title: Highly scalable generation of DNA methylation profiles in single cells

first: Ryan

middle: M

last: Mulqueen

address: 3181 SW Sam Jackson Park Rd

department: Molecular and Medical Genetics

institute: Oregon Health and Science

summary: We present a novel method: single-cell combinatorial indexing for methylation analysis (sci-MET), which is the first highly scalable assay for whole genome methylation profiling of single cells. We use sci-MET to produce 3,282 total single-cell bisulfite sequencing libraries and achieve read alignment rates of 68± 8%, comparable to those of bulk cell methods. As a proof of concept, we applied sci-MET to deconvolve the cellular identity of a mixture of three human cell lines. Next, we applied sci-MET to mouse cortical tissue, which successfully identified excitatory and inhibitory neuronal populations as well as non-neuronal cell types.

overall_design: Tissue culture cell lines (GM12878, Coriell; HEK293, ATCC CRL-1554; Primary Fibro., inguinal fibroblast, GM05756, Coriell, passage 7) were cultured and nuclei were isolated. The three cell lines were both pooled to serve as randomized controls, and processed independently, as described in the available Methods section.  Sixty-day-old C57BL/6J mice had their entire cortex isolated. Three biological replicates were independently processed as described in the available Methods section.  Additional files are available under BioProject ID: PRJNA397747 and Study Accession: SRP127273.

first: Dmitry

last: Pokholok

first: Steve

last: Norberg

first: Andrew

last: Fields

first: Daunchen

first: Kristof

last: Torkenczy

first: John

last: Sinnamon

first: Jay

last: Shendure

first: Cole

last: Trapnell

first: Brian

last: O'Roak

first: Zheng

last: Xia

first: Frank

last: Steemers

middle: C

last: Adey


title: Effect of ER-464195-01 on the gene expression in the intestine of mice.

status: Public on Apr 11 2018

city: Toyama

first: Jun-Dal

last: Kim

state: Toyama

address: 2630 Sugitani

department: Department of Complex Biosystem Research (CBR), Institute of National Medicine (INM)

country: Japan

institute: University of Toyama

type: Expression profiling by high throughput sequencing

summary:
Our previous study identified a small molecule, orally active ER-464195-01, inhibits the calreticulin binding to alpha integrins, and clearly suppresses the adherence ability of leukocytes. Our transcriptome analysis with the colon of dextran sodium sulfate (DSS)-treated mice reveals that the increased expression of pro-inflammatory genes was down-regulated by ER-464195-01.
Male Balb/c mice (7 weeks) were allowed free access to distilled water (Otsuka Pharmaceutical). ER-464195-01 (10 mg/kg) or vehicle was orally administered once per day for 6 days.

overall_design: Intestinal mRNA profiles of control (normal water) and ER-464195-01 mice were generated by RNA sequencing using the NextSeq 500 (Illumina)

first: Masayoshi

last: Ohkuro

first: Hayase

last: Mizukami

first: Akiyoshi

last: Fukamizu


title: Transcriptomic analysis of myosin IIa-deficient B cells

status: Public on Apr 14 2018

city: London

first: Robbert

last: Hoogeboom

address: 123 Coldharbour lane

country: United Kingdom

institute: King's College London

summary: Myosin IIa-deficient follicular B cells have a hyperactivated phenotype. To identify what pathways are regulated by myosin IIa, we performed RNA-seq of coding RNA on flow cytometry sorted follicular B cells from CD23Cre+Myh9fl/fl and CD23Cre+Myh9wt/fl mice.

overall_design: B220+AA4.1-CD23+CD21lo follicular B cells were sorted from 3 CD23Cre+Myh9fl/fl and 3 CD23Cre+Myh9wt/fl mice and mRNA was isolated and  sequenced.

first: R


title: Single cell analysis of Granuloma crypt epithelium

status: Public on Apr 12 2018

city: San Francisco

first: Ysbrand

last: Nusse

state: CA

address: 513 Parnassus Ave

country: USA

institute: UCSF

summary: The purpose of this study is to compare the cellular populations present within granuloma (Sca1+) and non-granuloma (Sca-1–) crypt epithelum arising from parasitic H polygyrus infections.

overall_design: Single cell transcriptomes of Sca-1+(Granuloma) and Sca-1–(Non-granuloma) were generated from infected mice and then re-merged for population comparison

first: Adam

last: Savage


title: Tet1 and Tet2 Maintain Mesenchymal Stem Cell Homeostasis via demethylation of P2rX7 promoter

city: Beijing

first: Ruili

last: Yang

address: 22# South Zhongguancun  Ave

country: China

institute: Peking University

summary: Ten-eleven translocation (Tet) family-mediated DNA oxidation represents a novel epigenetic modification capable of converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) to regulate various biological processes. However, it is unknown whether the Tet family affects mesenchymal stem cells (MSCs) or the skeletal system. Here we show that depletion of Tet1 and Tet2 resulted in impaired self-renewal and differentiation of bone marrow MSCs (BMMSCs) and a significant osteopenia phenotype. Mechanistically, Tet1 and Tet2 deficiency reduced demethylation of the P2rX7 promoter and thus downregulated exosome release, leading to intracellular accumulation of miR-297a-5p, miR-297b-5p, and miR-297c-5p. These miRNAs inhibited Runx2 signaling to impair BMMSC function. We show that overexpression of P2rX7 consistently rescued the impaired BMMSCs and osteoporotic phenotype in Tet1 and Tet2 double knockout mice. These results indicate that Tet1 and Tet2 play a critical role in maintaining BMMSC and bone homeostasis through epigenetic regulation of P2rX7 to control exosome and miRNA release. This newly identified Tet/P2rX7/Runx2 cascade may serve as a target for the development of novel therapies for osteopenia disorders.

overall_design: Compare the global gene expression of three control BMMSCs sample and three Tet1 siRNA and three Tet2 siRNA combinational treated-BMMSCs

first: Tingting

last: Yu

first: Songtao

last: Shi


title: An exploratory study to examine the effects of maternal choline supplementation during mouse pregnancy on placental epigenetic markers

status: Public on Apr 10 2018

city: Ithaca

first: Jennifer

middle: K

last: Grenier

state: NY

address: 526 Campus Rd

department: Biomedical Sciences

institute: Cornell University

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: ChIP-chip from Escherichia coli CC72 cells with RNAP β’ subunit before and after osmotic stress.

city: Tianjin

first: Zhe

last: Sun

address: 32 West 7th Ave

institute: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

type: Genome binding/occupancy profiling by genome tiling array

summary:
RNA polymerase (RNAP) is the key transcription machinery and its interaction with genomic DNA orchestrates gene expression in response to environmental cues. Dynamic interaction and localization between RNAP and nucleoid were observed before and after osmotic stress.
Chromatin immunoprecipitation (ChIP) of RNAP β’ subunit together with chromatin profiling by ChIP-on-chip analysis demonstrated the dynamics of genome-wide RNAP-DNA interactions during osmotic stress.

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

overall_design: Comparison of RNAP binding genes in Escherichia coli CC72 at early-exponential phase, and 10 min, 20 min and 45 min after osmotic stress.

first: Ding

middle: J

last: Jin


title: Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts [strain 2]

city: Würzburg

first: Anna

last: Maierhofer

address: Biozentrum, Am Hubland

department: Institute of Human Genetics

country: Germany

institute: University of Würzburg

summary: To study delayed genetic and epigenetic radiation effects, which may trigger radiation-induced carcinogenesis, we have established single-cell clones from irradiated and non-irradiated primary human fibroblasts. Stable clones were endowed with the same karyotype in all analyzed metaphases after 20 population doublings (PDs), whereas unstable clones displayed mosaics of normal and abnormal karyotypes. To account for differences in radiation sensitivity, all experiments were performed with two different embryonic fibroblast strains. After a single dose of 2 Gray more than half of the irradiated clones exhibited radiation-induced genomic instability (RIGI). Methylation arrays identified several hundred differentially methylated genes and nine  enriched pathways associated with in vitro ageing. Methylation changes in 259 genes and the MAP kinase signaling pathway were associated with delayed radiation effects (after 20 PDs). Collectively, our results suggest that both genetic and epigenetic changes occur in the progeny of exposed cells that were not damaged directly by irradiation, likely contributing to radiation-induced carcinogenesis.

