes mouse data.

overall_design: The CA1, CA3, DG and ACC of 20 mice (10 mice of 3 months of age and 10 of 16 months) pooled into 3 replicates per region for sequencing.

first: Ricardo

last: Castro Hernandez

first: Tea

last: Berulava

first: Tonatiuh

last: Peña Centeno


title: Transcriptomics of AML core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles

city: Providence

first: Patrycja

last: Dubielecka

state: RI

address: 1 Hoppin St., Coro West, 5th Fl., Suite 5.36

department: Division of Hematology/Oncology, Department of Medicine

institute: Warren Alpert Medical School of Brown University and Rhode Island Hospital

summary: While the bone marrow (BM) microenvironment is significantly remodeled in acute myeloid leukemia (AML), our understanding of chemotherapy-induced changes within the BM stroma and their involvement in disease recurrence remains limited. Molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma with non-hematopoietic cells supporting hematopoiesis. We assessed the effect of standard anthracycline- and cytarabine-based induction chemotherapy on both hematopoietic and non-hematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis to determine the transcriptomic profile and factors associated with the response to treatment. We compared matched human core BM biopsies at diagnosis and 2 weeks after standard cytarabine/anthracycline induction therapy in responders (<5% blasts present post-treatment ) and non-responders (>5% blasts present post-treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2(SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1), and osteonectin (SPARC) was observed in core BM biopsies from non-responders to induction chemotherapy. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML BM response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimized therapeutic strategies for AML.

overall_design: Comparative gene expression profiling analysis of responders and non-responders to standard induction therapy in AML. Total mRNA was extracted from paired FFPE core bone marrow biopsies obtained at diagnosis (responder n=6/nonresponder n=5) and 2-weeks post- treatment (responder n=6/nonresponder n=5) induction.

first: Diana

middle: O

last: Treaba

first: Dennis

last: Bonal

first: Mireia

last: Castillo-Martin

first: Anna

last: Chorzalska

first: Alissa

last: Oakes

first: Makalya

last: Pardo

first: Max

last: Petersen

first: Christoph

last: Schorl

first: Kelsey

last: Hopkins

first: Dean

last: Melcher

first: Ting

last: Zhao

first: Olin

last: Liang

first: Eui-Young

last: So

first: John

last: Reagan

first: Adam

last: olszewski

first: James

last: Butera

first: Douglas

last: Anthony

first: Peter

last: Rintels

last: Quesenberry


title: PHYTOCHROME-INTERACTING FACTOR 7-mediated epigenetic reprogramming promotes the transcriptional response to shade in Arabidopsis

city: Shanghai

first: Chuanwei

last: Yang

address: 2005 Songhu Road, Yangpu District

department: school of life sciences

institute: Fudan university

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ChIP-Seq were performed using 35S::GFP-ASF1A, pHTR13::HTR13-HA, pHTR5::HTR5-HA, and pHTR5::HTR5-HA/pif7-1 transgenic plants after white light and 1 hr shade treatment. Two biological replicates were prepared for each genotype of plants grown under light and shade conditions. High-confidence binding peaks with cut off fold change > 1.5 and P-value <0.01. Our data reveal that ASF1 directly regulating a subset of target genes of PIF7. Furthermore, shade-elicited gene activation is accompanied by H3.3 enrichment, which is mediated by the PIF7-ASF1 regulatory module.

overall_design: ChIP-seq was applied to comprehensively profile the ASF1A/H3.3/H3.1 landscape in response to shade exposure. 8-day-white-light grown seedlings were treated 1 hr shade before harvesting for pHTR13::HTR13-HA, pHTR5::HTR5-HA, pHTR5::HTR5-HA/pif7-1 DNA extraction using HA antibodies. 6-day-white-light grown seedlings were treated 1 hr shade before harvesting for 35S::GFP-ASF1A DNA extraction using GFP antibodies.

first: Lin

last: Li


title: Epigenetic reprogramming orchestrates transcriptional responses in shade avoidance syndrome of Arabidopsis

summary: For shade-intolerant plants, changes in light quality indicative of competition from neighboring plants trigger shade avoidance syndrome (SAS). PYHTOCHROME-INTERACTING FACTOR 7 is the major transcriptional regulator of SAS in Arabidopsis. However, the epigenetic reprogramming under shade is poorly understood. To identify the histone chaperone ASF1 and HIRA function during SAS, we performed transcriptome deep sequencing (RNA-seq) to search for differentially expressed genes (DEGs) by comparing transcript levels between Col-0 and pif7-1, asf1ab, or hira-1 seedlings during white light and shade conditions. Our data shown that histone chaperone ASF1, through interacting with PIF7 and helping of HIRA, positively regulates shade-induced genes expression.

overall_design: 6-day-white-light grown seedlings were treated with 1 hr shade before harvesting for RNA-seq.


title: Indian Hedgehog release from inflamed epithelia links TNF-alpha   to organ fibrosis

city: Edinburgh

first: Eoin

middle: D

last: O'Sullivan

address: 47 Little France Crescent

department: Centre for Inflammation Research

institute: University of Edinburgh

summary: In chronic kidney disease (CKD) and with ageing, individuals lose regenerative capacity after renal injury and are predisposed to progressive fibrosis and cardiovascular disease.  With ageing and CKD increased numbers of activated leukocytes are present in the circulation and within the kidney where they correlate with progressive fibrosis.   The potential role of activated leukocytes in mediating progressive renal and systemic fibrosis remains incompletely understood.  Here, we show that tumour necrosis factor alpha (TNFa) released with injury and aging promotes renal and cardiac fibrosis. We identify a Ubiquitin D expressing population of TNFa induced inflammatory proximal tubular epithelia (iPT) responsible for Indian Hedgehog release in aged and fibrotic kidneys. Indian Hedgehog production by iPT cells activates canonical Hedgehog signalling in Gli1+ stromal cells leading to activation, proliferation and fibrosis deposition.  Our data links the immune activation seen in aging and chronic kidney disease to cardio-renal fibrosis. This provides multiple targets for antifibrotic therapies which we validate in murine models of aging and kidney disease.

overall_design: Unbiased examination of single cell RNA-seq of injured murine kidney, control kidneys and BULK RNA-SEQ of IHH KO kidneys, paired controls, uninjured controls, post nephrectomy contralatearl kidneys , and post iri contralateral

first: David

last: Ferenbach


title: Transcriptomics of AML core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles [microarray]

first: Patrycja

last: Dubielecka

overall_design: Comparative gene expression profiling analysis of responders and non-responders to standard induction therapy in AML. Total mRNA was extracted from paired FFPE core bone marrow biopsies obtained at diagnosis (responder n=6/nonresponder n=5) and 2-weeks post-treatment (responder n=6/nonresponder n=5) induction.

