title: The CD90+CD39+ and CD90+CD39- fibroblast subtypes in human hypertrophic scar and normal skin

first: Xin

last: Huang

state: China

address: zhizaoju Road 639

institute: Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine

summary: To explore the functional difference between CD90+CD39+ and CD90+CD39- fibroblasts in human hypertrophic scar and normal skin, the gene expresson microarray was performed on Live CD49f- E-Cadherin- Lin- CD45- CD31- CD90+ CD39+ and Live CD49f- E-Cadherin- Lin- CD45- CD31-  CD90+ CD39- cells sorted from suspension disgested from three human hypertrophic scar samples; and Live CD49f- E-Cadherin- Lin- CD45- CD31- CD90+ CD39+ cells sorted from suspension disgested from three human normal skin samples

overall_design:
Three 10 cm × 10 cm samples of hypertrophic scar and three 5 cm × 5 cm samples of normal skin were cut into small pieces and put into Dispase II (Gibco, 17105041, 0.3% in DMEM) for 30min at 37℃ to allow the separation of the epidermis. Scar were then minced and digested with a modified protocol, namely, 2.5 mg/mL Collagenase I (Gibco, 17100017), 1 mg/mL Collagenase II (Gibco, 17101015), 1 mg/mL Collagenase IV (Gibco, 17104019), 0.2 mg/mL Hyaluronidase (Sigma, H3884) and 0.1 mg/mL DNase Ⅰ(Sigma, D4263) in DMEM + 2% P/S (Gibco, 15070-063) for 4h shaking in a 37°C waterbath.
Acquired cell suspension was filtered, lysed with ACK Lysis Buffer, stained with FVS780- APC-CY7 and Human Fc block. Afterwards, cells were stained with anti-human antibodies for 10min at 4°C: Lineage Cocktail-Pacific Blue (BD, 562722, 1:100), CD45- APC-CY7 (BD, 557833, 1:100), E-Cadherin-BV421 (BD, 743712, 1:100), CD31-PE-CY7 (BD, 563651, 1:100), CD49f/ITGA6-PE (BD, 555736, 1:50), CD90-BV510 (BD, 563070, 1:20), and CD39-PE-CF594 (BD, 563678, 1:100). After negative selection (singlets, FVS780-, CD49f-, E-Cadherin-, Lin-, CD45-, and CD31-), CD90+CD39+ and CD90+CD39- Fb from hypertrophic scar tissue and CD90+CD39+ Fb from normal skin were sorted using BD FACS Aria II. Sorted cells were collected in TRIzol™ Reagent (Invitrogen, Carlsbad, CA, USA) for RNA isolation and subjected to Agilent Human 4x44K Gene Expression Microarrays v2 according to Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology)

first: Tao

last: Zan


title: Next-generation sequencing analysis of microglia from AD and Calhm2 cKO/AD mice

status: Public on Jul 05 2021

city: Beijing

first: Jinbo

last: Cheng

state: Beijing

address: No. 27 Taiping Road, Haidian District Beijing

country: China

institute: Beijing Institute of Basic Medical Science

type: Expression profiling by high throughput sequencing

summary: To analyze the different genes in microglial Calhm2 cKO in normal and AD conditions.

overall_design: Microglial cells from 4 groups 6-month-old mice: WT mice, Calhm2 cKO mice, AT/AD mice and Calhm2 cKO/AD mice.

first: Yuan

last: Dong

first: Zengqiang

last: Yuan


title: MCF-7 cell RNA-seq analysis of PELP1-induced gene expression

city: Minneapolis

first: Carol

last: Lange

state: MN

address: 2231 6th ST SE

department: Masonic Cancer Center

country: USA

institute: University of Minnesota

summary: RNA-seq was performed on MCF-7 cells expressing vector control (LXSN), PELP1-wt, and PELP1-cyto

overall_design:
Cell lines were cultured under tumorsphere conditions for 3 days prior to RNA isolation. Comparisons 1) 3D_LXSN_7L vs. 3D_CytoPELP1; 2) 3D_LXSN_7L vs. 3D_WTPELP1_7L; 3)3D_CytoPELP1_7L vs. 3D_WTPELP1_7L

contributor: Artificial Intelligene (AI, www.artificialintelligene.com)

first: Thu

last: Truong

first: Julie

last: Ostrander


title: Molecular alterations induced by Yersinia pestis, dengue virus and Staphylococcus enterotoxin B under the background of battlefield-like stress (miRNA-Seq)

city: Fort Detrick

first: seid

last: muhie

state: MD

address: 568 Doughten Dr

department: Systems Biology Enterprise

institute: USACEHR

type: Non-coding RNA profiling by high throughput sequencing

summary:
Background: Severe stress can have drastic and systemic effects with dire implications on the health and wellbeing of exposed individuals.  Particularly, the effect of stress on the immune response to infection is of interest because of its implications for vaccine efficacy and treatment strategy during stressful scenarios. Battlefield-like stress has previously been shown to cause an anergic state in the immune system that persists following exposure to a potent mitogen.
Methods: Genome-wide alterations of gene expression were characterized using leukocytes collected from (15 informed and consented, all male) U.S. Army Ranger cadets immediately before and after training followed by exposure to a representative group of pathogens Yersinia pestis, dengue virus, Staphylococcus enterotoxin B (SEB).  We employed experimental and computational approaches in identifying altered molecules, processes, pathways, and functional and regulatory networks mediating the host’s response towards battlefield-like stress, in assessing protective immunity status of the stressed host towards infection, and in identifying pathogen-induced biomarkers under severe stress background.
Results: We observed predicted inhibition of pathways significantly associated with lymphopoiesis, wound healing, inflammatory response, lymphocyte activation, apoptosis, and predicted activation of oxidative stress.  Using weighted correlation network analyses, we showed preservation of these pathways across infection plus stress combinations.  Regulatory networks comprising a common set of upstream regulators: transcription factors, microRNAs and post-translational regulators (kinases, phosphatases) may be drivers of molecular alterations leading compromised protective immunity.  Other sets of transcripts were persistently altered in both the pre- and post-stress conditions due to the host’s response to each pathogen forming pathogen-specific molecular signatures with a potential to distinguish infection from that of severe stress.
Conclusion: Our results suggest that severe battlefield-like stress alters molecular players implicated in the developments of hematopoietic stem cells probably leading to depletion of cellular and molecular repertoires of protective immunity, membrane trafficking of recycling endosomes and membrane translocation of the antigen processing mechanisms leading to suboptimal antigen processing and presentation, cell adhesion leading to impaired formation of productive immunological synapse,  and inhibited T-cell activation.  These factors may collectively be responsible for compromised protective immunity (infection susceptibility, delayed wound healing and poor vaccine response), and cellular anergy.

overall_design: Subjects (RASP cadets) were exposed to eight weeks of severe stress (RASP training stressors). Plasma was obtained from whole blood samples collected immediately before and after training. MicroRNA were isolated from plasma and sequenced.

first: S

last: Muhie

first: A

last: Gautam

first: R

last: Hammamieh

first: M

last: Jett


title: Induction of a chromatin boundary in vivo upon insertion of a TAD border (ChIP-Seq)

city: Lausanne

first: Lucille

last: Lopez-Delisle

address: Station 19

country: Switzerland

institute: EPFL

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Mammalian genomes are partitioned into sub-megabase to megabase-sized units of preferential interactions called topologically associating domains or TADs, which are likely important for the proper implementation of gene regulatory processes. These domains provide structural scaffolds for distant cis regulatory elements to interact with their target genes within the three-dimensional nuclear space and architectural proteins such as CTCF as well as the cohesin complex participate in the formation of the boundaries between them. However, the importance of the genomic context in providing a given DNA sequence the capacity to act as a boundary element remains to be fully investigated. To address this question, we randomly relocated a topological boundary functionally associated with the mouse HoxD gene cluster and show that it can indeed act similarly outside its initial genomic context. In particular, the relocated DNA segment recruited the required architectural proteins and induced a significant depletion of contacts between genomic regions located across the integration site. The host chromatin landscape was re-organized, with the splitting of the TAD wherein the boundary had integrated. These results provide evidence that topological boundaries can function independently of their site of origin, under physiological conditions during mouse development.

overall_design: Analysis of CTCF and RAD21 binding by ChIP in developing mouse limbs upon ectopic integration of region CS38-40 (TgN(38-40))

first: Andréa

last: Willemin

first: Eddie

last: Rodríguez-Carballo

first: Denis

last: Duboule


title: Genome-wide analysis of the gene expression of different subtype macrophages

city: Taipei

first: Sung-Liang

last: Yu

address: Jen Ai Road Section1

department: Clinical Laboratory Sciences and Medical Biotechnology

country: Taiwan

institute: National Taiwan University

summary: Profiling the THP-1 derived macrophages, included M0, M1, M2a and M2c macrophages.

overall_design: Total RNA obtained from differentiated M0, M1, M2a and M2c macrophages

first: Yi-Jing

last: Hsiao


title: Molecular alterations induced by Yersinia pestis, dengue virus and Staphylococcus enterotoxin B under the background of battlefield-like stress (microarray)

summary: Genome-wide alterations of gene expression were characterized using leukocytes collected from (informed and consented, all male) U.S. Army Ranger cadets immediately before and after training followed by exposure to a representative group of pathogens Yersinia pestis, dengue virus, Staphylococcus enterotoxin B.  We employed experimental and computational approaches in identifying altered molecules, processes, pathways, and functional and regulatory networks mediating the host’s response towards battlefield-like stress, in assessing protective immunity status of the stressed host towards infection, and in identifying pathogen-induced biomarkers under severe stress background.  We observed predicted inhibition of pathways significantly associated with lymphopoiesis, wound healing, inflammatory response, lymphocyte activation, apoptosis, and predicted activation of oxidative stress.  Using weighted correlation network analyses, we showed preservation of these pathways across infection plus stress combinations.  Regulatory networks comprising a common set of upstream regulators: transcription factors, microRNAs and post-translational regulators (kinases, phosphatases) may be drivers of molecular alterations leading compromised protective immunity.  Other sets of transcripts were persistently altered in both the pre- and post-stress conditions due to the host’s response to each pathogen forming pathogen-specific molecular signatures with a potential to distinguish infection from that of severe stress.  Our results suggest that severe battlefield-like stress alters molecular players implicated in the developments of hematopoietic stem cells probably leading to depletion of cellular and molecular repertoires of protective immunity, membrane trafficking of recycling endosomes and membrane translocation of the antigen processing mechanisms leading to suboptimal antigen processing and presentation, cell adhesion leading to impaired formation of productive immunological synapse,  and inhibited T-cell activation.  These factors may collectively be responsible for compromised protective immunity (infection susceptibility, delayed wound healing and poor vaccine response), and cellular anergy.

overall_design: A total of 20 subjects were included in the pre- and post-RASP study. The microarray was done using two chanel custom arrays consisting of about 9000 probes.