overall_design:
Bisulphite converted DNA from 48 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip
starting culture/mass culture was analyzed as duplicate, 20 non-irradiated/control clones, 26 irradiated clones (10 unstable, 9 stable)

first: Julia

last: Flunkert

first: Steve

last: Horvath

first: Indrajit

last: Nanda

first: Thomas

last: Haaf


title: MCM2-7-dependent cohesin loading during S phase promotes sister-chromatid cohesion

city: Dallas

first: Hongtao

state: TX

address: 6001 Forest Park Rd

department: Pharmacology

institute: University of Texas Southwestern Medical Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We employ ChIP-seq methodology to study genome-wide distribution of MCM2, NIPBL and cohesin in thymidine-arrested cells

overall_design: Antibodies against MCM2, RAD21 or NIPBL were used to detect MCM2, RAD21 or NIPBL bound DNA in thymidine-arrested HeLa Tet-On cells

first: Ge

last: Zheng

first: Mohammed

last: Kanchwala

first: Chao

last: Xing


title: Cell specific response to IFNg

city: Boston

first: CBDM

last: Lab

state: MA

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

summary: We report cell specific responses to IFNg in 11 different peripheral immunocyte populations in the mouse. These cells represent the core ImmGen immunocyte lineage panel. Profiles from these were used to analyze cell specific responses to IFNg. In general a core set of ISG transcripts are induced in all cells. Smaller sets of ISGs were induced in a cell specific manner. In particular, splenic granulocytes and dendritic cells show restriced indcution of sets of ISGs.

overall_design: Male C57BL/6 mice were injected subcutaneously with 10,000U IFNg according to time course profile. Splenocytes/Peritoneal lavage suspensions were stained in multicolor antibody panels that were used to distinguish 11 different immunocyte populations (Immgen 11 cell set). These cells were sorted by FACS directly into TriZo and gene expression profiling was performed on Affymetrix Mouse Gene 1.0 ST Arrays.

first: Sara

last: Mostafavi

first: Hideyuki

last: Yoshida

first: Devapregasan

last: Moodley

first: Andrew

last: Rhoads

first: Adriana

last: Ortiz-Lopez

first: Liang

first: Diane

last: Mathis

first: Christophe

last: Benoist


title: Expression data from WT and MED12 KO SKOV3 and HO8910 cells

city: Guangzhou

first: Xiaolin

last: Luo

state: Guangdong

address: 651 Dongfeng East Road

department: Gynecology Oncology

institute: Sun Yat'sen University Cancer Center

summary:
Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecological  malignancies due to high rate of disease relapse. Disease relapse in cancer patients  after clinical remission are often referred to tumor dormancy. Here we identify the  RNA polymerase II transcriptional Mediator subunit 12 (MED12) as an important  molecular regulator of tumor dormancy. We found that MED12 knock-out (KO)  could induce dormancy of EOC cells in vitro and in vivo. Mechanistically, microarray  analysis showed that MED12 KO decreased the expression of EGFR.
Hierarchical cluster assays of the differentially expressed genes between MED12 WT and KO single clones of SKOV3 and HO8910 cells.

overall_design: MED12 WT and KO single clones of SKOV3 and HO8910 cells were selected for RNA extraction and hybridization on Affymetrix microarrays.

first: Liwu

last: Fu

first: Jihong

last: Liu


title: An exploratory study to examine the effects of maternal choline supplementation during mouse pregnancy on placental epigenetic markers (miRNA-seq data set)

first: Jennifer

last: Grenier

summary: This study sought to explore the effects of 4X maternal choline supplementation (MCS) on mRNA and miRNA abundance in mouse placentas collected on gestational day 15.5. These data advance our understanding about the importance of choline in reproductive health and will guide future mechanistic studies designed to elucidate the mechanisms mediating choline's beneficial effects.

overall_design: miRNA sequencing was performed on 6 placental samples (3 males and 3 females) from 1X control choline diet group and 6 placental samples (3 males and 3 females) from 4X choline diet group

first: Marie

middle: A

last: Caudill

first: Mark

middle: S

last: Roberson

first: Sze-Ting

last: Kwan


title: Genes linked to cervicovaginal integrity are dysregulated in DMPA but not COC contraception users

city: Norfolk

first: Irina

last: Zalenskaya

state: Virginia

address: 601 Colley ave

department: Ob/Gyn

institute: Eastern Virginia Medical School

summary: Injectable depot medroxyprogesterone acetate (DMPA) is one of the most popular contraception methods in areas of high HIV seroprevalence. Evidence is accumulating that use of DMPA might be associated with an increased risk of acquiring HIV-1; however, mechanisms of this association are not completely understood. We conducted a comparative whole genome transcriptome profiling of human ectocervical tissues before and after use of two hormonal contraception methods: injectable DMPA and not linked to increased risk of HIV acquisition combined oral contraceptive (COC). Microarray analysis identified 235 ectocervical genes altered in the DMPA users. The most striking effect of DMPA, but not COC, was a strong dysregulation of genes strategically involved in the maintenance of epithelial barrier function; the alterations were most likely due to the DMPA-induced estrogen deficiency. We also demonstrated inter-individual heterogeneity in gene expression profiles of the DMPA users that clustered into distinct groups. The differential response may explain divergence in reports on effects of DMPA use.  We propose that impairment of the cervicovaginal epithelial integrity in response to DMPA administration is the major mechanism underlying a potential increased risk of HIV-1 acquisition in DMPA users.

overall_design: Participants received hormonal contraception (HC) either DMPA (n=31) or COC (n=32). Ectocervical tissue biopsies were taken at baseline and after six weeks of HC use

first: Neelima

last: Chandra

first: Nazita

last: Yousefieh

first: Oluwatosin

middle: E

last: Adedipe

first: Suzanne

last: Jackson

first: Sharon

last: Anderson

first: Christine

last: Mauck

first: Jill

middle: L

last: Schwartz

first: Andrea

middle: R

last: Thurman

first: Gustavo

middle: F

last: Doncel


title: Single-cell transcriptomics of human oocytes: environment-driven metabolic competition and compensatory mechanisms during oocyte maturation

first: Yang

last: Yu

address: 49 North Garden Rd.,Haidian District

department: Ministry of Education Key Laboratory of Assisted Reproduction, and Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Center of Reproductive Medicine

institute: Peking University Third Hospital

summary:
Aim: The mechanisms coordinating maturation with an environment-driven metabolic shift, a critical step in determining the developmental potential of human in vitro matured (IVM) oocytes, remains to be elucidated. Here, we explored the key genes regulating human oocyte maturation using single-cell RNA sequencing and illuminated the compensatory mechanism from a metabolic perspective by analyzing gene expression.
Results: Three key genes that encode CoA-related enzymes were screened from the RNA sequencing data. Two of them, ACAT1 and HADHA, were closely related to the regulation of substrate production in the Krebs cycle. Dysfunction of the Krebs cycle was induced by decreases in the activity of specific enzymes. Further, the activator of these enzymes, the calcium concentration, was also decreased because of the failure of influx of exogenous calcium. Although release of endogenous calcium from the endoplasmic reticulum and mitochondria met the requirement for maturation, excessive release resulted in aneuploidy and developmental incompetence. High nicotinamide nucleotide transhydrogenase expression induced NADPH dehydrogenation to compensate for the NADH shortage resulting from the dysfunction of the Krebs cycle. Importantly, high NADP+ levels activated DPYD to enhance the repair of DNA double-strand breaks to maintain euploidy.
Innovation: The present study shows for the first time that exposure to the in vitro environment can lead to the decline of energy metabolism in human oocytes during maturation but that a compensatory action maintains their developmental competence.
Conclusions: In vitro maturation of human oocytes is mediated through a cascade of competing and compensatory actions driven by genes encoding enzymes.

overall_design: Investigated the transcriptome characteristics of human oocytes matured in vitro and in vivo to gain a transcriptome-level understanding of how oocytes mature and to illuminate the differences between human IVM and in vivo (IVO) matured oocytes at the transcript level.

first: Yong

last: Fan

first: Tao

last: Tan

first: Jie

last: Qiao

first: Hongcui

last: Zhao

first: Tianjie

last: Li

first: Yue

first: Changyu

first: Yali

first: Liang

last: Chang

first: Ning

last: Huang

first: Chang

first: Rong


title: Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency [RNA-seq]

first: dongwei

state: Guangdong province

address: 190 Kai Yuan Avenue, Science Park

department: South China Institute for Stem Cell Biology and Regenerative Medicine

institute: Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences

summary: Chemical induction of pluripotency (CIP) is an ideal way to reprogram cell fate, but remains poorly understood. Here we report the development of an efficient CIP protocol and the chromatin accessibility dynamics (CAD) during CIP by ATAC-seq. CIP first reorganizes the somatic genome to an intermediate state that must be resolved by primarily closing loci opened  earlier before reaching a pluripotent state with gradual opening of loci enriched with motifs for the OCT/SOX/KLF families. Bromodeoxyuridine, a critical ingredient of CIP, is responsible for both closing and opening critical loci, at least in part by preventing the opening of loci enriched with motifs for the AP1 family and facilitating the opening of loci enriched with SOX/KLF/GATA motifs. Our studies provide the first link between small molecules and nuclear architecture in the context of cell fate decisions.