first: Diana

last: Treaba

first: Dennis

last: Bonal

first: Mireia

last: Castillo-Martin

first: Anna

last: Chorzalska

first: Alissa

last: Oakes

first: Makalya

last: Pardo

last: Petersen

last: Olszewski


title: RNA-seq profiling of salivary transcriptome reveals potential diagnostic RNAs and oral microbial dysbiosis in oral squamous cell carcinoma

city: ropar

first: srivatsava

last: naidu

state: punjab

address: bhara phool

department: biomedical engineering

institute: indian institute of technology ropar

summary:
Background:
The differential abundance of cell-free RNAs in bodily fluids is emerging as a promising tool for the non-invasive molecular diagnosis of cancer. Human saliva is considered a promising source of non-invasive biomarkers of diagnostic value for oral cancer detection. This study aims to identify diagnostic potent salivary RNAs in oral squamous cell carcinoma (OSCC)-patients by RNA-Sequencing.
Method:
Unstimulated saliva was collected from 5 normal control (NC) individuals and 9 OSCC patients (PS) with prior consent and ethical committee approvals. Total RNA isolated from cell-free saliva (CFS) supernatant was used to prepare small RNA libraries and sequenced on the Ion Torrent S5 platform. The sequencing reads were aligned to the human genome (hg19) using Bowtie 2, and the differential expression analysis was performed using RUVSeq and DESeq2. Mapped reads were screened across miRBase (v22) annotations for miRNAs and Gencode (v19) annotation for other RNAs. Reads were quantified by the Featurecount (v1.4.6) module of the R-package. The microbial-RNA enrichment analysis was determined using the One Codex platform.
Result:
RNA-sequencing detected protein-coding transcripts (PCTs), long-intergenic RNAs (lincRNAs), microRNAs (miRNAs), small nuclear RNAs (snRNAs), transfer RNAs (tRNAs) and pseudogenes from the saliva of PS and HC samples. Transcriptome analyses revealed 89 PCTs, 18 lincRNAs and 6 miRNAs differentially expressed between PS and HC with a log2fold change ≥ 1 or ≤ -1 and p-value < 0.05. Gene ontology and pathway enrichment analyses indicated a significant correlation of the identified PCTs and miRNAs to various cancer-related pathways that may have implications in the pathogenesis of OSCC. Interestingly, unmapped non-human reads aligned to the microbial reference genomes. Further analyses of these microbial sequence reads revealed a significant microbial dysbiosis differentiating PS from HC. Metabolic pathways and functional analysis of the identified microbial phylotypes showed gene ontologies associated with inflammation, cell proliferation, ROS generation, and a range of metabolic processes.
Conclusion:
We report novel panels of differentially expressed PCTs, miRNAs and lincRNAs distinguishing PS from HC. Importantly, our results also provide evidence for oral microbial dysbiosis that appears to have pathological implications in OSCC. Summarily, this study provides a comprehensive landscape of salivary RNAs that can be exploited as non-invasive biomarkers for OSCC detection.

overall_design: Cell free salivary transcriptome profiling among 5 normal controls and 9 oral squamous cell carcinoma patients.

first: Sheetanshu

last: Saproo

first: Gaurav

last: Ahuja

first: Shashanka

last: Sarkar

first: Gouri

last: Dass

first: Chingmei

last: Konyak

first: Juhi

last: Tayal

first: Anurag

last: Mehta

first: Srivatsava

last: Naidu

first: Vishakha

last: Gautam


title: Transcriptomics of AML core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles by RNA-seq and microarray analysis

first: Patrycja

last: Dubielecka


title: DGAT1 is a bona fide oncogene stimulating cell growth and suppressing oxidative stress while enabling fatty acid accumulation

city: Manchester

first: Adam

last: Hurlstone

address: Michael Smith Building

institute: University of Manchester

summary: Forced over-expression of dgat1a using the minicoopR system increased the rate of tumour formation in tp53m214K/m214k; mitfa-/-; nras G12D. This was through increased TOR signalling and alterations in key metabolic pathways.

overall_design: Examination of the effect of dgat1a overexpression in NRAS driven Zebrafish Melanoma using the miniCoopR system.

first: Daniel

last: Wilcock

middle: F


title: Genome-wide transcriptional alterations during vascular growth in skeletal muscles of type 2 diabetic mice in response to femoral artery ligation

city: Kuopio

first: Minna

middle: U

last: Kaikkonen

department: A.I. Virtanen Institute, Department of Biotechnology and Molecular Medicine

institute: University of Eastern Finland

summary: Diabetes is a major risk factor of cardiovascular disease including coronary and peripheral arterial disease, attributed to impaired arteriogenesis and angiogenesis. Mechanisms behind them are poorly understood due to the complexity of these processes in presence of diabetes. To understand the cellular and molecular mechanisms underlying impaired adaptive vascular responses in type 2 diabetic (T2DM) mouse models of hindlimb ischemia using Genome-wide mRNA sequencing.In response to FAL, collateral density and lumen area were significantly reduced in adductor muscles of T2DM mice at day 7 and 14 days post-FAL. mRNA sequencing suggested an altered gene expression typical for monocyte and macrophage (Mϕ) subsets. Similarly, gene expressions typical of dendritic cell and lymphocyte were altered in adductor muscles of T2DM mice. Ischemic muscles of T2DM mice displayed impaired angiogenesis as revealed by endothelial staining. mRNA sequencing suggested downregulation of crucial genes implicated in angiogenesis and differential expression of genes typical for Mϕ subsets. Immunohistochemistry analysis corroborated with the mRNA sequencing analysis, suggesting an altered Mϕ polarization behind the impaired arteriogenesis and angiogenesis seen in T2DM mouse models of hindlimb ischemia.

overall_design: Hindlimb ischemia was induced by femoral artery ligation (FAL). Arteriole and capillary parameters together with blood flow were analyzed from normoxic adductor and ischemic gastrocnemius muscles respectively, at day 7 and 14 post-FAL. Total RNA was isolated from both adductor and gastrocnemius muscles at day 7 post-FAL and sequencing was performed with libraries prepared from Poly (A) RNA fraction, using Illumina Genome Analyzer II.

first: Babu

last: Mohan

first: Seppo

last: Ylä-Herttuala


title: ApoA-I nanotherapy rescues inadequate post-ischemic vascular adaptation in diabetic mice by modulating macrophage phenotype

first: Tiit

last: Örd

address: Neulaniementie 2

institute: A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland

summary: Purpose: To clarify cellular and molecular mechanisms underlying impaired post-ischemic adaptive vascular responses and to evaluate reconstituted HDL (rHDL)-ApoA-I nanotherapy to rescue the defect in type 2 diabetic (T2DM) mouse model of hindlimb ischemia (HLI). Methods: Mice with beta-cell specific over-expression of insulin-like growth factor-2 in atherosclerotic background (IGF-II/ LDLR-/-ApoB100/100) with T2DM features were used in the study with C57BL/6J mice fed with a regular chow-diet (R36, Lactamin) serving as controls. Mice aged between 16 to 20 weeks were used in the study . Mice were anaesthetized with 1.5-2% isoflurane/air mixture during the surgical procedure. Hair was removed from the operation site and cleaned with sterile water. Single incision was made just above medial thigh and superficial femoral artery was separated from femoral vein and nerve. HLI was induced by unilateral ligation of femoral artery distal to the origin of the profunda femoral artery. The skin was closed with interrupted silk sutures. Mice were  were administered with reconstituted ApoA-I nanoparticles with phosphatidyl serine core (rHDL) intravenously starting 2 days post-HLI with doses every 2 days until scarification with Saline injections serving as controls. Adductors muscle aortic endothelial cells (AECs) and Ischemic muscle Macrophages and Endothelial Cells (ECs) were isolated using a combination of magnetic assisted cell sorting and flow Cytometry using Macrophage (F4/80) and EC (CD-31) specific markers. Total RNA isolated from FACS sorted cells were used for RNA seq library preparation. Results: Cell-specific gene expression, flow cytometry and immunohistochemistry revealed a persistent Macrophage and Endothelial cell inflammation and a reduced anti-inflammatory M2-macrophage activation associated with impaired collateral remodelling and sprouting angiogenesis in T2DM mice. Furthermore, reconstituted HDL (rHDL)- ApoA-I was found to rescue impaired collateral remodelling and angiogenesis in T2DM mice through modulating EC and macrophage phenotypes. Conclusions: Our results suggest that an impaired collateral remodelling and sprouting angiogenesis in T2DM mice after HLI is associated with a persistent EC and macrophage inflammation and a reduced activation of anti-inflammatory M2-macrophage. Skewing the macrophage phenotype towards the pro-arteriogenic and pro-angiogenic anti-inflammatory M2-macrophages using reconstituted HDL (rHDL)-ApoA-I nanotherapy may serve as a potential therapeutic modality for T2DM PAD.