first: Seid

first: Ross

last: Campbell

first: Aarti

first: Rasha

first: Christiano

last: Cummings

first: David

last: Yang

first: Marti


title: Molecular alterations induced by Yersinia pestis, dengue virus and Staphylococcus enterotoxin B under the background of battlefield-like stress

first: seid

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

overall_design: Refer to individual Series


title: Single cell RNA-seq of tumor antigen specific CD8 Tissue Resident Memory (Trm) Cells

status: Public on Jul 04 2021

city: Lebanon

first: Aleksey

last: Molodtsov

state: NH

address: 1 Medical Dr

department: Micro Immuno

institute: Dartmouth College

summary:
Purpose: To characterize  the tumor-specific TRM cells in LNs, skin, lung and liver in mice with melanoma-associated vitiligo (MAV). scRNAseq was performed to identify populations of CD8 T cells with TRM transcriptional characteristics.
Methods: CD8+Thy1.1+ T cells were sorted from DLN, skin lung and liver of MAV mice and scRNA-sequenced
Results: Heterogenouse CD8 memory populations with both circulatating and resident phenotypes were identified.
Conclusions: Tumor-specific CD8 T cells form TRM responses in tumor-draining LNs.

overall_design: scRNA-seq of CD8+Thy1.1+ T cells from DLN, skin, lung and liver of  MAV mice 45 days post tumor surgery.

first: Mary

middle: J

last: Turk


title: Resident memory T cells in regional lymph nodes sustain immunity to cancer


title: Bulk RNA-seq of tumor antigen specific CD8 Tissue Resident Memory (Trm) Cells

summary:
Purpose: To characterize  the tumor-specific TRM cells in LNs, skin, lung and liver in mice with melanoma-associated vitiligo (MAV). Bulk RNA-seq was performed to identify populations of CD8 T cells with TRM transcriptional characteristics.
Methods: CD8+THy1.1+ T cells were sorted from DLN, skin lung and liver of MAV mice and bulk RNA-sequenced
Results: Heterogenouse CD8 memory populations with both circulatating and resident phenotypes were identified.
Conclusions: Tumor-specific CD8 T cells form TRM responses in tumor-draining LNs.

overall_design: bulk RNA-seq of CD8+Thy1.1+ T cells from DLN, skin, lung and liver of  MAV mice 45 days post tumor surgery.


title: scRNA/TCR-seq of CD8+CD44+ T cells from tumor draining lymph node and matched vitiligo-affected skin of MAV mice

summary:
Purpose: To identify the presence of endogenous tumor-specific TRM cells in LNs, scRNAseq was performed to identify populations of CD8 T cells with TRM transcriptional characteristics in DLNs.
Methods: CD8+CD44+ T cells were sorted from DLN and skin of MAV mice and scRNA-seq in parallel with scTCR-seq was performed.
Results: The twenty most expanded clonotypes identified in vitiligo-affected skin of these mice, eight matched to lymph nodes  and strikingly, those lymph node clonotypes that had matches in skin mapped almost exclusively to the LN TRM cluster
Conclusions: Endogenous tumor-specific CD8 T cells form TRM responses in tumor-draining LNs.

overall_design: scRNA/TCR-seq of CD8+CD44+ T cells from DLN and skin of  MAV mice 45 days post tumor surgery


title: INO80 promotes and safeguards bivalency [RNA-seq]

city: Research Triangle Park

first: Guang

last: Hu

state: NC

address: 111 TW Alexander DR

department: ESCBL

institute: National Institute of Environmental Health Sciences

summary: INO80 is involved in many chromatin-dependent functions. However, its role in pluripotency has not been fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the primed state. Mechanistically, INO80 co-occupied gene promoters that were bivalently marked by H3K4me3 and H3K27me3. Further, its occupancy was required for H3K27me3 installation and maintenance, as well as downstream gene repression. Finally, INO80 promoted H2A.Z occupancy at the bivalent domains, which in turn facilitated the polycomb repressive complex 2 (PRC2) recruitment. Together, our results identified the INO80-H2A.Z axis as an essential step for bivalent chromatin and poised gene expression and uncovered an epigenetic mechanism by which chromatin remodeling, histone variant, and histone modification coordinately control cell fate.

overall_design: Effects of INO80  deletion on the transcriptome (RNAseq) and epigenome (ChIP-seq, MNase-seq) in mouse naïve and primed pluripotent stem cells

first: Hongyao

first: Jiajia

last: Wang


title: INO80 promotes and safeguards bivalency [Mnase-seq]


title: INO80 promotes and safeguards bivalency [ATAC-seq]

overall_design: ATAC-Seq in  Naïve and Primed pluripotency cells


title: Differential effects of Am80 and calcipotriol administration on gene expression in CAFs

status: Public on Jul 03 2021

city: Nagoya

first: Tadashi

last: Iida

state: AICHI

department: Gastroenterology and Hepatology

country: Japan

institute: Nagoya university

summary: We investigated how Am80 differed from vitamin D analogues in terms of effects on gene expression in PDAC CAFs. we stimulated primary cultured PSCs isolated from three patients with PDAC (PSC163, PSC52, and PSC119) with dimethyl sulfoxide (DMSO, control), Am80, or calcipotriol. Gene expression profiles were then compared using microarray-based transcriptomic analysis. Extracting genes that were differentially expressed between Am80- and calcipotriol-treated cells revealed that Meflin expression was more significantly upregulated by Am80 than calcipotriol. Interestingly, the expression levels of fibroblast activation protein (FAP) and chemokine (C-C motif) ligand 2 (CCL2), which are markers of pCAFs, were higher in Am80-treated PSCs than calcipotriol-treated PSCs, whereas the expression levels of other pCAF marker genes, such as C-X-C motif chemokine ligand 12 (CXCL12), podoplanin (PDPN) and periostin (POSTN), were higher in calcipotriol-treated cells. These data suggested that Am80 and calcipotriol differentially regulated gene expression in PSCs.

overall_design: Primary cultured human PSCs isolated from three patients with PDAC (163, 52, and 119) were administered with DMSO (1 µM), calcipotriol (Cal, 1 µM), and Am80 (1 µM) for 48 h. Total RNA was then extracted, and gene expression microarray analysis was performed.

first: Yasuyuki

last: Mizutani

first: Iida

last: Tadashi


title: INO80 promotes and safeguards bivalency


title: HDACi promotes inflammatory remodeling of the tumor microenvironment to enhance epitope spreading and antitumor immunity

city: Hamilton

first: Yonghong

last: Wan

state: ON

address: 1280 Main St W

country: Canada

institute: McMaster University

summary: Adoptive cell therapy (ACT) with tumor-specific memory T cells has shown increasing efficacy in regressing solid tumors. However, tumor antigen heterogeneity represents a longitudinal challenge for durable clinical responses due to the therapeutic selective pressure for immune escape variants. Here, we demonstrate that delivery of class I histone deacetylase inhibitor, MS-275, promotes sustained tumor regression by synergizing with ACT in a coordinated manner to enhance cellular apoptosis.  We find that MS-275 alters the tumor inflammatory landscape to support antitumor immunoactivation through the recruitment and maturation of cross-presenting CD103+ and CD8+ dendritic cells and depletion of regulatory T cells. Activated endogenous CD8+ T cell responses against non-target tumor antigens was critically required for the prevention of tumor recurrence. Importantly, MS-275 alters the immunodominance hierarchy by directing epitope spreading towards endogenous retroviral tumor-associated antigen, p15E. Our data suggest that MS-275 multi-mechanistically improves epitope spreading to promote long-term clearance of solid tumors.

overall_design: Bulk tumor RNA obtained from mice treated with ACT (Tmem+VSV-gp33) or ACT+MS-275 1, 3, and 5 days post-treatment

first: Andrew

last: Nguyen

first: Louisa

last: Ho


title: Identification of the likely pathways downstream of Pdia4 in cancer stromal cells

city: Taipei city

first: Meng-Ting

state: Nankang

address: 128 Academia Road, Section 2, Nankang, Taipei

department: ABRC

institute: Academia Sinica

summary:
14151 genes whose signal intensity > 200
1554 genes whose expression ratio (WT/Pdia4-/-) was over 2.4 or below 0.4

overall_design: Affymetrix array analysis of the cancer stromata of GK1 tumor-bearing WT and Pdia4-/- (KO) mice.

first: Tzung-Yan

last: Chen


title: INO80 promotes and safeguards bivalency [ChIP-seq]


title: Single-cell RNA-seq profiling reveals novel insights in immune-complex deposition and epithelium transition in IgA nephropathy

status: Public on Jul 06 2021

first: Ping

last: Lu

address: 5 Yiheyuan Road

institute: Peking University

summary: IgA nephropathy represents the most prevalent chronic nephrosis worldwide. However, pathogenesis about IgA deposition and end-stage renal failure is still not well defined. Using single-cell RNA-seq, we identified the mesangial membrane receptor for IgA, which collaborates with increased extracellular matrix proteins and protease inhibitor to facilitate IgA deposition. Meanwhile, cell-cell interaction analysis revealed increased communications between mesangium and other cell types, uncovering how morbidity inside glomerulus spreads to whole kidney, which results in the genetic changes of kidney resident immune cells. Prominent interaction decreasing in intercalated cells leads to the discovery of a transitional cell type, which exhibited significant EMT and fibrosis features. Our work comprehensively characterized the pathological mesangial signatures, highlighting the step-by-step pathogenic process of IgA nephropathy from mesangium to epithelium.

overall_design:
In this study, we collected single cells from 13 IgAN patients’ renal biopsies and normal renal cells from 6 kidney cancer patients’ paracancerous tissues. As glomerulus are difficult to digest, we separately dissociated the glomerulus and the rest renal tissues. We applied CD326+ and CD14+ MACS to capture epithelium and macrophages, to cover the entire renal cell types, negative selected cells from MACS were also collected. Meanwhile, we isolated monocytes from 5 of the 13 IgAN patients and another 5 normal persons’ peripheral blood using CD14+ MACS

**Submitter declares that the raw data have been deposited in the Genome Sequence Archive for Human (https://bigd.big.ac.cn/gsa-human/) under submission number PRJCA003506.**

first: Fuchou

last: Tang


title: Analysis of the role of long non-coding RNAs involved in silica-induced pulmonary fibrosis of rat

city: Jinan

first: Gongchang

last: Yu

state: Shandong

address: NO.18877, Jingshi Road

department: Toxicology Department

institute: Shandong Academy of Occupational Health and Occupational Medicine

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

summary: Mounting evidence suggests that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of silicosis. In this study, Wistar rats were treated with an improved intubation method to ferry silicon dioxide (1mL per rat) into lung. Rats in the control group were injected with normal saline solution (1mL per rat). Twenty eight days after exposure, we examined the rats and found the formation of lung fibrosis in rats treated with silica. We then investigated the lncRNAs expression changes in lungs of silica-exposed rats and compared with that of rats in the control group. Treatment of silica leads to 682 lncRNAs differentially expressed including 300 upregulated and 382 down regulated compared to controls.