overall_design: expression profile of reprogramming cell were generated by RNA-seq, using illumina nextseq 500

first: Dongwei


title: Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency [ChIP-seq]

overall_design: H3K27ac binding profile of reprogramming cell were generated by ChIP-seq, using illumina nextseq 500


title: Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency


title: A whole genome-screening strategy to identify DNA2 binding sites and genomic regions that require DNA2 for replication

city: Duarte

first: Xiwei

last: Wu

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

institute: City of Hope National Medical Center

summary: We report that DNA2 predominantly bound to the centromeric α-satellite regions. The centromeric regions contained 58% of the DNA2-associated DNA, representing a 33.5-fold enrichment over genomic input. To define the under-replicated DNA regions in DNA2-null cells, we conducted whole-genome DNA sequencing of the under-replicated BrdU negative DNA. After normalization to genomic input DNA from the same cells, 12.9% of the peaks from the under-replicated DNA aligned with the centromeric DNA regions, representing an 8.5-fold enrichment. In addition, among the peaks that overlapped between the DNA2 pull-down and under-replicated regions, two thirds fell into the centromeric regions, representing a 48-fold enrichment.

overall_design: Examination of DNA2 binding sites in HCT-116 cells. Identifying BrdU negative DNA from DNA2-null cells. Computational analysis to find out the overlapping regions between these two pools.

first: Zhengke

first: Binghui

last: Shen


title: Bone density loci identified by genome-wide association studies segregate a lineage-specific PU.1-dependent gene regulatory network in osteoclasts [HsMmMicroarray]

city: Charleston

first: Michael

last: Ostrowski

state: SC

address: 86 Jonathan Lucas street

department: Biochemistry and Molecular Biology

institute: Medical University of South Carolina

summary:
Similar temporal expression kinetics of transcription factors in human and mouse osteoclast differentiation evaluated by microarray
We used the identical differentiation conditions to evaluate gene expression kinetics of mouse and human osteoclast differentiation by microarray

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Either, mouse myeloid precurosrs or human osteoclast precurosrs were treated with osteoclast differentiation medim for 36, 72 and 96 h and gene expression compared to precurosrs.

first: Sudarshana

last: Sharma


title: An exploratory study to examine the effects of maternal choline supplementation during mouse pregnancy on placental epigenetic markers (mRNA-seq data set)

first: Jennifer

last: Grenier

overall_design: mRNA sequencing was performed on 6 placental samples (3 males and 3 females) from 1X control choline diet group and 6 placental samples (3 males and 3 females) from 4X choline diet group

first: Marie

last: Caudill

first: Mark

last: Roberson

first: Sze-Ting

last: Kwan


title: Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency [ATAC-Seq]

overall_design: chromatin accessible profile of reprogramming cell were generated by ATAC-seq, using illumina nextseq 500


title: Distinct Transcriptomic and Exomic Abnormalities within Myelodysplastic Syndrome Marrow Cells

status: Public on Apr 09 2018

city: Stanford

first: Varsha

last: Rao

state: California

address: 300 Pasteur Drive

department: Genetics

institute: Stanford University

summary: Prior studies using DNA microarray platforms have shown alterations of gene expression profiles (GEPs) of marrow cells in myelodysplastic syndromes (MDS). Using the increased sensitivity and accuracy of high-throughput RNA sequencing (RNA-Seq) for detecting and quantifying mRNA transcripts, our study has demonstrated novel significant differences in GEPs between MDS and normal CD34+ marrow cells with 41 genes identified as disease classifiers. Additionally, two main clusters of GEPs distinguished patients based on their major clinical features, particularly between those whose disease remained stable (sMDS) vs patients whose illness transformed to acute myeloid leukemia within 12 months (tMDS). The genes whose expression was associated with disease outcome were involved in functional pathways and biologic processes highly relevant for MDS. Exomic analysis identified MDS-associated pathogenic mutations in virtually all patients tested. MDS subgroups with spliceosome mutations demonstrated distinct differential isoform usage and expression and consequent dysregulation of distinct biological functions. This combination of clinical, transcriptomic and exomic findings provides valuable molecular insights into the mechanisms underlying MDS and its progression to a more aggressive stage and also facilitates prognostic characterization of MDS patients.

overall_design: RNA-Seq was performed on CD34+ hematopoietic stem cells derived from healthy individuals and patients with myelodysplastic syndrome.

first: Peter

last: Greenberg


title: Expression data from SKOV3 KO1#_Vector and SKOV3 KO1#_MED12 rescued cells

first: Xiaolin

last: Luo

summary:
Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecological  malignancies due to high rate of disease relapse. Disease relapse in cancer patients  after clinical remission are often referred to tumor dormancy. Here we identify the  RNA polymerase II transcriptional Mediator subunit 12 (MED12) as an important  molecular regulator of tumor dormancy. We found that MED12 knock-out (KO)  could induce dormancy of EOC cells in vitro and in vivo. Mechanistically, microarray  analysis showed that MED12 KO decreased the expression of EGFR.
Hierarchical cluster assays of the differentially expressed genes between SKOV3 KO1#_Vector and SKOV3 KO1#_MED12 cells

overall_design: SKOV3 KO1#_Vector and SKOV3 KO1#_MED12 cells were selected for RNA extraction and hybridization on Affymetrix microarrays.

first: Liwu

last: Fu

first: Jihong

last: Liu


title: Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile (RNA-seq data set)

city: Oklahama city

first: David

last: Jones

address: 825 NE 13th st

department: Functional and chemical genomics

institute: Oklahama medical research foundation

summary: Screening and surveillance of colorectal cancers (CRCs) using advanced colonoscopy technologies have significantly reduced the incidence and mortality rates of CRCs in recent years. However, a significant portion of CRCs are still remained undiagnosed, especially those involving sessile serrated adenomas/polyps (SSA/P), most likely due to their flat shape and the excessive amounts of secreted mucin that cover the polyps, making them invisible for colonoscopy. Here, a potential alternative solution is the application of molecular markers enabling unambiguous characterization of SSPs. However, full implementation of this strategy requires availability of robust markers which are still lacking. In this work by comprehensive molecular analysis of several malignant and normal samples at the genome, methylome and transcriptome levels we show that activating mutation of BRAF-V600E drives the generation of a unique SSP-specific DNA methylation profile. As the result a robust set of DNA methylation markers showing significant (~3 to 30 fold) increase in their methylation levels, exclusively in SSP samples, are introduced. These markers can be of important clinical relevance, especially in early diagnosis of SSPs using non-invasive approaches such as fecal DNA testing.

overall_design: To study the effect of BRAF-V600E mutation on gene expression profile we performed high-throughput RNA sequencing (RNA-Seq) on BRAF-V600E positive and negative samples. The RNA-Seq was performed on all available samples, from the exact same list of samples we evaluated their DNA methylation profile in our study.

first: Somaye

last: Dehghanizadeh

first: Vahid

last: Khoddami

first: Timothy

last: Mosbruger

first: Sue

last: Hammoud

first: Kornelia

last: Edes

first: Therese

last: Berry

first: Michelle

last: Done

first: Wade

last: Samowitz

first: James

last: DiSario

first: Daniel

middle: G

last: Luba

first: Randall

middle: W

last: Burt


title: Transcriptomic analysis of microglia from 7 week Npc1 disease mouse model treated or not with 4000mg.kg-1 HPBCD

city: bethesda

first: antony

middle: cyril

last: cougnoux

state: md

address: BG 10-CRC RM 1-3288 10 CENTER DR

department: nichd

institute: nih

summary: the study aims to describe the modifications occurring the microglia isolated from NPC1 disease mouse model using a transcriptomic and phenotypic approach. The analysis is also performed on animal treated with the reference drug HPBCD, currently under clinical trial in human.

overall_design: FACS isolated cell from Npc1 KO and WT mice were harvested from 7-week-old animals treated with HPBCD (n=3/genotype) or vehicle (n=3/ genotype) or no treatment (n=5/ genotype). Transcriptome was analyzed for modified signaling pathway further validated by additional experiments.


title: Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts

first: Anna

last: Maierhofer

overall_design: Refer to individual Series


title: Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile (WGBS data set)

type: Methylation profiling by high throughput sequencing

overall_design: To provide a comprehensive high-resolution SSP-specific DNA methylome from the entire genome, we processed a SSP biopsy sample from one of the patients in our study (#P1-SSP3), confirmed by exome sequencing to harbor BRAFV600E mutation, paired with the grossly uninvolved (GU) colon sample and the blood sample from the same patient, and analyzed them, in parallel, with the whole-genome bisulfite sequencing (WGBS).


title: Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile (Exome-seq data set)

overall_design: To verify previous “targeted” mutational analysis reports claiming that BRAFV600E is the main mutation in common between different sessile serrated polyp (SSP) samples, we performed a comprehensive exome sequencing on DNA extracted from total of eight SSP samples from six patients diagnosed, via colonoscopy and pathological inspection of polyp sections, to have typical SSP type polyps in their colons. From five of these patients a single polyp was analyzed to study the common mutations between different affected individuals. From the sixth patient three different polyps were isolated from different locations of the colon, providing the opportunity to separately sequence the DNA extracted from all three samples to study the possible differences between different polyps of the same individual. In parallel to these eight SSP samples, exome sequencing was performed on corresponding available paired blood samples (from four patients; including the one with three different polyps) to filter the germline mutations and single nucleotide polymorphisms (SNPs).