overall_design: Macrophage and endothelial cell specific RNA sequencing was performed on 3-4 samples (Each sample was pooled from 3-4 mice) by 3’mRNA-Seq approach using single-end sequencing with 75 bp reads on an Illumina NextSeq500 instrument .

first: Mohan

last: Babu


title: Oregon-R: Control vs. Hdac1 mutant

city: Nankang, Taipei

first: Der-Hwa

last: Huang

state: Taiwan

address: 128, Sec2, Academia Rd.

department: Insititute of Molecular Biology

country: Taiwan

institute: Academia Sinica

summary:
Hdac1 mutant larvae comparing control WT larvae.
Goal was to determine the effects of Hdac1 mutation on global larval gene expression.

overall_design: Two-genotype experiment, WT vs. Hdac1 mutant. Biological replicates: 4 mutant/wild type replicates

first: Yuh-Long

last: Chang


title: Gene expression in SU-DIPG20, SU-DIPG31, SU-DIPG35, and SU-DIPG-48 primary human tumor samples

status: Public on Jan 29 2023

first: Michelle

last: Monje

address: 265 Campus Drive, SIM1 G3035

department: Neurology

summary: Gene expression in SU-DIPG20, SU-DIPG31, SU-DIPG35, and SU-DIPG-48 primary samples was quantified to gain insight into diffuse glioma biology.

overall_design: Bulk RNA sequencing was performed on primary human DIPG tumor tissue for patients "DIPG20", "DIPG31", "DIPG35" and "DIPG48". RNA was sequenced on the Illumina NextSeq 500 and fpkm values were quantified using featureCounts (Liao et al, 2014). Replicates were performed as indicated in the metadata spreadsheet. Due to patient privacy concerns, we are witholding raw sequencing files at this time.

first: Surya

last: Nagaraja

first: Michael

last: Quezada

first: Rebecca

last: Mancusi


title: Efficacy of JAK 1/2 inhibition in murine immune bone marrow failure

city: Bethesda

first: Shouguo

last: Gao

state: MD

address: 9000 Rockville Pike

department: NHLBI

institute: National Institutes of Health

summary:
Background and methods: Ruxolitinib (RUX) is a Jak inhibitor that is used in the treatment of intermediate or high risk myelofibrosis and resistant forms of polycythemia vera and graft-vs-host disease due to its efficacy of reducing inflammatory cytokines and markers of inflammation. We tested its potential therapeutic effects in murine models of immune-mediated bone marrow failure. RNA-Seq and analysis was performed using NEBNext Ultra™ II RNA Library Prep Kit for Illumina and Illumina Novaseq6000, according to the Institute's protocols.
Results: As both prophylaxis and therapy, Ruxolitinib improved pancytopenia and BM cellularity and decreased BM T cell infiltration in bone marrow failure mice. RNA sequencing demonstrated that Ruxolitinib suppressed expression of inflammtory genes in bone marrow infiltrated CD8 T cells compared with untreated mice.
Conclusion: Our results demonstrate that Ruxolitinib is highly effective in attenuation of disease and extending survival of immune-mediated bone marrow failure mice.

overall_design:
We compared transcriptomes of CD8 T cells in the bone marrow failure mice treated with and without Ruxolitinib, and described differentially expressed genes of these cells.
CD8+ T cells were isolated from bone marrow cells of Ruxolitinib-treated and control BMF mice using CD8 microbeads (Miltenyi Biotec, Auburn, CA). To get enough cells for RNA extraction, CD8+ T cells from every 2-3 mice in the same group were pooled together, we obtained 3 pools/group. Total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA), according to the manufacturer's instructions.

first: Xingmin

last: Feng


title: Oligoclonal T-cell expansion in a patient with bone marrow failure after CD19 CAR-T for Richter transformed DLBCL

summary:
Background and methods: CD19-directed CAR T-cell therapy (CAR-T) has emerged as a promising treatment modality for multiple B-cell malignancies. However, its utility is hampered by a unique toxicity profile that classically includes cytokine release syndrome (CRS) and neurotoxicity. Hematological toxicity represents a vexing and common side effect. Cytopenia can occur long after lymphodepleting chemotherapy and resolution of CRS and is often prolonged and biphasic in nature. Some patients have been described to develop severe bone marrow (BM) failure, which can predispose for severe infections and high non-relapse mortality. Overall, the underlying pathomechanisms of CAR-T-related hematotoxicity remain poorly understood. We performed flow cytometry, serum cytokine profiling, single cell transcriptome and TCR-sequencing of PBMCs from pre-/post-treatment sampels of a patient with DLBCL-type Richter Transformation receiving tisagenlecleucel in a standard-of-care setting and present one marrow failure.
Results: The advent of chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape of refractory B-cell malignancies. Real-world evidence has highlighted the high incidence of hematological toxicity, including prolonged and profound cytopenia. Here, we present the case of bone marrow failure in a 57-year old man with DLBCL-type Richter Transformation receiving tisagenlecleucel in a standard-of-care setting. The clinical course was notable for underlying bone marrow infiltration, severe cytokine release syndrome and multiple infectious complications in the setting of prolonged, profound neutropenia. Cytokine profiling revealed a signature consistent with acquired aplastic anemia – including downregulation of CD40-L and  EGF. Flow cytometric analyses demonstrated expansion of both CAR and non-CAR bearing CD8+ CD57+ T-cells in the bone marrow, an immunophenotype linked to oligoclonality in aplastic anemia. On a single-cell level, this was accompanied by T-cell receptor Vβ oligoclonal expansion and post-CAR clonal drift. Gene expression profiling revealed upregulation of exhaustion markers on (CAR) T-cells and decreased expression of genes involved in STAT signalling and inflammatory response.
Conclusion: In conclusion, this case highlights the complex nature of CAR-T-related hematological toxicity and introduces oligoclonal (CAR) T-cell expansion as a potential contributing pathomechanism.

overall_design: We applied scTCR-seq coupled with scRNA-seq to PBMCs obtained from a 57-year old man with DLBCL-type Richter Transformation receiving tisagenlecleucel in a standard-of-care setting and present one marrow failure.

first: Zhijie

last: Wu


title: Decoding changes in chromatin accessibility mediated by KDM6 histone demethylases [ATAC-seq]

city: Toronto

first: Alexander

middle: James

last: Murison

state: Ontario

address: 101 College Street, TMDT room 11-706

country: Canada

institute: University Health Network

summary: To interrogate the function of KDM6A and KDM6B in regulating genome accessibility, we generated U937 cell lines having knockdown of either KDM6A, KDM6B or both using lentiviral shRNA. We then profiled chromatin accessibility changes using ATAC-seq data obtained from the 3 knockdown cell lines compared with the wild type cell line.