overall_design: 6 rats were randomly divided into two groups, a control group and silicosis model group with 3 animals each. The two groups were treated with an improved intubation method to ferry drugs into lung under direct vision using self-made experimental equipment. After successfully setting up, the control rats were injected with normal saline solution (1mL per rat), and the model rats were injected with silicon dioxide (1mL per rat). Then these rats were fed normally for 28 days which the rat model of pulmonary fibrosis can be builded successfully. The body weight of rats in each group was monitored weekly. After treatment, the rats from each group were anesthetized by intraperitoneal injection of 10 % chloral hydrate (0.4 mL/100 g body weight). The rats lung tissues were randomly collected and stored at -80°C for lncRNA microarray expression analysis.

first: Linlin

last: Sai

first: Cunxiang

last: Bo

first: Yu

last: Zhang

first: Zhongjun

last: Du

first: Chao

last: Li

first: Zhenling

first: Qiang

last: Jia

first: Hua

last: Shao

first: Cheng

last: Peng


title: Next Generation Sequencing Analysis of  Transcriptomes in maculopapular drug rash with COVID-19 infection

city: Zurich

first: Ge

last: Tan

address: Winterthurerstrasse 190

department: Functional Genomics Center Zurich

institute: University of Zurich

summary:
Purpose: Investigate whether maculopapular drug rash with COVID-19 infection (COVID19-MDR) exhibits a distinct gene expression in the skin as compared to non-COVID19-MDR.
Methods:RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) skin biopsies from COVID-MDR (n=3), MDR (n=7), and Healthy control (n=5).  Library preparation for RNA-seq was performed by using the TruSeq Stranded RNA library preparation kit including polyA enrichment (Illumina) from total RNA. Sequencing was performed on the the Illumina NextSeq 500 platform with 75 cycles.
Results: RNA sequencing from lesional skin showed that pathways of cytolysis and eosinophil chemotaxis were activated in COVID MDR. Cytolysis/cellular defense response related genes, such as PRF1 (perforin), GZMA (Granzyme A) and GNLY (Granulysin), as well as eosinophil migration/lymphocyte chemotaxis genes, e.g. C-C Motif chemokine ligand 5 (CCL5), CCL13, were upregulated in COVID MDR.
Conclusions:  This study provide an opportunity to understand the pathogenesis of MDR with the COVID-19 infection.
We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

overall_design: Skin mRNA profiles of maculopapular drug rash with COVID-19, maculopapular drug without COVID-19, and healthy control

first: Marie-Charlotte

last: Brüggen

first: Yasutaka

last: Mitamura


title: Acute rhinovirus infection induces extensive antiviral and B cell receptor gene expression in B cells from asthmatics

summary: B cells of asthmatic patients have dysregulated expression of inflammatory cytokine, B cell receptor and antiviral genes at a steady-state. During experimental in vivo infection of human subjects with rhinovirus, interferon-induced antiviral response is exaggerated in B cells in asthmatic patients.

overall_design: Examination B cell-gene expression in asthmatic and healthy subjects before and after experimental rhinovirus infection.

first: Oliver

last: Wirz

first: Mübeccel

last: Akdis


title: IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy

city: Ann Arbor

first: Sunil

last: Tomar

address: 109 Zina Pitcher Place

institute: University of Michigan

overall_design: In this study we identified the IL-4 signaling-mediated regulation of small intestinal (SI) MMC9s using bulk RNAseq. We also identified the MMC9 transcriptome at the single cell level using single cell RNAseq. For the first goal, we employed an adoptive transfer model of food allergy, where ovalbumin (OVA)-sensitized wild type (WT) mice are reconstituted with bone marrow progenitors of either WT, IL-4RaY709F (IL-4RA gain of function mutant) or IL-4Ra-/- (loss of function mutant) mice, followed by 7 repeated oral Ova challenges (3 times per week). This approach generated SI-MMC9s of WT, IL-4RaY709F and IL-4Ra-/- origins. SI-MMC9s were sort purified as described in the study, and their gene expressions identified by bulk RNAseq. Each RNA sample was obtained by pooling from 12-20 biological replicates. For the second goal, we performed single cell RNAseq on 81 sort purified SI-WT MMC9s from mice that had undergone Ova sensitization, followed repeated oral Ova challenges. We obtained scRNA-seq reads from 81 SI Lin- IL-4GFPhigh IL-17RB2- c-Kit+ ST2+ β7-integrinlow cells that were aligned to the reference mouse genome (GRCm38) to obtain relative gene expression as RPKM as described in the bulk sequencing analyses.

first: Varsha

last: Ganesan

first: Ankit

last: Sharma

first: Chang

last: Zeng

first: Lisa

last: Waggoner

first: Andrew

last: Smith

middle: H

last: Kim

first: Paula

last: Licona-Limón

first: Richard

middle: L

last: Reinhardt

middle: A

last: Flavell

first: Yui-Hsi

last: Wang

first: Simon

middle: P

last: Hogan


title: Mechanical loading influences osteocytes and further regulates osteoblast metabolism and osteogenic differentiation(miRNA Array)

status: Public on Jul 02 2021

city: Guilin City

first: Cao

last: Zhen

address: 1 Zhiyuan Road, Lingui, Guilin, Guangxi 541004, P.R. China

institute: Guilin medical university

summary:
A previous study indicated that mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz for 8 h promoted osteogenesis and corresponding mechanoresponsive microRNAs (miRs) were identified in osteoblasts. However, in osteocytes, it has not been identified which miRs respond to the mechanical strain, and it is not fully understood how the mechanoresponsive miRs regulate osteoblast differentiation.
Mouse MLO-Y4 osteocytes were applied to the same mechanical tensile strain in vitro.MiR microarray and reverse transcription-quantitative polymerase chainreaction(RT-qPCR) assays were applied to select and validate differentially expressed miRs, and the target genes of these miRs were then predicted. In addition, the differentially expressed miRNAs in mechanically strained osteocytes in vitro were compared with those  in treadmill exercise of the bone tissues  .

overall_design: In this study, the osteocytes’biological response to a mechanical tensile strain of 2500με at 0.5 Hz for 8 h was investigated, and some novel mechanosensitive miRs were selected. In addition, the involvement of miR-29b in osteocytes’response to mechanical strain and osteoblast differentiation were studied. Total RNAs was extracted from MLO-Y4 osteocytes of mechanical loading .MiRNA microarray was used to acquire expression profiles of a total of 1900 unique miRNAs

first: Qiangcheng

first: Yang


title: Treadmill training regulates bone metabolism and osteogenic differentiation(miRNA Array)

summary:
MicroRNAs (miRNAs) are important in the process of exercise influencing bone metabolism. The present study aimed to detect and select differentially expressed miRNAs in the bone tissues of mice trained on a treadmill, predict the target genes of these differentially expressed miRNAs and lay a foundation for exploring the effect of treadmill training on bone metabolism through miRNAs.
In this experiment, after the mice were trained on a treadmill for 8 weeks, the mechanical properties of mouse femur bone were assessed, and the alkaline phosphatase (ALP) activity and osteocalcin (OCN) protein levels of the bone were assayed. miRNA microarray and reverse transcription-quantitative (RT-q)PCR were performed to select and validate differentially expressed miRNAs in the bone, and the target genes of these miRNAs were predicted with bioinformatics methods. In addition, the differentially expressed miRNAs in the bone tissues were compared with those in mechanically strained osteocytes in vitro.

overall_design: In the study presented here, 6 male BALB/c mice were randomly divided into the treadmill training group  and no treadmill training group. Total RNAs was extracted from femur of mice.MiRNA microarray was used to acquire expression profiles of a total of 1900 unique miRNAs

first: Zhen

last: Cao

first: Huan

last: Yang


title: The transcription factor BCL6 controls early development of innate-like T cells (RNA-seq)

city: Vari

first: Mihalis

last: Verykokakis

address: 34, Fleming street

country: Greece

institute: BSCR Alexander Fleming

summary: Innate T cells, including invariant natural killer T (iNKT) and mucosal-associated innate T (MAIT) cells, are a heterogeneous T lymphocyte population with effector properties pre-programmed during their thymic differentiation. How this program is initiated is currently unclear. Here, we show that the transcription factor BCL6 is transiently expressed in iNKT cells upon exit from positive selection and is required for their proper development beyond stage 0. Notably, development of MAIT cells is also impaired in Bcl6-deficient mice. We found that BCL6 regulates the expression of genes that are associated with the innate T cell lineage, including PLZF and its targets. BCL6 contributes to a chromatin accessibility landscape that is permissive for the expression of development-related genes and inhibitory for genes associated with naïve T cell programs. Our results reveal novel functions for BCL6 and indicate that this transcription factor controls the inception of innate T cell lineage programming.

overall_design: Tetramer+TCRb+CD24+CD44- (Stage 0, ST0) and Tetr+TCRb+CD24-CD44- (ST1) cells from the thymi of 4-7 pooled WT and CDCre;Bcl6F/F (Bcl6KO) mice were sorted and RNA was extracted for RNA-seq analysis. Similar populations were sorted and cells were porcessed for ATAC-seq.

first: Barbara

last: Kee

first: Mikael

last: Sigvardsson


title: Changes in splicing and neuromodulatory gene expression programs in sensory neurons with pheromone signaling and social experience

city: Chapel Hil

first: Corbin

last: Jones

address: 250 Bell Tower Dr

department: Biology

institute: UNC-CH

summary: Social experience and pheromone signaling in ORNs affect pheromone responses and male courtship behaviors in Drosophila, however, the molecular mechanisms underlying this circuit-level neuromodulation remain less clear. Previous studies identified social experience and pheromone signaling-dependent modulation of chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male behaviors. To identify the molecular mechanisms driving social experience-dependent neuromodulation, we performed RNA-seq from antennal samples of mutant fruit flies in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. We found that loss of pheromone detection differentially alters the levels of fruitless exons suggesting changes in splicing patterns. In addition, many Fruitless target neuromodulatory genes, such as neurotransmitter receptors, ion channels, and ion transporters, are differentially regulated by social context and pheromone signaling. Our results suggest that modulation of circuit activity and behaviors in response to social experience and pheromone signaling arise due to changes in transcriptional programs for neuromodulators downstream of behavioral switch gene function.

overall_design: RNA-seq using whole third segment antennae from Drosophila melanogaster. One biological replicate includes 180 antennae from 90 flies at 7 days old.