title: Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts [strain 1]

status: Public on Apr 07 2018

overall_design:
Bisulphite converted DNA from 48 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip
starting culture/mass culture was analyzed as duplicate, 12 non-irradiated/control clones, 34 irradiated clones (25 unstable, 9 stable)

first: Julia

last: Flunkert

first: Steve

last: Horvath

first: Indrajit

last: Nanda

first: Thomas

last: Haaf


title: Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile

first: David

last: Jones


title: Transcriptomic profile of TRIM8-driven neural stem cells

city: S. Giovanni Rotondo (FG)

first: Tommaso

last: Mazza

address: Viale Cappuccini, 1

department: Bioinformatics

country: Italy

institute: IRCCS Casa Sollievo della Sofferenza

summary: We recently described TRIM8, a nuclear E3 ubiquitin ligase, whose expression inversely correlates with glioma grade. TRIM8 restoration suppresses cell growth and induces a significant reduction of clonogenic potential in both U87MG glioblastoma and patients’ primary glioma cell lines.   Since E3 ubiquitin ligase proteins regulate carcinogenesis through the timely control of many cellular processes such as DNA damage response, metabolism, transcription, and apoptosis, we reasoned that TRIM8 activity might impact on cell transcriptome patterns, thereby promoting cancer development and progression. Therefore, we profiled the whole transcriptome of normal embryonic neural stem cells (eNSC) infected with a retrovirus expressing FLAG-Trim8 by using RNA-Seq.   RNA-Seq revealed 1365 differentially expressed transcripts of 912 genes. 723 of them (corresponding to 648 RefSeq genes) differed significantly of at least 1.5 folds (192 upregulated transcripts of 178 genes and 531 downregulated transcripts of 470 genes). 80 genes, among all differentially expressed genes, resulted to significantly enrich 18 pathways by IPA analysis. 53% of these genes (43 out of 80 genes) are related to cell-morphology, cell death and survival, with a preponderantly representation of signaling pathways related to neurotransmission and to CNS, including axonal guidance, GABA Receptor, ephrin B, synaptic long-term potentiation/depression, and glutamate receptor.   Specifically, our results substantiate the role of TRIM8 in the brain functions through the dysregulation of genes involved in different pathways, including JAK-STAT. Finally, we provided additional evidence about the existence of a functional interactive crosstalk between TRIM8 and STAT3 with possible implications in the development and progression of glioma.

overall_design: Profiling the transcriptome of TRIM8-expressing primary mouse embryonal neural stem cells using RNA-Seq

first: Lucia

last: Micale


title: Coactivator condensation at super-enhancers links phase separation and gene control

city: Cambridge

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

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


title: Transcriptome analysis of genetically matched human induced pluripotent stem cells disomic or trisomic for chromosome 21

city: Boulder

first: Christopher

last: Link

state: Colorado

address: 1725 Pleasant st

department: Integrative Physiology

institute: University of Colorado Boulder

summary: Trisomy of chromosome 21, the genetic cause of Down syndrome, has the potential to alter expression of genes on chromosome 21, as well as other locations throughout the genome. These transcriptome changes are likely to underlie the Down syndrome clinical phenotypes. We have employed RNA-seq to undertake an in-depth analysis of transcriptome changes resulting from trisomy of chromosome 21, using induced pluripotent stem cells (iPSCs) derived from a single individual with Down syndrome. These cells were originally derived by Li et al, who genetically targeted chromosome 21 in trisomic iPSCs, allowing selection of disomic sibling iPSC clones. Analyses were conducted on trisomic/disomic cell pairs maintained as iPSCs or differentiated into cortical neuronal cultures.

overall_design: 12 total polyA selected samples. 6 IPSC samples with 3 biological repeats for trisomic samples and 3 biological repeats for disomic samples. 6 IPSC derived neuronal samples with 3 biological repeats for trisomic samples and 3 biological repeats for disomic samples.

first: Patrick

last: Gonzales

first: Christine

last: Roberts

first: Virginia

last: Fonte

first: Connor

last: Jacobsen

first: Gretchen

middle: H

last: Stein

middle: D


title: Coactivator condensation at super-enhancers links phase separation and gene control (RNA-Seq)

summary: Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. We recently proposed that a phase-separated multi-molecular assembly underlies the formation and function of SEs.  Here, we demonstrate that the SE-enriched factors BRD4 and MED1 form nuclear puncta that occur at SEs and exhibit properties of liquid-like condensates. Disruption of BRD4 and MED1 puncta by 1,6-hexanediol is accompanied by a loss of BRD4 and MED1 at SEs and a loss of RNAPII from SE-driven genes. We find that the intrinsically disordered regions (IDRs) of BRD4 and MED1 are sufficient to form phase-separated droplets in vitro and the MED1 IDR promotes phase separation in living cells. The MED1 IDR droplets are capable of compartmentalizing BRD4 and other transcriptional machinery in nuclear extracts. These results support the idea that SEs form phase-separated condensates that compartmentalize the transcription apparatus at key genes, provide insights into the role of cofactor IDRs in this process, and offer new insights into mechanisms involved in control of key cell identity genes.

overall_design: polyA RNA-Seq in mouse embryonic stem cells

first: Benjamin

last: Sabari

first: Alessandra

last: Dall'Agnese

first: Ann

last: Boija

first: Isaac

last: Klein

first: Eliot

last: Coffey

first: Krishna

last: Shrinivas

first: Brian

last: Abraham

first: Nancy

last: Hannett

first: Alicia

middle: V

last: Zamudia

first: John

last: Manteiga

first: Charles

last: Li

first: Daniel

last: Day

first: Jurian

last: Schuijers

first: Eliza

last: Vasile

first: Sohail

last: Malik

first: Tong

middle: I

last: Lee

first: Ibrahim

last: Cisse

first: Robert

last: Roeder

first: Phillip

last: Sharp

first: Arup

last: Chakraborty


title: Far upstream element-binding protein 1 regulates LSD1 alternative splicing to promote terminal differentiation of neural progenitors

city: New york

first: Jihye

last: Paik

address: 1300 York Ave.

department: Pathology

institute: Weill Cornell Medical College

summary: Analysis of mouse neural progenitors with non targeting or FUBP1 targeting shRNA in proliferation and differentiation conditions

overall_design: 8 samples

first: Inah

last: Hwang

first: Tuo

last: Zhang


title: Coactivator condensation at super-enhancers links phase separation and gene control (ChIP-Seq)

first: Richard

last: Young

overall_design: ChIP-Seq for transcription co-factors and RNA Polymerase in mouse embryonic stem cells before and after treatment with 1,6-Hexanediol, a phase body-dissolving alcohol

first: Benjamin

last: Sabari

first: Alessandra

last: Dall'Agnese

first: Ann

last: Boija

first: Isaac

last: Klein

first: Eliot

last: Coffey

first: Krishna

last: Shrinivas

first: Brian

last: Abraham

middle: M


title: Treatment of rheumatoid arthritis patients with chaperonin 10

city: Brisbane

first: Andreas

last: Suhrbier

state: Queensland

address: 300 Herston Road, Herston

country: Australia

institute: QIMR Berghofer Medical Research Institute

summary:
Trial described in Lancet. 2006 Sep 2;368(9538):855-63. Rheumatoid arthritis patients were treated with chaperonin 10, with a dose of 10 mg rCpn10 i.v. twice weekly for 56 days. Blood samples were collected before and after treatment.
Microarray analysis described in Hirata et al., Early pregnancy factor, chaperonin 10 and rheumatoid arthritis; the story unravels. J Transl Sci 2018.

overall_design: Microarray data from 4 deidentified patients enrolled in the aforementioned trial. Samples were collected before and after treatment with CPN10, making a total of 8 samples.

first: Thiago

last: Hirata


title: Effect of KLK5 knockdown in PC9 lung cancer cells

status: Public on Apr 06 2018

city: Jacksonville

first: Derek

middle: Charles

last: Radisky

state: Florida

address: 4500 San Pablo Road

department: Cancer Biology

institute: Mayo Clinic

summary: Response of PC9 cells to infection with NT (control) lentivirus or one of two different KLK5 knockdown lentiviruses.

overall_design: Cells were plated in 35 mm dishes, infected with NT lentivirus, KLK5 KD #40 lentivirus (NM_012427.31248s1c1, Sigma), or KLK5 KD #42 lentivirus (NM_012427.3650s1c1, Sigma).

middle: C


title: Effect of KLK5 knockdown and PRSS3 knockdown in PC9 lung cancer cells


title: Effect of PRSS3 knockdown in PC9 lung cancer cells

summary: Response of PC9 cells to infection with NT (control) lentivirus or one of two different PRSS3 knockdown lentiviruses.

overall_design: Cells were plated in 35 mm dishes, infected with NT lentivirus, PRSS3 KD #C24 lentivirus (NM_002771.2-454s1c1, Sigma), or PRSS3 KD #337 lentivirus (NM_002771.3-337s21c1, Sigma), then harvested after 2 days.