overall_design: Comparative chromatin accessibility profiling by performing ATAC-seq in 3 replicates each of wild type U937 cells and its knockdown derivatives (shKDM6A, shKDM6B and shKDM6A+6B)

first: Amitava

last: Sengupta

first: John

middle: E

last: Dick


title: The SIRT6 Activator MDL-800 Extends Lifespan and Health-span of Mice

city: Hangzhou

first: Li

last: Shen

state: Zhejiang Province

address: 866 Yuhangtang Road

institute: Life Sciences Institute, Zhejiang University

summary: The prevention or delay of the onset of age-related diseases prolongs survival and improves quality of life while reducing the burden on the health care system. Activation of sirtuin 6 (SIRT6), an NAD+-dependent deacetylase, improves metabolism and confers protection against physiological and cognitive disturbances in old age. Here we show that MDL-800 is a specific SIRT6 activator that has health and lifespan benefits in adult mice fed a standard diet. We found extension in lifespan, delayed onset of age-related metabolic diseases, and improved general health in mice fed a standard diet after MDL-800 supplementation. Treatment with MDL-800 induced synthesis of anti-oxidation related proteins, and this rejuvenated HSCs and ISCs in aged mice. Inhibition of pro-inflammatory gene expression in both liver and muscle of MDL-800-treated animals was noted. MDL-800 lowered the level of NF-kB pathway and improved fatty acid metabolism in liver. Combined with our previous work, the current study further supports the beneficial effects of MDL-800 on health across the lifespan in mice.

overall_design: RNA-seq comparing mouse liver of MDL-800-treated animals and control animals.

first: Hu

last: Wang

first: Zhenyu

last: Ju


title: Effect of depletion of histone demethylases KDM6A and KDM6B on gene expression in the acute myeloid leukemia (AML) cell line U937 [RNA-seq]

summary: To investigate the function of KDM6A and KDM6B in regulating gene expression in AML, we generated U937 cell lines having knockdown of either KDM6A, KDM6B or both using lentiviral shRNA. Control cells were generated using a scrambled shRNA. We then profiled gene expression using RNA-seq data obtained from the 3 knockdown cell lines compared with the control cell line.

overall_design: Comparative gene expression profiling by performing RNA-seq in 3 replicates each of control U937 cells and its knockdown derivatives (shKDM6A, shKDM6B and shKDM6A+6B)


title: Differential Nodal level promotes mesendoderm cell fate segregation mediated by chromatin organization

status: Public on Jan 28 2023

city: Hang Zhou

first: Tao

last: cheng

state: Zhe Jiang

address: 886 Yuhangtang Road

department: Institute of genetics

institute: Zhejiang University

summary:
Purpose: To investigate the mechanism of prechordal plate and anterior endoderm separation .
Methods:  Nodal injected explants (injected with 10pg ndr2 mRNA) constructed from lft1 mutants and ndr1 morphants were harvested at 6hpf. Libraries were prepared using Chromium Controller and Chromium Single Cell 3’Library & Gel Bead Kit v3 (10x Genomics, PN-1000075) according to the manufacturer’s protocol for 10000 cells recovery. For single-cell multiomics, zebrafish embryos at 6 hpf were harvested.  Libraries were prepared using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle (10x Genomics, 4 rxns PN-1000285) according to the manufacturer’s protocol for 10000 cells recovery.
Results: A total of 10,614 single-cell transcriptomes and 4,335 multiomics were collected after stringent quality control measures.
Conclusions: A slight bias in Nodal signaling promotes a differential chromatin structure between prechordal plate and endoderm, which drives a differential expression of those key regulators, such as gsc and ripply1 in these two cell lineages, and further regulates mesendoderm cell fate separation.

overall_design: zebrafish Nodal explants constructed from lft1 mutants and ndr1 morphants were harvested at 6hpf for scRNA-seq. Zebrafish embryos were harvested at 6hpf for single-cell multiomics.

last: Cheng

first: Xiang

last: Liu


title: Gene expression profiling of HtrA2/Omi S400D mice liver

city: Chengdu

first: Haoran

last: Tai

state: Sichuan

address: #783 Xindu Avenue

department: Department of Histology and Embryology

institute: Chengdu Medical College

summary: To study the role of HtrA2/Omi S400 phosphorylation, we generated knock in mice with HtrA2/Omi S400D mutation which mimics constitutive S400 phosphorylation of HtrA2/Omi.

overall_design: Wildtype C57BL6/N mice, as well as mice with heterozygous or homozygous HtrA2/Omi S400D mutation were euthanized at the age of 2 months and liver was used for RNA sequencing.


title: ATAC-seq and RNA-seq

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

overall_design: Refer to individual Series


title: Expression of mRNA and lncRNA in human oral cancer cell SCC25 treated with tunicamycin

first: Chenzhou

address: Number 14, Unit 3, Renmin Nan Road,

institute: West china hospital of Stomatology, Sichuan university

summary: ER stress induced mRNA and lncRNA change has not been fully characterised yet in human cells. We used HTA 2.0 microarry to explore this topic.

overall_design: Human oral cancer cell SCC25 was treated with tunicamycin (5μg/ml) or DMSO (0.1%) for 6 hours, and the cells were collected for furter analysis. For each group, there are 3 independent biological replicates

first: Zhe


title: Effects of transient microglia depletion and repopulation on retinal gene expression in the diabetic murine retina

city: San Antonio

first: Kaira

middle: Adriana

last: Church

state: TX

address: One UTSA Circle 1.212

department: Molecular Microbiology and Immunology

institute: University of Texas at San Antonio

summary: Diabetic retinopathy, a microvascular disease characterized by irreparable vascular damage, neurodegeneration and neuroinflammation, is a leading complication of diabetes mellitus. Medical interventions slow the progression of disease. However, current therapies do not specifically target microglia, a cell type implicated in mediating disease development. Microglia-mediated inflammation in the diabetic retina is regulated via CX3CR1-FKN signaling, where FKN serves as a dampening signal for microglial activation. Studying this signaling axis is important as polymorphic variants of CX3CR1 are found in 25% of the human population, hCX3CR1I249/M280, resulting in a receptor with lower binding affinity for FKN. Furthermore, disrupted CX3CR1-FKN signaling in CX3CR1-KO and FKN-KO mice leads to exacerbated microglial activation, robust neuronal cell loss and substantial vascular damage in the diabetic retina. Thus, studies to characterize the effects of hCX3CR1I249/M280-expression in microglia-mediated inflammation in the diseased retina are potentially clinically relevant to identify microglia-specific therapies. Our results show that hCX3CR1I249/M280 mice are significantly more susceptible to microgliosis and production of Cxcl10 and TNFα under acute inflammatory conditions. This pathology is exacerbated under diabetic conditions and coincides with robust neuronal loss in comparison to CX3CR1-WT mice. Therefore, to further investigate the role of hCX3CR1I249/M280-expression in microglial responses, we pharmacologically depleted microglia using PLX-5622, a CSF-1R antagonist. PLX-5622 treatment led to a robust (~70%) reduction in Iba1+ microglia in all non-diabetic and diabetic mice. CSF-1R antagonism in diabetic CX3CR1-WT prevented TUJ1+ axonal loss, angiogenesis and fibrinogen deposition. In contrast, PLX-5622 microglia depletion in CX3CR1-KO and hCX3CR1I249/M280 mice did not alleviate TUJ1+ axonal loss or angiogenesis. Interestingly, PLX-5622 treatment reduced fibrinogen deposition in CX3CR1-KO mice but not in hCX3CR1I249/M280 mice, revealing that hCX3CR1I249/M280 receptor variant mice behave differently in terms of vascular pathology compared to CX3CR1-KOs. mRNAseq gene expression analysis in CX3CR1-WT retinal isolates revealed that PLX-5622-induced microglia depletion and repopulation induced a downregulation in genes associated with microglial activation and phagocytosis, B2m, Cx3cr1, and Trem2, and complement-associated synaptic pruning, C1qa, C1qb, and C1qc. Furthermore, mRNAseq analysis of PLX-5622 treated CX3CR1-WT retinas showed lower fold changes in genes encoding proinflammatory mediators (Cxcl10, Ccl2, Il6, Cxcl1, Selp, Il12b, Tnf, Cxcl2, Icam1 and Vcam1) in comparison to diabetic + normal chow mice.