first: Bryson

last: Deanhardt

first: Qichen

last: Duan

first: Chengcheng

last: Du

first: Charles

last: Soeder

first: Pelin

middle: C

last: Volkan


title: microRNAs and gene-environment interactions in autism: effects of prenatal maternal stress and the SERT gene on maternal microRNA expression

city: Kansas City

first: Zohreh

last: Talebizadeh

state: MO

address: 2401 Gillham Road

institute: Children's Mercy Hospital

summary:
In the study presented here, microRNA expression was profiled in blood samples from mothers of children with autism, with known stress exposure during pregnancy. 2500 mature microRNAs were examined.
Thirty-four maternal blood samples were examined in this study. Samples were divided into five groups based on maternal SERT genotypes (LL/LS/SS) and prenatal stress level (High/Low).

overall_design: The five comparison groups were: G1a (SS & High), G1b (LS & High), G2a (LL & High), G2b (SS & Low), and G3 (LL & Low).

first: David

last: Beversdorf


title: The m6A reader IMP2 directs autoimmune inflammation through an IL-17- and TNFa-dependent C/EBP transcription factor axis

city: Pittsburgh

first: Anita

last: Bansal

state: PA

address: Biomedical Science Tower

department: Immunology

institute: University of Pittsburgh

summary: Using IMP2-deficient mice to study the role of the RNA-binding protein (RBP) IMP2 in models of autoimmunity, namely autoimmune glomerulonephritis

overall_design:
Examination of IMP2 Binding proteins using RIP-seq and RNA-seq
RNA-seq and RIP-seq were both done on Mouse embryonic fibroblasts (MEFs):
For RNA-seq: Mouse embryonic fibroblasts (MEFs) were plated in 6-well tissue culture-treated dishes and placed in a 5% CO2 37C incubator overnight. MEFs were transfected with siRNA control or siRNA targeting IMP2 . After 24 hours, media was changed and cells were left either unstimulated, or stimulated with IL-17 (200 ng/mL) for 8 hours . Cells were trypsinized and RNA was harvested using Rneasy Mini Kit from Qiagen. RNA-seq libraries were prepared using standard Nextera XT Kit.
For RIP-seq: Mouse embryonic fibroblasts (MEFs) were plated in 150mm tissue culture-treated dishes and placed in a 5% CO2 37C incubator overnight. Cells were left either unstimulated or stimulated with IL-17 (200 ng/mL) for 3 hours . Cells were trypsinized and extracts were isolated with lysis buffer [100 mM KCl, 5 mM MgCl2, 10 mM Hepes (pH 7.0), 0.5% NP-40, and 1 mM dithiothreitol] with RNaseOUT (100 U/ml; Invitrogen). Buffers included a protease inhibitor cocktail (Sigma-Aldrich). Lysates were precleared with protein A agarose (Roche) and immunoprecipitated with Abs against IMP2 (MBL) or rabbit IgG control (MBL). Beads were washed with NT2 buffer and digested with DNase I (Roche Applied Science) and protease K (Sigma-Aldrich). Total RNA was extracted with acid phenol or TRIzol. RIP-seq libraries were prepared using SMART-Seq v4 Ultra Low Input RNA kit

first: Rami

last: Bechara

first: Sarah

last: Gaffen


title: Wound healing and anti-oxidant activities induced by Dersani Hydrogel with Alginate and Dersani Hydrogel ointments

city: Montevideo

first: Tatiana

last: Basika

state: Montevideo

address: Mataojo 2020

country: Uruguay

institute: Institut Pasteur de Montevideo

summary: Explore the role of these hydrogels in wound healing, this study assessed the effects of both, Dersani Hydrogel with Alginate (DHA) and Dersani Hydrogel (DH), in human skin keratinocytes and fibroblasts gene expression profiles in a wound healing context. Sodium alginate (SA) and culture medium were also included as controls.

overall_design: 1x106 HaCaT-SF or HDFn cells were seeded in 6 well plates and incubated at 37 °C until 70-80% confluence was reached. Then a gap (wound) was created by scratching the monolayer with a sterile 200 µl tip, then the cells were washed two times with phosphate buffered saline (PBS) 1X and incubated with the compounds DHA, DH and SA (0.625% w/v) dissolved in culture media containing 0.01% v/v Tween 20 during 12 h. Biological triplicates were analyzed.

first: Gonzalo

last: Grief

first: Carlos

last: Robello

first: Marcos

last: Giusti

first: Mariela

last: Bollati-Fogolín


title: Gene expression of type 1 regulatory T (Tr1) cells

city: Stanford

first: Molly

last: Uyeda

state: CA

address: 265 Campus Drive

institute: Stanford University

summary:
Purpose: To identify gene expression patterns in ex vivo isolated human Tr1 cells.
Method: RNA sequencing of total mRNA.
Results: Differential gene expression of Tr1 and non-Tr1 CD4+ T memory cells.
Conclusions: ex vivo type 1 regulatory T cells have a distinct gene expression profile compared to non-Tr1 CD4+ T cell memory cells.

overall_design: Peripheral blood mononuclear cells from 3 healthy human donors were sorted for CD3+/CD4+/CD45RA-/LAG3+/CD49b+ Tr1 cells and CD3+/CD4+/CD45RA-/LAG3-/CD49b- non-Tr1 T memory cells and subject to RNA-seq.

first: Maria

middle: G

last: Roncarolo


title: Increased expression of yes-associated protein/YAP and transcriptional coactivator with PDZ-binding motif/TAZ activates intestinal fibroblasts to promote intestinal obstruction in Crohn’s disease

city: Shanghai

first: Weijun

last: Ou

state: Shanghai

address: 1665, Kongjiang Road

institute: Xinhua Hospital, Shanghai Jiaotong University School of Medicine

summary: The results of this study indicate that the expression of YAP/TAZ was significantly upregulated in stenotic fibroblasts, which was associated with the YAP/TAZ target gene signature. YAP/TAZ knockdown suppressed the activation of intestinal fibroblasts. In intestinal fibroblasts, YAP/TAZ were activated by the Rho-ROCK1 signalling pathway. High YAP/TAZ expression was positively correlated with ROCK1 expression, which is a prognostic marker for intestinal obstruction in CD patients.

overall_design: Total RNA of human intestinal fibroblasts from 2 individual Crohn's disease patients. From every patient we used paired normal and stenotic intestines to isolate fibroblasts.

first: Weimin

last: Xu

first: Fangyuan

last: Liu

first: Yuegui

last: Guo

first: Zhenyu

last: Huang

first: Tienan

last: Feng

first: Chen-Ying

first: Peng


title: Next generation sequencing of miR6024 overexpressing tomato transgenics in control state and under Alternaria solani stress

city: Kolkata

first: Pallob

last: Kundu

state: West Bengal

address: P 1/12 CIT Scheme VIIM

department: Division of Plant Biology

country: India

institute: Bose Institute

summary: miR6024 overexpression may lead to changes in the transcriptome profile of tomato plants. Further changes may be noticed on infecting these plants with the necrotrophic pathogen Alternaria solani. These changes can only be gauged by carrying out a comparative transcriptome analysis with the wild type plants under similar conditions. We have used tomato (Pusa Ruby) for generation of miR6024 overexpressing transgenics. Disease study on these plants were carried out with the necrotrophic fungus A. solani. We carried an RNA-seq analysis using Illumina hiseq sequencing of 5 RNA libraries created from leaf tissues of wild type, OVX6024  transgenics and A. solani infected wild type and OVX6024 plants. The analysis revealed that 334 and 781 genes were significantly regulated in the transgenic plants and the infected transgenic plants respectively, with respect to their suitable  wild type controls. GO enrichment analysis and pathway analysis have been carried out as well. This work is supported by grants from DBT and SERB, GoI.

overall_design: Transcriptome profile of Indian tomato (Solanum lycopersicum) cultivar Pusa Ruby overexpressing miR6024 (and)infected with Alternaria fungus. A wild type control, two OVX6024 samples, A. solani infected wild type control (one) and OVX6024 (one) sample were analysed.

first: Sayani

last: Dey

first: P


title: RNA sequencing analysis of unchallenged, PBS injected and S. pneumoniae infected pcsk9 KO and WT zebrafish

city: Tampere

first: Markus

last: Ojanen

address: Arvo Ylpön katu 34

department: Faculty of Medicine and Health Technology

country: Finland

institute: Tampere university

summary:
Purpose: S. pneumoniae infection in both zebrafish larvae and adult fish can be used to model pneumococcal bacteremia. Here, we used unchallenged, PBS injected and S. pneumoniae (T4) infected adult zebrafish to study the genome-wide role of pcsk9 in vivo in steady-state and upon immune system stimulation.
Methods: Adult pcsk9 KO zebrafish and their WT siblings were infected with S. pneumoniae (3,370,000 CFU; SD 840,000 CFU) and their total liver RNA isolated at 1-day post infection. RNA-sequencing data was produced by NovaSeq. Data processing pipeline was built on Snakemake wrappers, and used STAR to quantify features from quality and adapter trimmed reads in gene level. Prior to the normalisation and differential gene expression analysis with DESeq2, the matrix of raw gene counts was prefiltered to keep only rows that have at least 10 reads total and then split by treatment to analyze each case separately. Result tables were annotated with biomaRt.
Results: Snakemake preprocessing pipeline measured 32,520 genes against GRCz11, ENSEMBL release 97. 20,709 of them was used for statistical testing. Full result tables for each treatment were ranked by p-value and the normalized DESeq2 abundances in samples were included. The genes with BH-adjusted p-value <0.05 was considered differentially expressed.
Conclusions: Our study identified up- and down-regulated genes in unchallenged, PBS injected and pneumococcal infected pcsk9 KO zebrafish liver compared to WT zebrafish.

overall_design: Total of 16 samples were analyzed: 4 samples collected from unchallenged fish (n=2 in both genotypes), 6 samples from PBS injected fish (n=3 in both genotypes) and 6 samples from S. pneumoniae infected zebrafish (n=3 in both genotypes).

first: Meeri

last: Pekkarinen

first: Dafne

last: Jacome Sanz

first: Marko

last: Pesu

first: Matti

last: Nykter


title: Arabidopsis MORC proteins function in the efficient establishment of RNA directed DNA methylation

city: Los Angeles

first: Zhenhui

last: Zhong

address: 610 Charles E Young Dr East

department: Department of Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

type: Methylation profiling by high throughput sequencing

summary: The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms.

overall_design: This dataset includes ChIP-seq, RNA-seq and BS-seq to understand the molecular mechanism of Arabidopsis MORC proteins in the efficient establishment of RNA directed DNA methylation.

first: Yan

last: Xue


title: RNA-seq profiling of RPE reprogramming

city: Baltimore

first: Seth

last: Blackshaw

address: 733 N Broadway

department: Neuroscience

institute: Johns Hopkins University

summary: The plasticity of the retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy (PVR), a defective repair process in humans. In contrast, in the embryonic chick, the RPE can be efficiently reprogrammed to regenerate a complete neural retina after surgical removal and when supplied an exogenous source of FGF2. Here, we analyzed discrete RPE cell populations during early times of transiently reprogrammed (RPE 6 hours post-retinectomy) and reprogrammed (RPE 6 hours post-retinectomy and FGF2 treatment) cells, using laser capture microdissection followed by RNA sequencing (LCM-seq) and computational analysis.