title: RNA sequencing of brown adipose tissue of lean and obese mice

city: Oxford

first: Haifei

last: Shi

state: Ohio

address: 700 E High St

department: Biology

institute: Miami University

summary: We report differential expressed genes in brown adipose tissue of mice fed with a low-fat diet or a high-fat diet.

overall_design: Methods: Male C57BL/6J mice at age of 6 weeks - 10 weeks fed either a low-fat diet or a high-fat diet.  mRNA profiles were generated by deep sequencing, in triplicates, using Illumina NextSeq500 sequencing system.  The sequence reads that passed quality filters were analyzed at the gene level with DESeq2.

first: Jingyi

last: Cao

first: Qi

last: Zhu

first: Lin

last: Liu

first: Bradley

middle: J

last: Glazier

last: Hinkel

first: Chun

last: Liang


title: Machine learning predicts individual cancer patient responses to therapeutic drugs with high accuracy

city: Atlanta

first: Cai

last: Huang

state: Georgia

address: North Ave NW

institute: Georgia Institute of Technology

summary: Samples of primary tumors collected from 23 ovarian cancer patients

overall_design: 23 ovarian cancer patients were snap frozen in liquid nitrogen within one minute of surgical removal and transferred to the lab for laser capture microdissection of cancer cells

first: Evan

middle: A

last: Clayton

first: Lilya

last: Matyunina

first: L

last: DeEtte McDonald

first: Benedict

middle: B

last: Benigno

first: Fredrik

last: Vannberg

first: John

last: McDonald


title: Layer-specific molecular expression in neocortical astrocytes

city: Tokyo

first: Yukiko

last: Gotoh

state: Tokyo

address: 7-3-1 Hongo, Bunkyo-ku

institute: University of Tokyo

summary: Protoplasmic astrocytes in layers II to VI of the mammalian neocortex have historically been thought to comprise a homogeneous population. Given that layer-specific neuronal subtypes play essential roles in cortical circuitry, astrocytes might also be expected to support and modify this circuitry in a layer-specific manner. In order to investigate whether protoplasmic astrocytes exhibit layer-specific heterogeneity, we compared the gene expression profiles of astrocytes between upper layers (layers II to IV) and deep layers (layers V and VI). Although most genes known to be preferentially expressed in astrocytes (astrocyte-enriched genes) were equally expressed between upper-layer astrocytes and deep-layer astrocytes, some such genes (astrocyte-enriched genes or genes with known function in astrocytes) were significantly enriched in upper-layer astrocytes or deep-layer astrocytes.

overall_design: With the use of fluorescence-activated cell sorting (FACS), we prepared upper-layer astrocytes and deep-layer astrocytes from the corresponding dissected layers of the somatosensory cortex of Aldh1l1-eGFP mice, in which all astrocytes are expected to be labeled with GFP. The meninges, layer I, and the corpus callosum were removed from upper- and deep-layer tissue samples. In addition, parts of layers IV and V were lost during separation of these layers in such a way as to prevent cross-contamination between the upper- and deep-layer samples. Total RNA from upper-layer astrocytes and deep-layer astrocytes (n = 3 brains from 4-week-old male mice) was isolated from sorted cells with TRIzol (Invitrogen) or RNAiso Plus (Takara) and was then subjected to reverse transcription with the use of a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech). Bar-coded libraries were prepared with a Nextera XT DNA Library Preparation Kit (Illumina), and single-end 36-bp sequencing was performed with a HiSeq 2500 instrument (Illumina).

first: Darin

last: Lanjakornsiripan

first: Baek-Jun

last: Pior

first: Daichi

last: Kawaguchi

first: Shohei

last: Furutachi

first: Yutaka

last: Suzuki


title: RNA-seq analysis of Retinoic Acid and Non-treated Control hiPSCs

city: Chicago

first: Dawood

last: Darbar

state: IL

address: 840 S. Wood St. 920S CSB

department: Cardiology

institute: University of Illinois at Chicago

summary: The goal of this experiment is to identify down stream gene networks differentially regulated upon treatment of retinoic acid in hiPSC derived cardiomyocytes after 30 days of differentiation

overall_design: Examination of 3 RA treated and 3 CT hiPSCs after 30 days of differentiation

first: Erin

last: Lambers


title: Gene expression in Listeria- or vaccine-elicited T cells

city: Aurora

first: Ross

middle: M.

last: Kedl

state: CO

department: Immunology and Microbiology

institute: University of Colorado Anschutz Medical Campus

summary: B6 mice were immunized with either 2000 cfu  of ova expressing Listeria monocytogenes (LM-ova) or 100ug whle ovalbumin adjuvanted with 50ug polyIC + 50ug anti-CD40 (FGK45).  7 days later, tetramer staining CD8+ T cells from the spleen were flow sorted to purity (~98%), the RNA isolated, reverse transcribed and the resulting cDNA analyzed by gene array MoGene-1_0-st-v1 gene expression determined in naïve (CD44lo) and day 7 post Listeria chalelneg  or combined adjuvant vaccine immunization (internally referred to as "synergy").

overall_design: Gene expression profiling of CD44lo T cells (naïve), d7 tetramer staining CD8+ T cells from Listeria-treated or vaccine-treated B6 mice.

first: Lindsay

last: Edwards


status: Public on Apr 05 2018

city: St. Louis

first: Maxim

last: Artyomov

state: Missouri

address: 425 S Euclid Ave

department: Pathology & Immunology

institute: WASHINGTON UNIVERSITY IN ST LOUIS

summary: Host and genetic viral adaptations enable development of an immunocompetent mouse model of Zika virus infection. This study explored the effect of wildtype Zika virus (ZIKV) and mouse adapted ZIKV infection on the transcriptional profile of mouse adult neuronal stem cells.

overall_design: Mouse adult neuronal stem cells were isolated from 4-6 week old C57BL/6 mice, and infected with wildtype ZIKV-Dakar or ZIKV-Dakar NS4B G18R.  scRNA-seq was performed according to 10x Genomic Technology.

first: Matthew

last: Gorman

first: Konstantin

last: Zaitsev

middle: N

first: Michael

last: Diamond


title: An Evolutionarily-Conserved Long Noncoding RNA Myolinc Regulates muscle differentiation [array_overexpression]

city: Copenhagen SV

first: Shizuka

last: Uchida

email: heart.lncrna@gmail.com, suc@dcm.aau.dk

address: Frederikskaj 10B, 2. (building C)

department: Department of Clinical Medicine

country: Denmark

institute: Aalborg University

summary: Myogenesis is a complex process required for skeletal muscle formation during embryonic development and for regeneration and growth of myofibers in adults. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) play key roles in regulating cell fate decision and function in various tissues. However, the role of lncRNAs in the regulation of myogenesis remains poorly understood. In this study, we identified a novel muscle-enriched lncRNA called "Myolinc (AK142388)", which we functionally characterized in the C2C12 myoblast cell line. Myolinc is predominately localized in the nucleus, and its levels increase upon induction of the differentiation. Knockdown of Myolinc impairs the expression of myogenic regulatory factors and formation of multinucleated myotubes in cultured myoblasts. Myolinc also regulates the expression of Filip1 in a cis-manner. Similar to Myolinc, knockdown of Filip1 inhibits myogenic differentiation. Furthermore, Myolinc binds to TAR DNA-binding protein 43 (TDP-43), a DNA/RNA-binding protein that regulates the expression of muscle genes (e.g. Acta1 and MyoD). Knockdown of TDP-43 inhibits myogenic differentiation. We also show that Myolinc-TDP-43 interaction is essential for the binding of TDP-43 to the promoter regions of muscle marker genes. Finally, we show that silencing of Myolinc inhibits skeletal muscle regeneration in adult mice. Altogether, our study identifies a novel lncRNA that controls key regulatory networks of myogenesis.

overall_design: Using C2C12 cells, we performed microarray experiments (n=2) for each time point and condition.

first: Giuseppe

last: Militello


title: An Evolutionarily-Conserved Long Noncoding RNA Myolinc Regulates muscle differentiation [array_knockdown]

overall_design:
Using C2C12 cells, we performed microarray experiments (n=2) for each time point and condition.
“Day_2” dataset: siMyolinc-knockdown C2C12 cells on day 2 of the differentation were compared to C2C12 cells transfected with siRNA against scramble control (siScr) and differentiated for 2 days.
“Undiff-Day_2” dataset: siMyolinc-knockdown C2C12 cells on day 2 of the differentation were compared to normally cultured C2C12 cells in the growth medium (“Undiff”), one and two days after the differentiation (“Day_1” and “Day_2”, respectively).