overall_design: Microglia were pharmacologically depleted in 6-wks diabetic CX3CR1-WT mice using PLX-5622 and analyzed at three different timepoints: 1) at 6-wks of diabetes (6D) to establish the retinal transcriptome prior to microglia depletion, 2) after 2-wks microglia depletion (PLX-treatment), and 3) after 2-wks of microglia repopulation (PLX-recovery). At each timepoint, ND, diabetic normal chow (NC) treated, and NC recovery were included.

first: Astrid

last: Cardona

first: Sean

last: Vargas

first: Yufeng

first: Zhao

last: Lai

first: Yi

last: Zhou

first: Teresa

last: Johnson-Pais

first: Leslia

last: Linehan


title: Expression data from MDA-MB-231 breast cancer cell lines with DAXX overexpression (OE) and DAXX SUMO interacting motif mutant (DSM) overexpression (DSM OE)

first: Daiqing

last: Liao

state: FL

address: 2033 Mowry Rd

department: Anatomy and Cell Biology

institute: University of Florida

summary: DAXX was originally discovered as a context-dependent regulator of cell death or survival. Emerging evidence suggests that DAXX has an oncogenic role in diverse cancer types. However, the molecular mechanisms underlying DAXX's oncogenic function remain to be defined. We used Affymetrix Human Transcriptome Array 2.0 (HTA-2_0) to analyze genes that were up or downregulated upon DAXX OE and DSM OE.

overall_design: Both DAXX OE and DSM OE MDA-MB-231 cells were seeded at 2 x 10^5 cells in 10 cm culture plate. Stable expression of cDNA was established through lentiviral transduction of MDA-MB-231 cell line and puromycin (2 µg/mL) selection. Three biological replicates were applied.

first: Iqbal

last: Mahmud


title: Relief of extracellular matrix deposition repression by downregulation of IRF1-mediated TWEAK/Fn14 signaling in keloids

city: Guangzhou

first: Gu

last: Jingjing

address: No2. Road Lujing

institute: Dermatology Hospital, Southern Medical University

summary: Keloids represent a fibrotic disorder characterized by the excessive deposition of extracellular matrix (ECM). However, the mechanisms by which ECM deposition in keloids is regulated remain elusive. Here, we found that the expression of both TWEAK and its cognate receptor Fn14 was significantly downregulated in keloids and that TWEAK/Fn14 signaling repressed the expression of ECM-related genes in keloid fibroblasts. The IRF1 gene was essential for this repression, and the TWEAK/Fn14 downstream transcription factor P65 directly bound to the promoter of the IRF1 gene and induced its expression. Furthermore, in keloid patients, the expression of TWEAK and Fn14 was negatively correlated with that of ECM genes and positively correlated with that of IRF1. These observations indicate that relief of TWEAK/Fn14/IRF1-mediated ECM deposition repression contributes to keloid pathogenesis, and the identified mechanism and related molecules provide potential targets for keloid treatment in the future.

overall_design: mRNA profiles of primary keloid fibroblasts with or without TWEAK stimulation and primary keloid fibroblasts with or without Fn14-siRNA transfection.

first: Jing-Jing

last: Gu

first: Cheng-Cheng

last: Deng

first: Qing-Lan

last: Feng

first: Ding-Heng

last: Zhu

first: Qing

first: Zhili

last: Rong

first: Bin

last: Yang


title: high concentration of insulin treated human ovarian granulosa cells(KGN cells)

first: Wenjun

last: Wang

address: Huangpu District Fangxie Lu 419

institute: Obstetrics and Gynecology Hospital, Fudan University

summary: we explore the effects of high concentration of insulin on ovarian grauulosa cells, we then performed gene expression profiling analysis using data obtained from RNA-seq at one time points.

overall_design: KGN cell was treated with solvent, 0.5 ug/ml insulin and 1ug/ml insulin

first: Hongna

last: Gao


title: RNA-seq reveals Nup62 as a potential regulator for cell division after traumatic brain injury in mice hippocampus

first: Jianwei

last: Zhao

address: 600 Yishan Road

department: Department of Neurosurgery

institute: Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary: To investigate the hippocampus impairment  of traumatic brain injury (TBI)，we adopted the controlled cortical impact of C57B6/J mice as the TBI model. Hippocampal specimens were removed for sequencing 24 h after the models were established. In this study, Illumina RNA-seq technology was used to determine the gene expression profile in the hippocampus after TBI.

overall_design: Comparative gene expression profiling analysis of RNA-seq data between hippocampal tissues dissected from TBI and sham group 24 h after surgery

first: Shiwen

last: Chen

first: Wenyu


title: Transcriptomic analysis of WT, Tfam+/-, Zbp1-/- and Tfam+/-Zbp1-/- mouse embryonic fibroblasts (MEFs)

city: Bar Harbor

first: Phillip

last: West

state: ME

address: 600 Main St

institute: The Jackson Laboratory

summary: The goal of this study was to compare the gene expression profiles of WT, Tfam+/-, Zbp1-/- and Tfam+/-Zbp1-/- MEFs.

overall_design: MEFs were isolated from embryonic day 14.0 embryos and passaged twice to generated enough cells for RNA seq. Total cellular RNA from MEFs was prepared using the Quick-RNA microprep kit (Zymo Research) and subjected to library preparation and RNA sequencing at the Experimental Genomics Core of the Texas A&M Institute for Genome Sciences and Society. FASTQ files were normalized, processed, and analyzed using BaseSpace Sequence Hub (Illumina). The STAR algorithm of RNAseq Alignment V2.0.0 software was utilized to align the results to the Mus musculus/mm10 (RefSeq) reference genome, then RNA-seq Differential Expression V1.0.0 software was used to obtain differential gene expression values and determine statistically significant changes in gene expression via pairwise comparison of genotypes.

first: Yuanjiu

last: Lei


title: Non-protective immune imprint underlies failure of S.  aureus IsdB vaccine

city: San Diego

first: Nathan

middle: E.

last: Lewis

address: 9500 Gilman Dr.