overall_design: RPE from chicken embryos were isolated via the Veritas Laser Capture Microdissection system in triplicate across three conditions: 1) intact embryonic day 4 (E4) RPE, 2) E4 RPE 6 hours post retinectomy (transiently reprogrammed RPE; t-rRPE), and 3) E4 RPE 6 hours post retinectomy and FGF2 treatment (reprogrammed RPE; rRPE). Total RNA extraction was performed using PicoPure RNA Isolation Kit (Arcturus, Applied Biosystems, Foster city, CA), including a treatment with DNase I. Intact, total RNA was reverse-transcribed using an Oligo(dT) primer, and limited cDNA amplification was performed using the SMARTer® Ultra® Low Input RNA Kit for Sequencing – v4  (Takara Bio USA, Inc., Mountain View, CA). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA). Libraries were sequenced as single-end 75 base pair reads on an Illumina NextSeq500 to generate 44-68 million single-end 75 bp reads for each sample. Reads were quality trimmed and adapter sequences removed using Trim Galore v0.6.1 and Cutadapt v1.18. Trimmed reads were aligned to Gallus gallus genome GRCg6a with Ensembl annotation 98 using STAR v2.7.3. Assembly and quantification of transcripts was performed with Stringtie v2.0.4 and normalization of gene counts was performed with DESeq2 v1.22.2.

first: Agustin

last: Luz-Madrigal

first: Jared

last: Tangeman

first: Sutharzan

last: Sreeskandarajan

first: Erika

last: Grajales-Esquivel

first: Lin

first: Chung

last: Liang

first: Panagiotis

last: Tsonis

first: Katia

last: Del Rio-Tsonis


title: Genome-Wide Profiling of DNA Methylation and Gene Expression induced by Alpha-Synuclein in Murine Microglia [methylation]

city: Grand Forks

first: Junguk

last: Hur

state: ND

address: 1301 N. Columbia Rd. Stop 9037

department: Biomedical Sciences

institute: University of North Dakota

summary: BACKGROUND: Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. However, the influence α-syn has on DNA methylation and gene expression patterns for other genes has not been thoroughly explored. Our objective is to determine gene expression and methylation changes in microglia induced by aggregate α-syn.METHODS: Transgenic mThy1-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-induced changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data.RESULTS: Microglial DNA isolated from our 3-month cohort had 5,315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3,742 DMGs and 3,766 DEGs in 13-month mThy1-Asyn mice.These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months.CONCLUSION: We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression.

overall_design: We performed transcriptomic and methylomic analysis of murine brain homogenate subjected to microglia selection enrichment from mice overexpressing full-length human wild-type α-syn under the murine Thy-1 promoter on the X chromosome. Transcriptomic and methylomic profiles were analyzed for biological meaning using Reactome pathway database, Gene ontology, and Kyoto Encyclopedia of Genes and Genomes on differentially expressed genes (DEGs) and differentially methylated genes (DMGs). Pathways were also examined by enrichment network anlaysis and MCODE to identify clusters of highly connected pathways. HEM samples are mice overexpressing human alpha-synuclein while WT mice are  controls. The corresponding background of these mice is the B6D2F1/J mice (stock# 100006) from The Jackson Laboratory (Bar Harbor, ME). There are eight 13 month old HEM mice and four 13 month wild-type controls. There are four 3 month HEM mice and three 3month wild-type controls.

first: James

middle: E

last: Porter

first: Brett

last: McGregor

first: Othman

last: Ghribi

first: Kai

last: Guo

last: Schommer

first: M

middle: O

last: Raihan


title: Genetic variants associated with development of colorectal cancer, type 1 diabetes, Hodgkin lymphoma and  Diffuse large B-cell lymphoma

city: Warszawa

first: Krzysztof

last: Goryca

address: Banacha 2c

department: CeNT

country: Poland

institute: Uniwersytet Warszawski

summary: In our genome-wide association study, we searched for an association of genetic variants with colorectal cancer, type 1 diabetes, Hodgkin lymphoma and  Diffuse large B-cell lymphoma among Polish population.

overall_design:
A total of  72 of 24-DNA sample pools from gender-matched individuals (18 colorectal cancer, 9 Hodgkin lymphoma, 8 Diffuse large B-cell lymphoma, 9 type 1 diabetes and 28 controls) were included in the study. Genomic DNA was extracted from whole blood treated with EDTA. Aliquots of DNA samples that passed quality control tests were combined according to diagnosis and gender in equimolar concentrations to obtain pools of DNA samples, adjusted to a final concentration of 50 ng/ml in Tris-EDTA buffer (pH 8).
Please note that no data processing was applied and thus only theta values from unprocessed file were used as a signal.
The main reason for not normalizing the data was that it was derived from pooled samples. Data normalization algorithms were in general optimized for genotype calling (which is impossible for these samples), and thus may introduce possible artifact introduction. Thus "the less the better" approach was made with the data analysis. PCA hasn't revealed any considerable stratification. Raw theta values were used as an input for Student's t-test. Clusters of significant probes were selected for validation in individual patients.

first: Filip

last: Ambrożkiewicz

first: Aneta

last: Bałabas

first: Patrycja

last: Cybula

first: Michalina

last: Dąbrowska

first: Ewa

last: Hennig

first: Jakub

last: Karczmarski

first: Anna

last: Kluska

first: Maria

last: Kulecka

first: Michał

last: Mikula

first: Jerzy

last: Ostrowski

first: Agnieszka

last: Paziewska

first: Magdalena

last: Piątkowska

first: Karolina

last: Skubisz

first: Natalia

last: Żeber-Lubecka

first: Bożena

last: Cukrowska

last: Paszkiewicz

first: Tomasz

last: Olesiński


title: RNA-seq expression profiling of terminal end buds and ducts from mouse mammary glands

city: Parkville

first: Gordon

middle: K

last: Smyth

state: Vic

address: 1G Royal Pde

department: Bioinformatics

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: The mammary glands of adult female mice were divided into ductal tissue and terminal end buds (TEBs). Basal and luminal epithelial cells were FACS sorted and RNA extracted for 75bp paired-end RNA-seq profiling using an Illumina NextSeq 500 sequencer.

overall_design: Tissue was extracted from three independent mouse pools resulting in n=3 biological replicates for basal cells from ducts, basal cells from TEBs, and luminal cells from TEBs and n=2 biological replicates for luminal cells from ducts.

first: Yunshun

last: Chen

first: Bhupinder

last: Pal

first: Jane

last: Visvader


title: Orc1, Orc2 and Orc4 ChIP-seq in Torulaspora delbrueckii

city: Buffalo

first: Laura

middle: N

last: Rusche

state: NY

address: 109 Cooke Hall, UB North/Amherst Campus

department: Biological Sciences

institute: State University of New York at Buffalo

summary: Chromatin Immunoprecipitation followed by high throughput sequencing to identify the location of Orc1, Orc2 and Orc4 genomic enrichment.

overall_design: This data represents two technical replicates for each IP sample (Orc1-MYC, Orc2-V5 and Orc4-V5), one mock IP in an untagged strain and two genomic input samples.

first: Haniam

last: Maria

first: Shivali

last: Kapoor

first: Tao

last: Liu


title: AC2P20 selectively kills M. tuberculosis at acidic pH by depleting free thiols

city: East Lansing

first: Robert

middle: B.

last: Abramovitch

state: MI

address: 567 Wilson Road

department: Microbiology and Molecular Genetics

institute: Michigan State University

summary: In this study, AC2P20 was prioritized for continued study to test the hypothesis that it was targeting Mtb pathways associated with pH-driven adaptation. RNAseq transcriptional profiling studies showed AC2P20 modulates expression of genes associated with redox-homeostasis. Gene enrichment analysis revealed that the AC2P20 transcriptional profile had significant overlap with a previously characterized pH-selective inhibitor, AC2P36. Like AC2P36, we show that AC2P20 kills Mtb by selectively depleting free thiols at acidic pH. Mass spectrometry studies show the formation of a disulfide bond between AC2P20 and reduced glutathione, supporting a mechanism where AC2P20 is able to deplete intracellular thiols and dysregulate redox homeostasis.

overall_design: Examination of M. tuberculosis treated with 20 uM AC2P20 for 24h hours at pH 5.7 as compared to a DSMO control

first: Shelby

last: Dechow

first: Garry

middle: B

last: Coulson

first: Michael

middle: W

last: Wilson

first: Scott

middle: D

last: Larsen


title: Gene expression analysis of neutrophils, Ym1+Ly6Chi monocytes and Ym1-Ly6Chi  monocytes

city: Yokohama

first: Hidehiro

last: FUKUYAMA

state: Kanagawa

address: 1-7-22, Suehirocho, Tsurumi-ku

department: IMS

institute: RIKEN

summary: mRNA expression profiles between Ym1+Ly6Chi monocytes and Ym1-Ly6Chi monocytes from LPS-treated mice were analyzed by RNA-sequencing

overall_design: mRNA profiles of neutrophils, Ym1+Ly6Chi monocytes and Ym1-Ly6Chi  monocytes from 7-weeks-old Ym1-Venus (+/Ym1Venus) mice were generated by deep sequencing, using HiSeq 1500 sequencer (Illumina).