title: Targeted RNA-Seq of Human Naïve CD4+ cells

city: Bethesda

first: Julie

middle: E

last: Niemela

state: MD

address: 10 Center Dr

department: Dept. of Lab Medicine

institute: NIH Clinical Center

summary: Purpose: The purpose of this RNA-seq experiment was to perform a correlation analysis of mRNA expression levels in naïve CD4+ T cells and LPS-stimulated monocytes from patients with an IKZF1 mutant haploinsufficient phenotype (H167R-a and H167R-b are two siblings carrying IKZF1 p.H167R mutation) versus patients with an IKZF1 mutant dominant negative phenotype (C1, G1) along with five healthy normal controls (HC).

overall_design: Method: Targeted RNA-seq library preparation was carried out using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies) which is designed to profile over 20,000 distinct human RefSeq genes using a highly multiplexed amplification method. Each amplicon represents a unique targeted gene (one transcript per gene). The average size of each amplicon is ~150 bp. For library preparation, each sample was run in duplicate and a cDNA library was first generated from a minimum of 10 ng of total RNA. The cDNA was barcoded and amplified using Ion AmpliSeq technology, and the amplified cDNA Libraries were evaluated for quality and quantified using Agilent Bioanalyzer high sensitivity chip. Libraries were then diluted to 100 pM and pooled equally, with four individual samples per pool. Pooled libraries were amplified and enriched using the Ion Chef System (Life Technologies). Templated libraries were then sequenced on an Ion Torrent Proton sequencing system (Life Technologies), using Ion PI HiQ kit and chip version 3.

first: Sergio

last: Rosenzweig

first: Hyesun

last: Kuehn

first: Jennifer

middle: L

last: Stoddard


title: Epigenome analysis of normal and T cells from patients with myasthenia gravis

city: Chang Sha

first: jingjiao

last: chen

state: Hu Nan

address: Renmin

institute: xiangya hospital

summary: We quantified different methylcytosine genes in CD4+T cells from patients with MG and healthy controls using Infinium HumanMethylation450 BeadChip Assays. Cells were isolated from the peripheral venous blood of 6 MG patients and 6 healthy donors.

overall_design: Bisulphite converted DNA from the 12 samples were hybridised to the Illumina Infinium HumanMethylation450 BeadChip

first: Jingjiao

last: Chen

first: Jing

last: Li


title: Intron retention induced by microsatellite expansions as a disease biomarker.

city: Seattle

first: James

middle: Douglas

last: Thomas

state: WA

address: 1100 Fairview Ave. N., B3-111

department: Computational Biology

institute: Fred Hutchinson Cancer Research Center

summary: Microsatellite expansions often occur in non-coding regions of the genome. In this study, we test their effect on host transcript RNA processing.

overall_design: RNA-seq on affected tissues and peripheral blood from control and affected patients.

first: Łukasz

last: Sznajder


title: RNA-seq analysis of osteogenic differentiating mouse wild type (WT) and Zfp384 (Nmp4) knockout mesenchymal stem/progenitor cells (MSPCs)

city: Indianapolis

first: Joseph

last: Bidwell

state: Indiana

address: 635 Barnhill Dive

department: Anatomy and Cell Biology

institute: Indiana University School of Medicine

summary: A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor Nmp4 respond to several classes of osteoporosis drugs with enhanced bone formation compared to wild type (WT) animals. To address how loss of Nmp4 maximizes anabolic output we used RNA-seq and network analyses, along with biochemical and bone mechanical measurements to define this anti-anabolic axis. Several independent preparations of expanded mesenchymal stem/progenitor cells (MSPCs) were derived from individual Nmp4-/- and WT mice. The Nmp4-/- cells exhibited accelerated and enhanced mineralization. Loss of Nmp4 significantly altered the expression of over 5,000 genes. While Nmp4 status did not alter the mRNA expression of Runx2 and Sp7, key regulators of osteoblast differentiation, Nmp4-depletion enhanced expression of genes that drive osteogenesis and conversion to aerobic glycolysis, a key step in bone anabolism. Functional studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion. Several canonical pathways sensitive to Nmp4 status controlled protein production and delivery and as predicted the Nmp4-/- cells showed elevated collagen translation and secretion. Expression of matrix genes that contribute to bone material-level mechanical properties were elevated in Nmp4-/- cells. Mechanical analysis of femurs from Nmp4-/- mice treated with osteoporosis therapies resulted in enhanced bone material properties compared to WT mice. In conclusion, disabling Nmp4 converts the osteoblast into a super-secretor by metabolically reprogramming the cell to support the concomitant elevated matrix production and expansion of the cell’s delivery capacity. Resolving this pathway will guide advances in osteoporosis therapy.

overall_design: To compare transcriptome profiles of non-differentiating and osteogenic-differentiating WT and Nmp4-/- MSPCs, cells were seeded into 12-well plates at either 10,000 cells/well (25 cells/mm2) or 25,000 cells/well (62 cells/mm2). The cells seeded at the lower density were maintained in Mesencult MediaTM + Mesencult Stimulatory SupplementTM (non-differentiating medium) for 3 days post-seeding and then harvested for total RNA. Cells plated at the higher density were maintained in α-MEM complete medium throughout the experiment. At 48 hours post-seeding the medium was refreshed with the ascorbic acid, dexamethasone, and BGP supplement. These cells were harvested at 7 days post-seeding as early osteogenic cells. For each condition there were four technical replicates and 1 biological replicate.

middle: P

first: Paul

last: Childress


title: Transcriptome analysis of a MECP2 conditional KO mouse model

city: SYDNEY

first: Irina

last: VOINEAGU

address: High ST

institute: UNSW

summary: Rett syndrome is a severe neurodevelopmental condition that rsults primarily from mutations in the MECP2 gene. MECP2 is known to function as both a transcriptional activator and transcriptional repressor. However, it remains unclear how transcriptional dysregulation resulting from MECP2 mutations lead to the Rett syndrome phenotype. Multiple mouse models have been generated to investigate the function of MECP2 in vivo. Remarkably, despite the neurodevelopmental phenotype characteristic of Rett syndrome, temporal conditional MECP2 knock-out mouse models with MECP2 deletion induced postnatally recapitulate the Rett-syndrome-like phenotype in mouse. Here we investigated gene expression changes in 22-weeks old mice following conditional MECP knock-out at 12 weeks by RNA-sequencing. Consistent with previous data, we identify mild gene expression changes following MECP2 knock-out. These data could prove valuable in future studies comparing conditional MECP2 knock-out at distinct time points and in additional brain regions, and can also serve for investigating alternative splicing changes resulting from MECP2 conditional deletion.

overall_design: RNA-samples from the cerebellum (CB ) and cerebral cortex (CTX) of 4 wild-type and 4 conditional MECP2-KO mice were analysed by RNA-seq.

last: Voineagu


title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development (HPC-5F RNAseq)

city: Cambridge

last: Guo

state: MA

address: 415 Main St

institute: Broad Institute

summary: While gene expression dynamics have been extensively catalogued during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs. Our findings indicate that HSCs manifest distinct alternative splicing patterns in key hematopoietic regulators. Detailed analysis of the splicing dynamics and function of one such regulator, HMGA2, identified an alternative isoform that escapes miRNA-mediated targeting. We further identified the splicing kinase CLK3 that, by regulating HMGA2 splicing, preserves HMGA2 function in the setting of an increase in let-7 miRNA levels, delineating how CLK3 and HMGA2 form a functional axis that influences HSC properties during development. Collectively, our study highlights molecular mechanisms by which alternative splicing and miRNA-mediated post-transcriptional regulation impact the molecular identity and stage-specific developmental features of human HSCs.

overall_design: RNA-seq of HPC-5F cells transduced with a control (CTRL), HMGA2-L (LONG), HMGA2-S (SHORT) or CLK3 ORF lentiviral over-expression vectors.

first: Guo

last: Michael

first: Cacchiarelli

last: Davide

first: Cesana

last: Marcella


title: SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis [ChIP-Seq]

city: Vienna

first: Matthias

last: Muhar

address: Campus-Vienna-Biocenter 1

country: Austria

institute: Research Institute for Molecular Pathology

summary: Defining direct targets of transcription factors and regulatory pathways is key to understanding their role in physiology and disease. Here we combine SLAM-seq, a novel method for direct quantification of newly synthesized mRNAs, with pharmacological and rapid chemical-genetic perturbation to interrogate primary transcriptional targets of BRD4 and MYC and define the response to BET bromodomain inhibitors (BETi). While BRD4 acts as a global co-activator of Pol2-dependent transcription in a BET bromodomain-dependent manner, therapeutic BETi doses deregulate a small set of hypersensitive target genes. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator that controls basic metabolic processes such as ribosome biogenesis and de-novo purine synthesis across diverse cancer contexts. Beyond defining primary regulatory functions of BRD4 and MYC in cancer, our study establishes a simple, robust and scalable approach to dissect direct transcriptional targets of any gene or pathway.

overall_design: Baseline gene expression profiling of clonal cell lines engineered to express AID-tagged proteins and unedited controls by 3'mRNA sequencing.