department: Pediatrics

institute: University of California, San Diego

summary: The failure to date of all S. aureus vaccine trials has been a conundrum and underlines fundamental flaws in current translational models. A major difference between humans and laboratory animals is the early and frequent exposure of humans to S. aureus compared to animals, which is not accounted for in vaccine studies. In recapitulating the failed IsdB human vaccine trial, we showed that mice previously infected with S. aureus do not mount a protective vaccine antibody response, unlike naïve mice. S. aureus infection induces robust but non-neutralizing anti-IsdB antibodies that target different but overlapping IsdB epitopes compared to IsdB vaccine. In the setting of prior S. aureus infection, however, IsdB vaccine recalls the non-protective humoral response which further competes and reduces efficacy of protective vaccine-generated specific antibodies.  Vaccination against the heme-binding domain of IsdB or select IsdB epitope can overcome interference by S. aureus infection. Using an adoptive transfer model, we showed that human serum antibodies against IsdB and another vaccine target, ClfA, are also non-protective and blocked anti-staphylococcal immunity conferred by passive immunizations. Overall, our study demonstrates proof of concept that failure of anti-S. aureus active and passive immunizations could be explained by non-protective imprint of prior host-pathogen interaction.

overall_design: Single-cell immune profiling of the S.  aureus IsdB vaccine. In this study, we conducted BCR sequencing for three different samples: LAC (LAC-infected mice given adjuvant alone), LAC/IsdB (LAC-infected mice vaccinated with IsdB) and N/IsdB (Naïve mice vaccinated with IsdB).

first: George

middle: Y

last: Liu


title: Characterization of the ZFX family of transcription factors that bind downstream of the start site of CpG island promoters [EPIC array]

city: Los Angeles

first: Weiya

middle: Stephanie

last: Ni

state: California

address: 1450 Biggy Street

institute: University of Southern California

type: Methylation profiling by genome tiling array

summary: Our study focuses on a family of ubiquitously expressed human C2H2 zinc finger proteins comprised of ZFX, ZFY, and ZNF711. Although their protein structure suggests that ZFX, ZFY, and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11-13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C2H2 zinc finger proteins.

overall_design:
EPIC array in HEK293T kidney cells

The GEO Series accession numbers for the associated RNA-seq, ChIP-seq, ChIP-exo study is GSE145160.


title: Single-cell RNA-Seq Profiling of the Transcriptomic Response   to RB Loss in the Retinoblastoma Cell of Origin

first: David

last: Cobrinik

address: 4661 Sunset Blvd

institute: Childrens Hospital Los Angeles

summary: We applied single cell RNA-sequencing to define transcriptomic and cell cycle changes that follow RB loss in prenatal cone photoreceptor precursors, the cell of origin of retinoblastoma.

overall_design: We transduced intact retina and isolated cone precursors with a cone-specific H2B-cerulean fluorescent protein (CFP) reporter vector, and transduced prospectively isolated cones with YFP-marked lentivirus carrying either of two RB1 shRNAs or a SCR control shRNA. CFP+YFP+ cells were re-isolated by FACS for scRNA-seq at multiple time points over 15 days.

first: Sunhye

last: Lee

first: Kevin

last: Stachelek


title: Xrn1 modulates the dynamic osmostress response at a transcriptional and translational level

city: Barcelona

first: Juana

last: Díez

state: Barcelona

address: Carrer del Dr. Aiguader 88

department: Department of Experimental and Health Sciences

institute: Universitat Pompeu Fabra

summary: To study the role of the exonuclease Xrn1 in gene expression dynamics under osmotic stress conditions, we performed RNA-seq in Xrn1-depleted cells. By using an auxin-inducible degron, we were able to study immediate effects of Xrn1 depletion in gene expression dynamics. Therefore, we could overcome experimental limitations associated to stable deletion mutants.

overall_design: RNA was obtained as described in (Ingolia, 2009), from BY25598 cells (Nishimura and Kanemaki, 2014) modified to express an Xrn1 protein tagged with an auxin inducible degron (AID). Libraries were obtained by the TrueSeq Stranded mRNA Sample kit (Illumina) from cells  treated with auxin for 30 minutes (t30) and compared from libraries from untreated controls (t0). After 30 minutes of treatment, Xrn1 protein was reduced to less than 10%. Stressed samples were treated with 0.4 M NaCl for 10 and 30 min in cells treated with auxin (Xrn1-AID) or untreated with auxin (Xrn1-WT). Strains were grown to logaritmic phase and collected at approximately 0,5 OD.

first: Leire

last: de Campos-Mata

first: Jennifer

last: Jungfleisch

first: René

last: Böttcher


title: Gene expression profile during Ebselen treatment in budding yeast

city: Lucknow

first: Prabhat

last: Singh

state: Uttar Pradesh

address: MG Marg

department: Toxicology

institute: Central Drug Research Institute

summary: Ebselen is synthetic selenium containing organic compound. It exhibits glutathione peroxidase (GSH-Px) like activity hence acts as a potent antioxidant therefore has been widely used in the treatment of various diseases. In contrast, certain studies also demonstrate its toxic nature; however its mechanism of action is not well understood. Presently, Ebselen has been used at  100 µM concentration for the elucidation of its molecular and cellular mechanism of action. Microarray analysis identified the differentially expressed genes in 100 µM Ebselen concentration.

platform_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae

overall_design: Total RNA was isolated from 100 µM Ebselen and DMSO (control) treated yeast  (Saccharomyces cerevisiae) cell culture by heat/freeze method as described previously (Schmitt et al., 1990) with slight modification. Whole genome expression was performed on Affymetrix GeneChipYeast Genome 2.0 Arrays (Affymetrix, Santa Clara, CA) as described in GeneChip® 3’ IVT Express Kit User Manual. Briefly, RNA was reverse transcribed to synthesize First-Strand cDNA that was primed with T7 oligo (dT) primer to synthesize cDNA containing a T7 promoter sequence. Second-strand cDNA Synthesis was performed to convert the single-stranded cDNA into double stranded DNA (dsDNA) which acted as a template for transcription. The reaction employed DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA. In vitro transcription was performed to synthesize biotin-modified aRNA with IVT labeling master mix generates multiple copies of biotin-modified aRNA from the double stranded cDNA templates; this is the amplification step. aRNA Purification removes unincorporated NTPs, salts, enzymes, and inorganic phosphate to improve the stability of the biotin-modified aRNA. Fragmentation of labeled aRNA prepares the target for hybridization to GeneChip® 3’ expression arrays. The labeled cRNA was purified, fragmented, and hybridized to the arrays at 45°C for 16 h with constant rotational mixing at 60 rpm. Washing and staining of the arrays was performed using the Affymetrix GeneChip Fluidics Station 450. Arrays were scanned using an Affymetrix GeneChip Scanner 3000 and GCOS software. The data set was analyzed using trial version of Gene Spring software 7.2 (Silicon Genetics, Palo Alto, CA). The Affymetrix CEL files were normalized. Statistically significant differentially expressed genes were identified through t-test with a Benjamini & Hochberg test correction.

first: Raghuvir

middle: S

last: Tomar

first: Srikanta

middle: K

last: Rath


title: Multi-omics profiling of cholangiocytes reveals chromatin state dynamics during hepatic cystogenesis in polycystic liver disease (RNA-Seq Illumina)

status: Public on Jan 27 2023

city: Tianjin

first: Jiayuan

state: State...