last: Fukuyama

first: Naoki

last: Ikeda

first: Masato

last: Tanaka


title: Differentially expressed genes in CSRP2BP knockdown HTR-8 cells

status: Public on Jul 01 2021

city: Guangzhou

first: Chonghui

last: Hu

state: Guangdong

address: 106 Zhongshan Er Road

department: Department of General Surgery

institute: Guangdong Provincial People's Hospital

summary: CSRP2BP plays an important role in cell cycle progression, apoptosis and mammalian development, CSRP2BP knockdown by specific shRNA (CSRP2BP-shRNA) significantly inhibited the proliferation, migration, and invasion of HTR-8 trophoblast cells, and placental explant outgrowth, while CSRP2BP overexpression did the opposite. To elucidate the mechanisms by which CSRP2BP regulated the function of trophoblast cells, we performed microarray analysis to compare the transcription profiling between the HTR-8-shNC and HTR-8-shCSRP2BP cells

overall_design: Microarray analysis analysis was performed using 3 independent replicates. HTR8_shcon is control group, HTR8_shBP is experimental group

first: Yunjian

last: Wei

first: Zhen

last: Wang

first: Yanlin

last: Ma

first: Qi

last: Li


title: Transcriptional profiling of mouse auditory nerve development

city: Charleston

first: Jeremy

last: Barth

state: SC

address: 173 Ashley Ave, BSB 627

institute: MUSC

summary: Overall goal of the study is to identify the molecular regulators of auditory nerve development

overall_design: Three experimental samples each (n=3) of mouse auditory nerve developmental stages postnatal (P) days P3, P7, P14, P21 and adult were analyzed

first: Hainan

last: Lang

first: Clarisse

last: Paganiban

first: Kenyaria

last: Noble


title: Transcriptional profiles associated with Context dependent behavior in Rock X Sand F1 in Lake Malawi Cichlids

city: atlanta

first: Jeffery

last: Streelman

address: 950 atlantic drive

institute: georgia institute of technology

summary: We sought to evaluate the brain gene expression profiles of male courtship display. To assess male display and courtship behavior, we designed a courtship preference assay. We evaluated social interactions between males and females using a 40 gallon tank design with a ‘rock’ habitat at one end and ‘sand’ at the other, separated by glass bottom. When parental rock species (Petrotilapia nigra (TaxId 526958), Maylandia zebra (TaxId 106582), Labeotropheus feulliborni) are placed in this tank paradigm, males court females over the rocks. Males of sand species (Mchenga conophorus, Aulonocara baenschi (TaxId 143496), Tramitichromis intermedius (TaxId 323801)) court females over sand and construct species appropriate bowers. When single rock x sand F1 males were placed in this set up with F1 females, males invariably courted females over the ‘rock’ habitat, suggesting genetic dominance. When two rock x sand F1 males were allowed to compete for F1 females in this tank paradigm, something interesting happened. One male, typically the larger, courted females over the rock habitat, and the other simultaneously constructed bowers to court females in the sand. We detected no difference in GSI (gonadal somatic index) between F1 males behaving as ‘socially rock’ vs. ‘socially sand.’ This observation of divergent behavior among interacting F1 brothers suggests an interaction between the genome and the social environment in these males.

sample_organism: Petrotilapia nigra x Aulonocara baenschi

sample_organism: Maylandia zebra x Tramitichromis intermedius

sample_organism: Labeotropheus fuelleborni x Mchenga conophoros

overall_design:
Whole brains of F1 males tested singly (n=2 lone) as well as F1 brothers assayed in dyads (n=4 dyads) were dissected after sacrifice during courtship and interrogated using RNA-seq. Individuals from the same cross are from the same brood/clutch of hybrid progeny i.e. are siblings.
Please note that the dyads are brothers and the 'LONE' samples are the F1 males tested singly. Sub and Dom were internal naming conventions, behavioral context is the key distinction between the individuals (whether they perform sand like or rock like behavior); SUB if the individual was behaving on sand, DOM if the individual was behaving on rock and LONE if it was tested singly.

first: Chinar

last: Patil

first: Kawther

last: Abdilleh


title: Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer

city: La Jolla

first: Gene

last: Yeo

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

institute: UCSD

summary: We found in this study through targeted CRISPR-Cas9 screening of RBPs that disrupting YTHDF2-dependent degradation of its target transcripts triggers apoptosis of MYC-dependent cancer cells and tumors. Enhanced CLIP (eCLIP), m6A-sequencing (m6A-seq) and YTHDF2 knockdown followed by RNA-seq analysis of YTHDF2-m6A-mRNA interactions revealed extensive associations with mRNAs encoding MAPK pathway genes that when stabilized, induce epithelial-to-mesenchymal transition and stress-induced apoptosis. scRibo-STAMP profiling of translating mRNAs revealed global alterations in the translatome of single cells in YTHDF2-depleted solid tumors. Thus, our work highlights the therapeutic relevance of RBPs by uncovering the critical role of YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven cancers.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: We constructed and applied an RBP targeting CRISPR-Cas9 library to MYC-inducible human mammary epithelial cells 8 and 16 days after transduction by sequencing of gDNA in biological duplicate. YTHDF2 binding and m6A profiles were examined using eCLIP-seq and m6A-seq by immunoprecipitating mRNA fragments bound to either endogenous YTHDF2 or m6A antibody in biological duplicates with input controls. In addition, we examined changes in mRNA expression following depletion of YTHDF2 using two independent shRNAs in biological duplicate. Lastly, we identified translational changes in the YTHDF2-depleted solid tumors by directing C-to-U editing of translating transcripts by transducing cells with RPS2-APOBEC1 fusions prior to subcutaneous xenograft.

first: Jaclyn

middle: M

last: Einstein

first: Mark

last: Perelis

first: Isaac

last: Chaim

first: Julia

last: Nussbacher

first: Alexandra

middle: T

last: Tankka

first: Brian

last: Yee

first: Assael

last: Madrigal

first: Archana

last: Shankar


title: Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

city: Dalseonggun, Daegu

first: Yongcheol

last: Cho

state: Daegu

address: 333, Techno jungang-daero, Hyeonpung-eup, Dalseong-gun

department: Department of Brain Sciences

country: South Korea

institute: DGIST

summary: Gene expression profiling in response to nerve injury has been mainly focused on protein functions of coding genes to understand mechanisms of axon regeneration and to identify targets of potential therapeutics for nerve repair. However, the protein functions of several highly injury-induced genes including Gpr151 for regulating the regenerative ability remain unclear. Here we present an alternative approach focused on non-coding functions of the coding genes, which led to the identification of the non-coding function of Gpr151 RNA interacting with RNA-binding proteins such as CSDE1. Gpr151 promotes axon regeneration by the function of its 5’- untranslated region (5’UTR) and expression of an engineered form of the 5’UTR improves regenerative capacity in vitro and in vivo in both sciatic nerve and optic nerve injury models. Our data suggest that searching injury-induced coding genes potentially functioning by their non-coding regions is required for the RNA-based gene therapy for improving axon regeneration.

overall_design: Adult mouse L4,5 DRG tissues, RiboTag mice crossed with Advilllin-Cre or CD-1 mice, Embryonic E13.5 DRG culture with control or 5'UTRm-overexpression, CSDE1-ip

first: Jinyoung

last: Lee

first: Jung

last: Shin

first: Bohm

first: Hyemin

last: Kim

first: Yewon

last: Jeon

first: Minjae

last: Kwon

first: Eunsoo

last: Jang

first: Yeonsoo

last: Oh


title: Transcriptome analysis of interleukin-4 induced intestinal organoids from Japanese monkey

first: Takaho

middle: A.

last: Endo

address: 1-7-22 Suehiro, Tsurumi

summary:
Several chemosensory cells exist throughout gastrointestinal tracts to sense nutrition and contaminants such as bacteria or parasites. Although rodent tissues are extensively used as a human gut model, recent studies show that the chemical sensing system in rodents differs from that in humans. Thus, experiments using rodents would not be the first choice. Nonhuman primates in biomedical research are valuable animal models to advance our understanding of biological responses in humans since availability of human tissues are limited. It has been reported that the 3D organoid culture produces functional gastrointestinal epithelial cells in vitro and can be generated from animal tissues. We have generated intestinal chemosensory cells from Japanese monkey using an organoid culture system. We could maintain monkey intestinal organoids in the proliferation medium for over one year.
In this study, we aim to see changes in gene expression when monkey intestinal organoids were stimulated by interleukin (IL)-4. Previous studies revealed that these intestinal organoids express cytokine receptors such as IL-4R and response to IL-4 supplementation. We stimulated organoids with IL-4 for 72 hrs followed by total RNA purification and RNAs were subjected to transcriptome analysis

overall_design: Monkey organoids were cultured in differentiation induction (DI) medium with or without IL4.

first: Ken

last: Iwatsuki


title: RNA methylation in diabetes [MeRIP-seq]

city: shanghai

first: xinping

last: she

state: shanghai

address: shanghai

department: Ophthalmology

institute: Department of Ophthalmology,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine

summary:
N6-methyladenosine (m6A) is one of the most prevalent and abundant epigenetic modifications in various fundamental bioprocesses. We hypothesized that m6A-mediated inflammation pathway contributes to diabetes. Total RNA was extracted from the retinas of wide type rat and their littermates with diabetes by STZ injection. A total amount of 1 μg RNA per sample was used as an input for the RNA sample preparations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia), the reagents used in library preparation are NEBNext® Ultra RNA Library Prep Kit for Illumina (NEB, USA) .After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
The MeRIP-seq was carried out in Novogene (Beijing, China). Briefly, 2 μg total RNA was extracted from the retinas of both wide type mice and their littermates with diabetes. The integrity and concentration of extracted RNA was detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare), respectively. Fragmented RNA (~100 nt) was incubated for 2 hours at 4 ℃ with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated RNA or input was used for library construction with Ovation SoLo RNA-Seq System Core Kit (NuGEN). The library preparations were sequenced on Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with three independent biological replicates.

overall_design: Differentially expressed mRNA and m6A peaks were explored by RNA-seq combined with MeRIP-seq in triplicates using Illumina novaseq 6000

first: Chuandi

last: Zhou

first: Xinping

last: She


title: RNA methylation in diabetes

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

overall_design: Refer to individual Series


title: Next Generation Sequencing to determine the roles of RNA-binding proteins PCBP1 and PCBP2 on murine erythropoiesis

city: Philadelphia

first: PAUL

last: JEWELL

address: 3451 Walnut Street Philadelphia

institute: Biociphers

summary: E14.5 fetal liver cells from CD-1 mice were enriched for hematopoietic progenitors. The purified hematopoietic progenitor cells were infected immediately with retroviruses encoding shRNAs that target Pcbp1, Pcbp2, as well as two sets of control shRNAs; Luciferase and scrambled sequence. The transduced cells were grown for 48 hours. These cells were collected (‘Day 0’ cells), or washed and resuspended in differentiation medium  and incubated for an additional 2 days (‘Day 2’ cells).   1ug of total RNA isolated from ‘Day 0’ and ‘Day 2’ cells from each Pcbp1 or Pcbp2 depleted sample was used for cDNA library construction after polyA selection. A total of 9 samples were generated from each of the two time points: 3 controls; Pcbp1 knockdowns with three distinctly targeting shRNAs (sh1,3,4); Pcbp2 knockdown with three distinctly targeting shRNAs (sh1,3,4). Sequencing was carried out using a 150nt paired-end sequencing protocol.

overall_design: RNA-seqs to examine the impact of PCBP1 and PCBP2 on murine erythropoiesis

first: Xinjun

last: Ji

first: Anupama

last: Jha

first: Jesse

last: Humenik

first: Louis

middle: R

last: Ghanem

first: Andrew

last: Kromer

first: Christopher

last: Duncan-Lewis

first: Elizabeth

last: Traxler

first: Mitchell

middle: J

last: Weiss

first: Yoseph

last: Barash

first: Stephen

last: Liebhaber


title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

city: Pittsburgh

first: Megan

middle: E.