first: Anja

last: Ebert

first: Tobias

last: Neumann

first: Christian

last: Umkehrer

first: Corinna

last: Wieshofer

first: Anna

last: Obenauf

first: Johannes

last: Popow

first: Stefan

last: Ameres

last: Zuber


title: Acquired resistance to MEK-CDK4/6 inhibitor combinations in cutaneous melanoma

city: Philadelphia

first: Andrew

last: Aplin

state: PA

address: 233 South 10th St.

institute: Thomas Jefferson University

summary: Continuous MEK and CDK4/6 inhibition is effective in pre-clinical models, nevertheless, some tumors acquire resistance that was associated with enhanced phospho S6. To characterize the mechanism mediating the upregulation of mTOR-S6 pathway in these tumors, we performed RNA sequencing and targeted panel sequencing on xenograft tumors that progressed on either MEK plus CDK4/6 inhibitors (ComboR) or control diet. Gene set variance analysis (GSVA) revealed distinct gene expression signatures between the two ComboR tumors indicating heterogeneity. However, one of the 44 shared pathways between ComboR1 and ComboR2 included upregulation of an AKT pathway gene signature. By targeted sequencing and RNA sequencing, we observed high copy number change of NRAS in ComboR1 and a third tumor sample isolated from a continuous MEK inhibitor-intermittent CDK4/6 inhibitor schedule. NRAS was confirmed to mediate increased mTOR-S6 pathway activation suggesting that while distinct mechanisms are employed by melanoma tumors to circumvent MEK plus CDK4/6 inhibition, those resistance pathways eventually converge upon phosphorylation of S6 protein.

overall_design: DNA or RNA were obtained from progressing WM1366 melanoma xenograft tumors treated with either control diet or the combination of MEK plus CDK4/6 inhibitor diet.

first: Jessica

last: Teh

first: Phil

middle: F

last: Cheng

first: Timothy

last: Purwin


title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development [ChIP-seq]

first: Michael

last: Guo

overall_design: ChIP-seq of HPC-5F cells transduced with a control (CTRL), V5-tagged HMGA2-L (LONG) or HMGA2-S (SHORT) ORF lentiviral over-expression vectors. V5 antibody (V5), K4me2 antibody (k4me2) were used for ChIP alongside with input control whole cell extract (WCE).

first: Kendell

last: Clement

first: Davide

last: Cacchiarelli


title: Macrophage microRNA-150 promotes pathological angiogenesis as seen in age-related macular degeneration: part II

city: Saint Louis

first: Jonathan

last: Lin

state: MO

address: 660 S. Euclid Ave, Campus Box 8096

institute: Washington University School of Medicine

summary: We identified that microRNA-150 is upregulated in aged macrophages of diverse origins and therefore may mechanistically skew them towards a disease-promoting phenotype. The purpose of this experiment was to identify how the murine transcriptome changes with microRNA-150 overexpression.

overall_design: We prepared 12 samples (N=6/group; microRNA-150_overexpression versus negative control). Quality control revealed improper fragmentation of one sample, which we omitted from further analysis. This sample has not been included in these records.


title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development [BM low-input mRNA-seq]

overall_design: RNA-seq of BM-HSC cells transduced with a control (CTRL), HMGA2-S+3'UTRwt (SHORT) or CLK3 lentiviral over-expression vectors.

first: Marcella

last: Cesana


title: A CLK3-HMGA2 alternative splicing axis impacts human hematopoietic stem cell molecular identity throughout development [HSC and PROG RNA-seq]

overall_design: RNA-seq of hematopoietic stem cells (HSC) and progenitor cells (PROG) from fetal liver (FL), cord blood (CB) and bone marrow (BM) and control HPC cells differentiated from IPSC cells.


title: Expression data of RANKL-deficient CCR6+ ILC3

first: Jennifer

middle: Kaoru

last: Bando

address: 660 South Euclid Ave

institute: WUSM

summary: Microarrays were used to determine transcriptional differences between CCR6+ ILC3s isolated from RorccreTnfsf11fl/fl and Tnfsf11fl/fl small intestine lamina propria.

overall_design: RNA was extracted from CCR6+ ILC3s isolated from adult mouse RorcCreTnfsf11fl/fl or Tnfsf11fl/fl small intestine lamina propria.

first: Marco

last: Colonna

middle: K


title: Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine

status: Public on Apr 04 2018

city: Hangzhou

first: Yuanhui

last: Mao

state: Zhejiang

address: 1369 West Wenyi Road

institute: Zhejiang University

summary: Amino acid availability regulates translation through the action of the GCN2 and mTORC1 pathways. Low amino acids activate the eIF2α kinase GCN2 through binding of uncharged tRNAs to a histidyl-tRNA synthetase−related regulatory domain. Once activated GCN2 phosphorylates eIF2α, inhibiting ternary complex formation and translation initiation. Recent studies show that mTORC1 is particularly sensitive to arginine and leucine status, with a deprivation of these amino acids leading to a strong inhibition of mTORC1 that prevents the phosphorylation and inactivation of the translational repressor 4EBP1. Though amino acids are known regulators of translation, the effects that deficiencies of specific amino acids have on translation have yet to be determined. We demonstrate that deprivation of leucine or methionine results in large inhibitory effects on translation initiation and on polysome formation that are not replicated by overexpressing non-phosphorylatable 4EBP1 or a phosphomimetic eIF2α. Our results demonstrate that a lack of either leucine or methionine has a major impact on mRNA translation, though they act by quite different mechanisms. Leucine deprivation appears to primarily inhibit ribosome loading, whereas methionine deprivation appears to primarily impair start site recognition. These data point to a unique regulatory effect that methionine status has on translation initiation.

overall_design: Ribosome profiling of single Amino acid starvation

first: Kevin

last: Mazor

first: Leiming

last: Dong

first: Shu-Bing

last: Qian

first: Martha

middle: H

last: Stipanuk


title: RNA sequencing to study transcriptomic changes in DLD-1 (colorectal adenocarcinoma) cells exposed to soft polyacrylamide matrices (~2 kPa and ~55 kPa) for short time scale of 90 minutes

city: Pune

first: Kundan

last: Sengupta

state: Maharashtra

address: Dr. Homi Bhabha Road, Near NCL Innovation Park, Pashan

country: India

institute: Indian Institute of Science Education and Research (IISER), Pune

summary:
Aim: To examine transcriptional changes in DLD-1 cells exposed to softer matrices (2 kPa and 55 kPa) and identify the chromosomes that are enriched with maximmally deregulated genes
Methods: DLD-1 cells (otherwise growing on stiff tissue culture plastic substrates) were exposed to softer matrices for 90 minutes and to collagen coated glass coverslips (served as control) served as control)
Results: RNA sequencing revealed nearly equivalent transcriptional deregulation in cells on both the polyacrylamide matrices (783 genes up and 872 genes down on 2 kPa, 649 genes up and 783 genes down on 55 kPa) when compared to cells on glass. Additionally, GO classification revealed that unique sets of transcriptionally deregulated genes (log fold≥2) belonged to pathways associated with transcription regulation, chromatin organization, cell cycle and DNA damage/repair
Results: We identified chromosomes 1, 2, 3, 6, 7, 10, 12, 14, 17 and 19 to be maximally enriched with the deregulated genes on softer matrices (log fold≥2), while chromosomes 13, 18 and 21 showed minimal enrichment of deregulated genes. We also examined the spatial organization of chromosome 1, 18 and 19 territories in cells on softer matrices (using 3D-FISH) and observed that these chromosomes were mislocalized away from their conserved nuclear locations
Conclusions: Our study reports the transcriptomic changes in DLD-1 cells upon lowering of extracellular substrate stiffnes and its impact on the spatial positioning of chromosome territories

overall_design: RNA Seq profiles for DLD-1 cells on soft polyacrylamide matrices of ~2 kPa and ~55 kPa (reference - glass) were generated across 2 independent biological replicates using Illumina HiSeq platform

first: Roopali

last: Pradhan

first: Devika

last: Ranade


title: A20 regulates canonical wnt-signaling through an interaction with RIPK4

city: Los Angeles

first: Ling

last: Shao

state: CA

address: 2011 Zonal Avenue, 600

department: Department of Medicine

institute: University of Southern California

summary: The role of A20 in regulating canonical wnt signaling was investigated in colon cancer cell lines (RKO) by RNAseq

overall_design: Examination of gene-transcriptional changes in colon cancer cell lines stimulated by wnt3a for 24 hours

first: Brooke

last: Nakamura


title: Deciphering the reading of the genetic code by near-cognate tRNA

city: ORSAY

first: Pierre

last: Bertin

address: Batiment 400, Université Paris Sud

country: France

institute: Institut de Biologie Intégrative de la Cellule

summary: Some codons of the genetic code can be read not only by cognate, but also by near-cognate tRNAs. This flexibility is thought to be conferred mainly by a mismatch between the third base of the codon and the first of the anticodon (the so-called wobble position). However, this simplistic explanation underestimates the importance of nucleotide modifications in the decoding process. Using a system in which only near-cognate tRNAs can decode a specific codon, we investigated the role of six modifications of the anticodon, or adjacent nucleotides, of the tRNAs specific for Tyr, Gln, Lys, Trp, Cys and Arg in Saccharomyces cerevisiae. Modifications almost systematically rendered these tRNAs able to act as near-cognate tRNAs at stop codons, even though they involve non-canonical base-pairs, without markedly affecting their ability to decode cognate or near-cognate sense codons. These findings reveal an important effect of modifications to tRNA decoding with implications for understanding the flexibility of the genetic code.