address: Heping

institute: Tianjin Medical University

summary: Through genome-wide profiling of chromatin dynamics, we revealed a profound increase of global chromatin accessibility and a switch from H3K9me3 to H3K9ac on cis-regulatory DNA elements of cyst-associated genes during cystogenesis. In an integrated epigenomic and transcriptomic analysis, we identified a transcription factor network associated with dynamic shifts in chromatin states. Functionally, we showed that inhibition of H3K9ac acetyltransferase or H3K9me3 demethylase restored chromatin states and gene expression program to slow cyst growth in PLD mice.

overall_design: RNA-seq in primary WT and PLD cholangiocytes.

first: Rongjie

last: Ji


title: Multi-omics profiling of cholangiocytes reveals chromatin state dynamics during hepatic cystogenesis in polycystic liver disease (RNA-Seq BGI)

last: Chen


title: Multi-omics profiling of cholangiocytes reveals chromatin state dynamics during hepatic cystogenesis in polycystic liver disease (ChIP-Seq)

overall_design: Chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) in primary WT and PLD cholangiocytes.


title: Time series RNA-seq of the Drosophila melanogaster female response to mating and Sex Peptide.

city: New York

first: Sofie

last: Delbare

state: NY

address: 435 East 30th Street

institute: NYU Langone

summary: Sex Peptide, a seminal fluid protein of Drosophila melanogaster males, elicits an array of post-mating responses in females, including decreased receptivity to re-mating, and increased egg laying, activity and food intake, with a preference for protein-rich food. To determine how one protein can have such widespread effects, we set out to dissect the genetic architecture of the female’s response to Sex Peptide, to determine whether Sex Peptide alters the expression of several regulators targeted to specific post-mating responses or acts on a pleiotropic regulator that controls multiple responses. We performed bulk RNA-seq of female heads at 10 time points within the first 24 hours after mating, sampling virgin females, females mated to control males and females mated to Sex Peptide-null males. Using this high-resolution time series, we identified mating- and Sex Peptide-dependent differentially expressed genes and discovered the presence of differentially used exons. We constructed gene regulatory networks using clustering and motif enrichment analyses, and identified cell types in which these changes might take place using deconvolution of our bulk RNA-seq dataset. One key network included metabolic genes which might change in expression in the female’s fat body. A second network included genes with neuronal functions, whose changes might be located in neurons or sensory organs in the female’s head. Within these networks we identified known molecular regulators of the circadian clock. Further, we found that many differentially expressed genes, and some differentially used exons, followed a circadian rhythm in virgin females, and that this rhythm was altered after mating with a Sex Peptide- male.

overall_design: Bulk RNA-seq was performed on the heads of D. melanogaster virign females, females mated to SP-null males and females mated to wildtype males. Samples were collected at 30 minutes and 1, 2, 3, 4, 5, 6, 8, 12 and 24 hours after mating. Virgin females were collected in parallel at the same time of day to avoid confounding of mating effects and circadian effects. The entire time series experiment was repeated 3 times, yielding 3 biological replicates.

middle: N

first: Sara

last: Venkatramen

first: Kate

last: Scuderi

first: Martin

middle: T

last: Wells

first: Mariana

last: Wolfner

first: Sumanta

last: Basu

first: Andrew

middle: G

last: Clark


title: RNA mis-splicing sensitizes SETD2-mutant kidney cancer cells to DNMTi (ChiP-Seq and RNA-Seq)

city: Grand Rapids

first: Hyo Sik

last: Jang

state: Michigan

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

summary: The dysregulation of the histone H3 lysine 36 (H3K36) methyltransferase, SETD2, is associated with worse clinical outcomes and metastasis in clear cell Renal Cell Carcinoma (ccRCC). Here, we reveal that kidney cancer cells displaying diminished H3K36me3 levels (SETD2 deficiency) show increased sensitivity to the anti-tumor effects of the DNA hypomethylating agent 5-aza-2’-deoxycytidine (Decitabine/DAC). DAC treatment induced stronger viral mimicry activation and immunostimulatory signals by higher transposable element (TE) expression in SETD2-mutant cancer cells. Surprisingly, we demonstrate that the increased TE abundance in SETD2-knockout (SETD2-KO) kidney cancer cells is substantially derived from mis-spliced products induced by DAC treatment. Epigenetic profiling suggests that differential DNA methylation, H3K36me3, and H3K9me3 marks across exons and intronic TEs might contribute to elevated mis-splicing rates specifically in the SETD2 loss context. Finally, SETD2 dysregulation also sensitized tumors in vivo to combinatorial therapy of DAC and immune checkpoint inhibitors highlighting the translational potential for this precision medicine.

overall_design: Two biological replicates of Total RNA-seq generated for ACHN, Caki-1, Caki-2, A-704, A-498, 769-P, 786-O (EV), 786-O (SETD2 KO) treated with 300nM decitabine (DAC). Two biological replicates of H3K4me3/H3K27ac/H3K27me3/H3K9me3/H3K36me3/Input ChIP-seq generated for 786-O (EV) and 786-O (SETD2 KO) treated with 300nM DAC.

first: Hyo

first: Hong-tao

last: Li

first: Gangning

last: Liang

first: Peter

middle: A

last: Jones


title: Buffering of transcription rate by mRNA half-life is a conserved feature of Rett syndrome models

first: Marat

last: Mufteev

address: 686 Bay St.

department: Molecular Genetics

institute: University of Toronto

summary: Models of MECP2 dysfunction in Rett syndrome (RTT) assume that transcription rate changes directly correlate with altered steady-state mRNA levels. However, limited evidence suggests that transcription rate changes are buffered by poorly understood compensatory post-transcriptional mechanisms. Here we measure transcription rate and mRNA half-life changes in RTT patient neurons using RATE-seq, and re-interpret nuclear and whole-cell RNAseq from Mecp2 mice. Genes are dysregulated by changing transcription rate only or half-life only and are buffered when both are changed. We utilized classifier models to understand the direction of transcription rate changes based on gene-body DNA sequence, and combined frequencies of three dinucleotides were better predictors than contributions by CA and CG. MicroRNA and RNA-Binding Protein (RBP) motifs were enriched in 3ʹUTRs of genes with half-life changes. Motifs for nuclear localized RBPs were enriched on buffered genes with increased transcription rate. Our findings identify post-transcriptional mechanisms in humans and mice that alter half-life only or buffer transcription rate changes when a transcriptional modulator gene is mutated in a neurodevelopmental disorder.

overall_design: Human isogenic iPSC-derived WT and RTT neurons were subjected to RATE-seq and miRNA-seq to characterize changes in transcription rate and RNA stability.

first: Deivid

middle: C

last: Rodrigues

first: Olivia

last: Rissland

first: Michael

last: Wilson

first: James

last: Ellis


title: CCDC25 Regulates Neurogenesis During the Brain Devolopment

city: Beijing

first: Jianwei

last: Jiao

state: Beijing

address: 1 Beichen West Road

department: State Key Laboratory of Reproductive Biology

institute: Institute of Zoology, Chinese Academy of Sciences

summary:
Purpose:T To investigate the role of CCDC25 in the development of cortical nerves
Methods:  mRNA from E13 neural steneural stem cell were obtained. Culture in vitro for 4 days, Agilent 2100 Bioanalyze was used to quality controlled and quantified. then, mRNA was converted to cDNA and bound the library. RNA-sequencing analysis was used by the Illumina HiSeq 2500 platform in Annoroad Genomics.
Results: Approximately  one thousand transcripts showed differential expression between the control and knock down brain cortex, with a fold change ≥2 and p value <0.05.Geneontology analysis of the up-regulated genes showed obvious enrichment of biological processes related to development process and regulation of cell communication.The down-regulated genes exhibited enrichment of biological processes related to cell movement. These results reflected ccdc25 plays a vital role in cortex development.
Conclusions:  Endothelial Arid1a RNA-seq would provide a overall understanding how endothelial Arid1a regulates the Balance among angiogenesis, neurogenesis, and astrogenesis in the developing Brain

overall_design: mRNA profiles of control and knockdown were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.