last: Yates

address: 5051 Centre Ave

institute: University of Pittsburgh

summary: Molecular chaperones prevent the accumulation of toxic proteins in the cell, but when chaperones are overwhelmed, the rapid modulation of proteostasis pathways provide corrective responses. One molecular chaperone in particular, Hsp70, plays a central role in both cellular protection and the response to proteotoxic insults. This is perhaps most evident in cancer cells, some of which overexpress Hsp70 and thrive even when harboring high levels of misfolded, aggregated, and/or unassembled protein complexes. In an effort to define how cells compensate for compromised proteostasis, we examined the adaptive response in breast cancer cells after challenge with a specific Hsp70 inhibitor, MAL3-101 and further define the pathways involved in the differential MAL3-101 response between sensitive and resistant cells.

overall_design: RNA-Seq expression profiling of two triple negative breast cancer cell lines treated and untreated with Hsp70 inhibitor, MAL3-101, in triplicates

first: Sara

last: Sannino

first: Mark

last: Schurdak

first: Steffi

last: Oesterreich

first: Adrian

middle: V

last: Lee

first: Peter

last: Wipf

first: Jeffrey

middle: L

last: Brodsky


title: Pulmonary fibrosis distal airway epithelia are dynamically and structurally dysfunctional

city: Aurora

first: Ivana

last: Yang

state: CO

institute: University of Colorado Anschutz Medical Campus

summary: To identify genes regulating the jamming transition in healthy distal airway epithelia compared to the dysregulated state in idiopathic pulmonary fibrosis epithelia (IPF) we analyzed RNA from three (3) healthy and four (4) IPF patient cells grown at air-liquid-interface (ALI). This bulk analysis of 3 timepointes (days 4, 8, and 14 of ALI) spanning the jamming transition revealed an enrichment for ERBB- and YAP-related genes.

overall_design:
Illumina TruSeq library prep was performed on 500 ng from each sample (1 per donor, per timepoint) ran on the NovaSEQ 6000 with 80 million reads per sample.
***Raw data disclaimer: Unable to provide raw sequencing files due to conflict with privacy protection for human subjects.***

first: Ian

last: Stancil

first: Jacob

last: Michalski

first: Duncan

last: Davis-Hall

first: Hong

last: Chu

first: Jin-Ah

last: Park

first: Chelsea

last: Magin

first: Bradford

last: Smith

first: Evgenia

last: Dobrinskikh

first: David

middle: A

last: Schwartz


title: High-resolution transciptome mapping and N6-methyladenosine modification mapping of RAW264.7 cell lines treated with TCP, TBP or TIP.

city: nanjing

first: Zhang

last: Jun

state: jiangsu

address: longmiandadao

institute: China Pharmaceutical University

summary:
Purpose:Detect transcriptome-wide mapping of m6A modifications by MeRIP-seq between normal and chemical-treated RAW264.7 cells.
Methods:Total RNA was extracted using TRIzol Reagent followed by chemical fragmentation.  The fragmented RNA was incubated with anti-m6A antibody. The desired m6A-enriched RNA eluate was used to construct cDNA library in parallel with input control.
Conclusion: Our study revealed the difference of m6A-methylated RNA between normal and TCP/TBP/TIP-treated cells by RNA-seq and MeRIP-seq technologies.

overall_design: Compare the difference of m6A-methyalated RNA between normal and TBP/TCP/TIP treated samples.

first: Linyuan

last: Huang

first: Jun

last: Zhang


title: Epigenome analysis of prostatectomy specimens and samples of prostate cancer bone metastases

city: UMEÅ

first: Pernilla

last: Wikström

address: Department of Medical biosciences, Buildning 6M, 2nd floor

department: Medical biosciences, Pathology

country: Sweden

institute: Umeå University

type: Methylation profiling by genome tiling array

summary: DNA methylation profiling of paired samples of non-malignant and malignant prostate tissue obtained from prostatectomy speciments (n=12) and of bone metastasis tissue samples obtained from separate prostate cancer patients (n=70). The Illumina Infinium Methylation EPIC arrays were used to obtain genome-wide DNA methylation profiles across approximately 850,000 CpGs in each sample. The bone metastasis samples were obtained from castration-resistant (n = 52) and untreated (n = 14) prostate cancer patients as well as from prostate cancer patients treated for a short period with castration therapy (n = 4).

overall_design: Bisulphite converted DNA from the 94 samples were hybridised to the Illumina Infinium Methylation EPIC arrays.

first: Erik

last: Ylitalo

first: Elin

last: Thysell

first: Mattias

last: Landfors

first: Maria

last: Brattsand

first: Emma

last: Jernberg

first: Sead

last: Crnalic

first: Anders

last: Widmark

first: Magnus

last: Hultdin

last: Bergh

first: Sofie

last: Degerman


title: Combinatorial analyis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons

city: Higashi-ku, Fukuoka

first: Kazuya

last: Ichihara

address: 3-1-1, Maidashi

institute: Kyushu University

summary: Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A, and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.

first: K

first: A

last: Matsumoto

last: Nakayama


title: Transcriptome-scale analysis for RNAs of medium length, designated as melRNA-seq [RppH-treated]

city: Nagoya

first: Kenji

last: Ichiyanagi

address: Furo-cho, Chikusa-ku

department: Graduate School of Bioagricultural Sciences

institute: Nagoya University

summary: Small interspersed elements (SINEs) is transcribed by RNA polymerase III (Pol III) to produce RNAs of typically 100 to 500 nucleotides in length. Although the abundance of SINE RNAs can be analyzed by Northern blotting and primer extension, the nature (sequence, exact length, and genomic origin) of these RNAs cannot be revealed by these methods. Moreover, mRNA sequencing (mRNA-seq) is not able to distinguish bona fide SINE RNAs and SINE sequences present in longer transcripts.  To elucidate the abundance, source loci, and sequence nature of SINE RNAs, we have established a deep sequencing method, designated as melRNA-seq (medium length RNA-seq), which can determine whole-length sequences of RNAs. The total RNA samples were treated with 5' Pyrophosphohydrolase (RppH), which allowed ligation of an RNA adaptor to the 5’ end of intact SINE RNAs. Another adaptor was ligated to the 3’ end, followed by reverse transcription, PCR amplification, size selection, and single-end deep sequencing. Analysis of two biological replicates of RNAs from mouse spermatogonia showed high reproducibility of the SINE expression data both at family level and locus level. Therefore, this new method can be used for the quantification and detailed sequence analysis of medium length non-coding RNAs, such as rRNA, snRNA, tRNAs, and SINE RNAs. The dynamic range is much wider than Northern blotting and primer extension.

overall_design: This novel sequencing method has been designed for expression analysis of medium length (50-300 bp)  RNAs, including those of short interspersed elements (SINEs), a class of retrotransposons.  After converting the 5' end of transcribed RNAs into a ligatable end, libraries were prepared by a protocol almost same as that for small RNA sequencing.  Single-end 300 bp sequencing  yielded sequences of the whole length of RNAs.

first: Yoshinobu

last: Mori


title: Contribution of Dysregulated Metabolic Signature in HIV-associated Immune Reconstitution Inflammatory Syndrome

city: Salvador

first: Artur

middle: Trancoso Lopo

last: Queiroz

address: Rua Waldemar Falcão, 121

country: Brazil

institute: Instituto Gonçalo Moniz

summary: In this study, we sought to evaluate the metabolic perturbations in IRIS across longitudinal time points using an unbiased plasma metabolomics approach as well as integrated analyses to include plasma inflammatory biomarker profile and whole blood transcriptome

overall_design: Different expression between IRIS and Non IRIS patients prospectively

first: Irini

last: Sereti

first: Luxin

last: Pei

first: Bruno

last: Andrade

first: Kyioshi

last: Fukutani


title: Inactivation of the Euchromatic Histone-lysine N-methyltransferase 2 Pathway in Pancreatic Epithelial Cells Antagonizes Cancer Initiation and Pancreatitis-Associated Promotion by Altering Growth and Immune Gene Expression Networks

city: Milwaukee

first: Gwen

last: Lomberk

state: WI

address: 8701 W Watertown Plank Rd

department: Surgery

institute: Medical College of Wisconsin

summary: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, painful disease with a 5-year survival rate of only 9%. Recent evidence indicates that distinct epigenomic landscapes underlie PDAC progression, identifying the H3K9me pathway as important to its pathobiology. Here, we delineate the role of Euchromatic Histone-lysine N-Methyltransferase 2 (EHMT2), the enzyme that generates H3K9me, as a downstream effector of oncogenic KRAS during PDAC initiation and pancreatitis-associated promotion. EHMT2 inactivation in pancreatic cells reduces H3K9me2 and antagonizes KrasG12D mediated acinar-to-ductal metaplasia (ADM) and PanIN formation in both the Pdx1-Cre and P48+/-Cre KrasG12D mouse models. Ex vivo acinar explants also show impaired EGFR-KRAS-MAPK pathway mediated ADM upon EHMT2 deletion. Notably, KrasG12D increases EHMT2 protein levels and EHMT2-EHMT1-WIZ complex formation. Transcriptome analysis reveals that EHMT2 inactivation upregulates a cell cycle inhibitory gene expression network that converges on the Cdkn1a/p21-Chek2 pathway. Congruently, pancreas tissue from KrasG12D animals with EHMT2 inactivation have increased P21 protein levels and enhanced senescence. Furthermore, loss of EHMT2 reduces inflammatory cell infiltration typically induced during KrasG12D-mediated initiation. The inhibitory effect on KrasG12D-induced growth is maintained in the pancreatitis-accelerated model, while simultaneously modifying immunoregulatory gene networks that also contribute to carcinogenesis.  This study outlines the existence of a novel KRAS-EHMT2 pathway that is critical for mediating the growth-promoting and immunoregulatory effects of this oncogene in vivo, extending human observations to support a pathophysiological role for the H3K9me pathway in PDAC.

overall_design: Pancreas from 17 animals (in duplicate or triplicate) were analyzed for transcriptomic changes as corresponds with different genotypes of the animals.  Consideration of the effects of G9a knock out alone in the pancreas were compared with control animals at the age of 4 weeks.  Additionally, effect of G9a knockout on the development of acinar to ductal metaplasia and PanINs, through the expression of KrasG12D variant was compared to the KrasG12D variant alone.  Finally, treatment of the PDAC KrasG12D and KrasG12D with G9a knockout animals with caerulein modeled the acceleration of PDAC by inducing chronic pancreatitis.

first: Raul

last: Urrutia


title: Overexpression of IGF-1 During Early Development Expands the Number of Mammary Stem Cells and Primes them for Transformation

city: Bryan

first: Robin

last: Fuchs-Young

state: TX

address: 8441 Riverside Pkwy

department: Molecular Cellular Medicine

institute: Texas A&M University Health Science Center

summary: In this paper we report transcriptomic profiles of mammary stem cells (MaSCs) from female BK5.IGF1 transgenic and age-matched WT mice, generated by single-cell RNA-sequencing technology. We found that IGF-1 stimulated quiescent MaSCs to enter the cell cycle and increased their expression of genes involved in proliferation, plasiticity, tumorigenesis, invasion, and metastasis.