first: Chong

last: Wang


title: EGFR and E-cadherin coregulated genes mediate the growth and metastasis of gastric cancer cells by ceRNA networks

city: Zhengzhou

first: Yuan

last: Yuan

state: Henan

address: No. 3, Fukang Front Street, Erqi District, Zhengzhou, Henan Province

institute: The Fifth Affiliated Hospital of Zhengzhou University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: EGFR and E-cadherin are key genes closely related with gastric cancer. EGFR overexpression while E-cadherin deletion are characteristic partners in certain types of gastric cancer. Discovering their co-regulatory mechanisms will contribute to provide molecular basis and newer therapeutic strategy for this type of gastric cancer.In this study, E-cadherin overexpression and EGFR knockdown were performed in gastric cancer cells, and their effects on cell growth and metastasis were confirmed. Gene microarrays were used to analyze the changes of gene expression profile. A group of molecular targets co-regulated by EGFR and E-cadherin were found, including lncRNAs, circRNAs and mRNAs. Total 1204 DEGs are related to EGFR knockdown, while 3882 DEGs related to E-cadherin overexpression. Among their downregulated mRNAs, GLI2 and CEACAM6 were identical targets with most significance. Furthermore, 274 overlapping DEGs were screened as potential co-regulatory targets of EGFR and E-cadherin.With GLI2 and CEACAM6 as co-targets, we mapped two representative ceRNA networks to throw light on the co-regulation mechanism of EGFR and E-cadherin in regulating gastric cancer cells.

overall_design: 9 samples were analyzed, 3 for control, 3 for  E-cadherin overexpression, 3 for  EGFR knockdown

first: Xiyun

last: Jia

first: Jinfeng

last: Du

first: Fanghui

last: Peng

last: Ba

first: Feiyan

first: Zisen

last: Zhang


title: microRNA profiling in the plasma of hospitalized COVID-19 patients

city: Luxembourg

first: Yvan

last: Devaux

address: 120 route d'Arlon

country: Luxembourg

institute: LIH

summary: Coronavirus disease 2019 (COVID-19) can be asymptomatic or lead to a wide spectrum of symptoms, ranging from mild upper respiratory system involvement to acute respiratory distress syndrome, multi-organ damage and death. In this study, we explored the potential of microRNAs (miRNA) in delineating patient condition and in predicting clinical outcome. Analysis of the circulating miRNA profile of COVID-19 patients, sampled at different hospitalization intervals after admission, allowed to identify miR-144-3p as a dynamically regulated miRNA in response to COVID-19.

overall_design: Plasma miRNA profiles of the COVID-19 patients sampled at different hospitalization intervals after admission with different clinical outcomes (alive or dead)

first: Fabio

last: Martelli

first: Simona

last: Greco

first: Alisia

last: Madè


title: Maternal Immune Activation perturbs fetal hematopoietic development and causes persistent changes to postnatal immunity [scRNA-Seq']

city: SALT LAKE CITY

first: Anna

last: Beaudin

state: Utah

institute: University of Utah

summary: Adult hematopoietic stem cells (HSCs) respond directly to inflammation and infection, resulting in both acute and persistent changes in quiescence, mobilization, and differentiation.  Here we show that fetal HSCs respond to maternal inflammation in utero, and the fetal response drives long-term changes to postnatal hematopoiesis and immunity. Heterogeneous fetal hematopoietic stem and progenitor cells (HSPCs) show divergent responses to maternal immune activation (MIA), including changes in quiescence, expansion, and immune cell output.  Single cell transcriptomic analysis of fetal HSPCs reveals specific upregulation of inflammation-responsive genes in discrete populations, in response to upregulated IFNα and IL-1α in the fetal liver cytokine milieu. Postnatally, MIA caused the inappropriate expansion and persistence of transient progenitors, concomitant with increased cellularity and hyper-responsiveness of fetal-derived immune cells.  Our investigation demonstrates how inflammation in utero can direct the trajectory of hematopoiesis and immunity by reshaping fetal HSC establishment.

overall_design: 6 samples (3 Saline, 3 pIC)

first: April

last: Apostol


title: Multi-omics profiling of cholangiocytes reveals chromatin state dynamics during hepatic cystogenesis in polycystic liver disease (ATAC-Seq)

first: Jiayuan

last: Chen

overall_design: Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) in primary WT and PLD cholangiocytes.

first: Rongjie

last: Ji


title: NONO knockdown RNA-seq and neuroblastoma PARCLIP analysis of NONO

city: Parkville

first: Jack

middle: Aatto Laasonen

last: Cooper

state: Victoria

address: 49 College Crescent

country: Australia

institute: University of Melbourne

summary: siRNA knockdown of NONO mRNA followed by RNA-seq was carried out in the KELLY neuroblasotma cell line. In order to determine potential regulatory targets of NONO protein, we performed differential expression analysis. To identify the RNA targets of NONO in neuroblastoma, we carried out PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) using an antibody recognising NONO protein.

overall_design: In the NONO knockdown experiment, four siRNA controls were sequenced and compared to eight NONO knockdown samples (two sets of four samples each using a distinct NONO siRNA). In the PARCLIP experiment (a separate sequencing run), one sample in each of KELLY, BEC2 and SKNAS cell lines was cross-linked and the PAR-CLIP protocol performed before sequencing. One sample cultured under normal conditions and without treatment was sequenced with the PAR-CLIP for each of the three cell lines.

first: Archa

last: Fox

first: Alina

last: Naveed

first: Simon

last: Kobelke


title: RNA mis-splicing sensitizes SETD2-mutant kidney cancer cells to DNMTi (Methylation)

first: Hyo Sik

last: Jang

overall_design: Infinium MethylationEPIC BeadChip assay generated for  786-O (EV) and 786-O (SETD2 KO) treated with 300nM Decitabine.

first: Hyo

first: Hong-tao

last: Li

first: Gangning

last: Liang

first: Peter

last: Jones


title: Shade represses photosynthetic genes by disrupting the DNA binding of GOLDEN2-LIKE1

city: Daejeon

first: Namuk

last: Kim

address: Daehakro 291

country: South Korea

institute: KAIST

summary: The goal of this study is to identify transcriptome changes by the RPGE overexpression or glk1/glk2 double mutant under white light. We compared the transcriptome of RPGE2-OX (35Spro::GFP-RPGE2) with wild type (Col-0) and glk12 (glk1/glk2 double mutant) with wild type in continuous white light to investigate the expression changes of genes which are related with chloroplast development and the relationship between RPGE and GLK in the chloroplast development process.

overall_design: Transcriptomes of RPGE2-OX, glk1/2 double mutant and wild-type Arabidopsis seedlings were compared.

first: Jinkil

last: Jeong

first: Jeongheon

first: Giltsu

last: Choi


title: RNA mis-splicing sensitizes SETD2-mutant kidney cancer cells to DNMTi

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

overall_design: Refer to individual Series