overall_design: Examination of transcriptomic profiles in MaSCs from BK5.IGF-1 and WT female mice.

first: Linjie

last: Luo

first: Andres

last: Santos

first: Kranti

last: Konganti

first: Andrew

last: Hillhouse

first: Ramesh

last: Gunaratna

first: Isabel

middle: U

last: Lambertz

first: Yuanning

last: Zheng

first: David

last: Threadgill


title: Muscle Overload scRNA-seq

city: Lexington

first: Kevin

last: Murach

state: KY

address: 900 South Limestone CTW 445

department: Physical Therapy

institute: University of Kentucky

summary: The goal of this project was to determine which mononuclear cell populations satellite cells communicate with throughout muscle during synergist ablation functional overload.  Mouse plantaris muscles from tamoxifen-treated Pax7-tdT, tdT, and Pax7-DTA mice were overloaded for 4-14 days, and a subset of mononuclear cells were submitted for sc-RNA-seq.

overall_design: Pooled plantaris muscles from multiple mice in each condition were used for scRNA-seq. A single cell suspension was made, and a subset of cells were isolated via fluorescent activated cell sorting to probe for the transfer of reporter transcript from satellite cells.

first: Bob

last: Policastro

first: Charlotte

last: Peterson


title: RNAseq samples of CNQX treated Arabidopsis roots regenerating at different time points.

city: New York

first: Kenneth

middle: David

last: Birnbaum

address: 12 Waverly Place

department: Biology

institute: New York University

summary: Wounding is a trigger for both regeneration and defense in plants, but it is not clear if the two responses are linked by common activation or represent potential tradeoffs. While plant glutamate-like receptors (GLRs) are known to mediate defense responses, we implicate GLRs in regeneration through dynamic changes in chromatin and transcription in reprogramming cells near wound sites. Here, we show that genetic mutation and pharmacological inhibition of GLRs increases regeneration efficiency in multiple organ repair systems and plant species. Perturbation of GLR-mediated function, possibly by affecting Calcium fluxes, speeds cell division and re-specification of the stem cell niche while dampening a subset of defense responses. We show that the GLRs work through salicylic acid (SA) signaling in their effects on regeneration, with mutants in the SA receptor NPR1 partially resistant to GLR perturbation and hyper regenerative. These findings reveal a conserved mechanism that regulates a tradeoff between defense and regeneration and also offer new strategies to improve regeneration in agricultural and conservation.

overall_design: Transcriptomic data of CNQX treated roots regenerating at 30 min, 4h, 14h and 16h after root tip excision

first: Marcela

last: Hernandez-Coronado

first: Poliana

last: Coqueiro

first: Pui-Leng

last: Ip

first: Custódio

middle: O

last: Nunes

first: Ramin

last: Rahni

first: Michael

last: Wudick

last: Lizzio

first: José

last: Feijó


title: WD repeat domain 5 promotes chemoresistance and Programmed Death-Ligand 1 expression in prostate cancer

city: Guangzhou

first: Qianghua

last: Zhou

address: 107th Yanjiangxi Road, Guangzhou

institute: sun yat-sen university

summary: Our results demonstated that OICR-9429 a inhibitor of WDR5, could supress the proliferation and chemo-resistance of prostate cancer cells. To explore the molecular mechanism underlying OICR-9429-induced WDR5 inhibition in Prostate cancer cells,  we conducted a genome-wide RNA-sequencing  to compare gene expression profiles between OICR-9429 treated DU145, PC3-3 cells and their control cells

overall_design: After treated with OICR-9429 or DMSO  for 48h, respectively. The RNA of the corresponding treated cells (including DU145 and PC-3) was extracted and sequenced on Illumina HiSeq 2500 platform and 100-bp paired-end reads were generated.

first: Tianxin

last: Lin

first: Jian

last: Huang


title: Transcriptome analysis of streptococus mutans UA159 and SMU_1995c knock out strain

city: Wenzhou

first: Yangyang

last: Pan

address: 183 guangchang Road, Lucheng District

department: Pediatric Dentistry,

institute: Wenzhou Medical University

summary: Zinc  is an essential divalent trace metal for living cells. Intracellular zinc homeostasis is critical to the pathogenicity and virulence of bacteria, however the strategies of zinc homeostasis maintain in Streptococcus mutans, an important causative pathogen of dental caries, is unknown. In this study, we examined the function of an ABC transporter AdcCBA and its transcription factor AdcR in S. mutans.  Using RNA-Seq analysis, LacZ-reporter studies and gel mobility shift assay, we  showed that a MarR family transcription factor AdcR directly regulates the activity of promoters of adcRCB and adcA in response to the concentration of zinc in their environmental niches. Deletion of adcR increases the sensitivity of S. mutans to excessive zinc supply. Taken together, our finding suggests that Adc regulon, consists of a zinc uptake transporter AdcCBA and a zinc-responsive repressor AdcR, plays a prominent role in the maintenance of intracellular zinc homeostasis of S. mutans.

overall_design: mRNA profiles of wild type UA159 and  its  SMU_1995c deletion mutant

first: Yuqing

last: Li

first: Jing

last: Zou


title: Testing the impact of Type I Restriction Modification Systems on Gene Expression in Uropathogenic Escherichia coli

city: Singapore

first: Swaine

last: Chen

state: Singapore

address: 60 Biopolis Street, Genome, #02-01

department: Medicine

country: Singapore

institute: National University of Singapore and Genome Institute of Singapore

summary:
Background: DNA methylation influences transcriptional regulation, and therefore cellular phenotype, across all domains of life. The regulatory role of DNA methylation and other epigenetic marks also extends to virulence; both orphan methyltransferases and those from restriction modification systems (RMSs) have been co-opted to regulate virulence in many bacteria. However, functional characterisation of DNA methylation mediated by archetypal Type I RMSs remains limited.
Results: We elucidated the methylation specificity of the Type I RMS in UTI89, a prototypical cystitis strain of E. coli. Deletion of this RMS led to loss of methylation of >750 sites across the genome but no detectable effect on gene expression, virulence, or growth in a screen of 1190 conditions. Similarly, deletion of the endogenous Type I RMS in two other E. coli strains with different methylation specificities also had no effect on gene expression or any growth phenotype. Finally, introduction of these two different RMSs into UTI89 also resulted in no detectable change in gene expression or growth phenotypes. These results stand in sharp contrast with many reports of RMSs regulating gene expression in other bacteria, leading us to propose the concept of “regulation avoidance” for these E. coli Type I RMSs. We hypothesize that regulation avoidance is a consequence of evolutionary adaptation of both the RMSs and the E. coli genome.
Conclusions: Our results provide a clear and currently rare example of regulation avoidance for Type I RMSs in multiple strains of E. coli, further study of which may provide deeper insights into the evolution of gene regulation and horizontal gene transfer.

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

overall_design: RNA-seq of E. coli strains with different methylation systems, including knockouts and heterologously complemented

first: Kurosh

middle: S

last: Mehershahi


title: Transcriptomic analysis of D458 medulloblastoma cells treated with 10nM THZ1 or DMSO in comparison to shNull and shCDK7-1,-2,-3 genetic knockdown cell lines

first: Rajeev

last: Vibhakar

department: Pediatrics/Hematology/Oncology

summary: The goal of this study was to first determine the effect of CDK7 chemical inhibition (THZ1) on global gene expression. Then to observe and compare to the transcriptomic profile of CDK7 genetic knockdown in D458 medulloblastoma cells.

overall_design: RNA-seq was performed using the illumina HiSEQ 4000 HT for 1x50 sequencing.  High quality base calls at 95% ≥ Q30 were obtained with 53M-69M single reads.  Statistical analysis of count data was performed with DESeq2 R package.

first: Bethany

last: Veo

first: Katrina

last: Diener

first: Dong

last: Wang


title: RNA methylation in diabetes [RNA-Seq]

first: xinping

last: she

summary:
N6-methyladenosine (m6A) is one of the most prevalent and abundant epigenetic modifications in various fundamental bioprocesses. We hypothesized that m6A-mediated inflammation pathway contributes to diabetes. Total RNA was extracted from the retinas of wide type rat and their littermates with diabetes by STZ injection. A total amount of 1 μg RNA per sample was used as an input for the RNA sample preparations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia), the reagents used in library preparation are NEBNext® Ultra RNA Library Prep Kit for Illumina (NEB, USA) .After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
The MeRIP-seq was carried out in Novogene (Beijing, China). Briefly, 2 μg total RNA was extracted from the retinas of both wide type mice and their littermates with diabetes. The integrity and concentration of extracted RNA was detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare), respectively. Fragmented RNA (~100 nt) was incubated for 2 hours at 4 ℃ with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated RNA or input was used for library construction with Ovation SoLo RNA-Seq System Core Kit (NuGEN). The library preparations were sequenced on Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with three independent biological replicates.

overall_design: Differentially expressed mRNA and m6A peaks were explored by RNA-seq combined with MeRIP-seq in triplicates using Illumina novaseq 6000

first: Chuandi

first: Xinping

last: She


title: CRISPR/Cas9 inducible UCP1 knockout in brown adipose tissue of adult mice

city: Ann Arbor

first: Ormond

last: MacDougald

address: 2800 Plymouth Rd

department: Molecular & Integrative Physiology

institute: University of Michigan

summary: Mice expressing Cas9 were administered AAV8-UCP1 sgRNA to the brown adipose tissue for inducible knockout. Mice were housed at 22ºC for two weeks and then were sacrificed. Brown adipose tissue (BAT) was harvested and RNAseq was performed to determine transcriptional changes stemming from inducible UCP1 knockout.

overall_design: Mice expressing Cas9 were injected with 10^12 vg/mL AAV8-UCP1 sgRNA or AAV8-Control sgRNA directly into BAT. Mice were kept at 22ºC for 14-days and were sacrificed and mRNA isolated from BAT and used for RNAseq.

first: Steven

last: Romanelli

first: Alan

middle: C

last: Rupp

first: Akira

last: Nishii


title: Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes of Melanoma Cell MUM2B overexpressing Empty Vector Or LACTB

city: Shanghai

first: Yawen

last: Ma

address: No.833 Zhizaoju Road

department: ophthalmology

institute: Shanghai Ninth people's hospital

summary: Methods: mRNA profiles of MUM2B cells stably overexpressing empty vector or LACTB were generated by deep sequencing, in triplicate, using Illumina. featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of two groups was performed using the DESeq2 R package(1.16.1). qRT–PCR validation was performed using TaqMan and SYBR Green assays

overall_design: mRNA profiles of MUM2B cells stably overexpressing empty vector or LACTB were generated by deep sequencing using Illumina.

first: Renbing

last: Jia