overall_design: Riboseq was done to study the readthrough after stop codons in WildType and Mutant.

first: Sandra

last: Blanchet

first: David

last: Cornu

first: Isabelle

last: Hatin

first: Henri

last: Grosjean

first: Olivier

last: Namy


title: Targeted RNA-Seq of Human LPS-stimulated Monocytes

first: Julie

last: Niemela

overall_design: Method: Targeted RNA-seq library preparation was carried out using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies) which is designed to profile over 20,000 distinct human RefSeq genes using a highly multiplexed amplification method. Each amplicon represents a unique targeted gene (one transcript per gene). The average size of each amplicon is ~150 bp. For library preparation, each sample was run in duplicate and a cDNA library was first generated from a minimum of 10 ng of total RNA. The cDNA was barcoded and amplified using Ion AmpliSeq technology, and the amplified cDNA Libraries were evaluated for quality and quantified using Agilent Bioanalyzer high sensitivity chip. Libraries were then diluted to 100 pM and pooled equally, with four individual samples per pool. Pooled libraries were amplified and enriched using the Ion Chef System (Life Technologies). Templated libraries were then sequenced on an Ion Torrent Proton sequencing system (Life Technologies), using Ion PI HiQ kit and chip version 3.

first: Sergio

last: Rosenzweig

first: Hyesun

last: Kuehn

first: Jennifer

last: Stoddard


title: Functional studies of missense TREM2 mutations in human stem cell-derived microglia

status: Public on Apr 03 2018

city: St Louis

first: Sabine

last: Dietmann

address: 4565 McKinley Avenue

summary: The derivation of microglia from human pluripotent stem cells provides systems for understanding microglial biology and enables functional studies of neurological disease-causing mutations.  We describe a robust method for the derivation and maintenance of microglia from human stem cells, which are phenotypically and functionally comparable to primary human microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which are causal for a frontotemporal dementia-like (FTD-like) syndrome and Nasu-Hakola disease (NHD). While many ligands and functions for TREM2 have been described, it is not known how TREM2 signalling dysregulation affects specific elements of microglial biology to influence disease pathogenesis. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not properly trafficked to the plasma membrane of patient-derived microglia. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the late adult-onset of disease in individuals with these mutations.

overall_design: Primitive macrophage precursors (iPMP) and microglia (iMG) were derived from induced pluripotent human stem cells. Total RNA from each cell type was isolated from three independent differentiations of two genetic backgrounds (AD3-4 and NDC1.2).

first: Philip

last: Brownjohn

first: Rick

last: Livesey


title: Bioinformatics analysis of transcriptome related to blood stasis syndrome in diabetes mellitus patients

city: Guangzhou

first: Ruixue

last: Chen

state: Guangdong

address: Huangpu Avenue West 601

department: School of Chinese Medicine

institute: Jinan University

summary: In traditional Chinese medicine (TCM), blood stasis syndrome (BSS) is mainly manifested by the increase of blood viscosity, platelet adhesion rate and aggregation, and the change of microcirculation, resulting in vascular endothelial injury. It is an important factor in the development of diabetes mellitus (DM). According to the differences in the internal and external environment of the individual disease, BSS were divided into qi-deficiency and blood stasis syndrome (QDBS), qi-stagnation and blood stasis syndrome (QSBS), cold-coagulation and blood stasis syndrome (CCBS), heat-accumulation and blood stasis syndrome (HABS). The aim of this study was to screen out the potential candidate mRNAs in DM patients with BSS by high-throughput sequencing (HTS) and bioinformatics analysis. CRL-1730 human umbilical vein endothelial cells (HUVECs) were incubated with 10% human serum to establish models of DM with BSS, DM without BSS (NBS) and normal control (NC). Total RNA of each sample was extracted and sequenced by the Hiseq2000 platform. Differentially expressed mRNAs (DE-mRNAs) were screened between samples. On the basis of mRNA expression profiles, four comparisons were made, including QDBS vs NBS and NC, QSBS vs NBS and NC, CCBS vs NBS and NC, HABS vs NBS and NC. Then, comparisons with P values <0.05 (Fisher's exact test), false discovery rates (FDR) <0.01 and |log2 ratios|≥1 were considered as DE-mRNAs in BSS. This study screened out the DE-mRNAs in DM patients with BSS by HTS and bioinformatics analysis.

overall_design: Bioinformatics analysis of transcriptome in DM patients with BSS by establishing cell models of DM with BSS.

first: Meixia

last: Fang


title: Genome-Wide Mapping and Interrogation of the Nmp4 Antianabolic Bone Axis

first: Joseph

last: Bidwell

summary: PTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4-/- mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8+ T cells. To determine whether the Nmp4-/- phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4-/- mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling onNmp4chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 weeks old) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4-/- bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4-/- mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8+ T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4-/- MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4-/- MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.

overall_design:
MC3T3-E1 subclone 4, PTH therapy, Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq)
Cells from ATCC (MC3T3-E1 subclone 4) were maintained in a-MEM supplemented with 100-IU/mL penicillin, 100-µg/mL streptomycin, 25-µg/mL amphotericin, 2mM L-glutamine (Gibco BRL), ascorbic acid (50 µg/mL; Sigma-Aldrich), and 10% fetal bovine serum (Sigma-Aldrich). Cells were seeded into twenty-one 150-mm plates at an initial density of 50,000 cells/plate (320 cells/cm2) and maintained in a-MEM complete medium plus ascorbic acid. On day 14 post-seeding, cells were treated with 25nM hPTH (1–34) or vehicle control for 1 hour before harvest.

first: Paul

last: Childress

first: Yunlong

last: Liu


title: An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters

first: Erica

last: Carpenter

address: #8-104, 3400 Civic Center Blvd

department: Hematology and Oncology

institute: University of Pennsylvania

summary: We demonstrate the feasibility of performing single cell RNA sequencing on cells isolated from whole blood using a novel flow cytometry-based platform

overall_design: Whole transcriptome sequencing of single and pooled cells from a mouse pancreatic cancer cell line to validate rare cell pre-enrichment and isolation approach

first: Neha

last: Bhagwat

first: Quanli

last: Wang

first: Tony

last: Berno

first: Keely

last: Dulmage


title: SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis [Quant-Seq]

first: Matthias

last: Muhar

first: Anja

last: Ebert

first: Tobias

last: Neumann

first: Christian

last: Umkehrer

first: Corinna

last: Wieshofer

first: Anna

last: Obenauf

first: Johannes

last: Popow

first: Stefan

last: Ameres

last: Zuber


title: RNA-seq analysis of RALD iPSCs after in vitro differentiation

status: Public on Apr 01 2018

city: Tsukuba

first: Kenji

last: Kubara

state: Ibaraki

address: Tokodai 5-1-3

department: Tsukuba Research Laboratories

institute: Eisai Co., Ltd.

summary: We investigated roles of KRAS on stemness maintenance and differentiation propensity in the context of induced pluripotent stem cells (iPSCs) using isogenic KRAS mutant (G13C/WT) and wild-type (WT/WT) iPSCs from the same RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD) patients. RNA-seq analysis was conducted to compare gene expression profiles of WT/WT and G13C/WT iPS cells after in vitro differentiation. We found some differences of gene expression profiles regarding stemness and linage markers in the two genotypes.

overall_design: To investigate the changes of stemness and linage markers between KRAS mutant and wild-type iPSCs after differentiation, the iPSCs were differentiated for 16 days . Two clones were used for each genotype (WT/WT and G13C/WT) before and after differentiation, resulting in total 8 conditions.

first: Kazuto

last: Yamazaki

first: Yasuharu

last: Ishihara

first: Huan-Ting

last: Lin

first: Masashi

last: Ito

first: Kappei

last: Tsukahara

first: Masatoshi

last: Takagi

first: Makoto

last: Otsu


title: Comparison of gene expression profiles of ex vivo sorted Common Lymphoid Progenitors (CLPs) and in vitro cultured cCLPs.

city: Berlin

first: Pawel

last: Durek

address: Charitéplatz 1

institute: Deutsches Rheuma-Forschungszentrum

summary: A common lymphoid progenitor cell culture system was established, maintaining a multipotent, proliferating state of CLPs. Cells can be cultured and expanded in vitro for several months, maintaining their in vitro and in vivo lymphoid differentiation potential. We were interested, whether cCLP are changing, with regards to their gene expression program, compared to in vivo CLPs, during their expansion in culture. Therefore we compared the gene expression profile of cultured cCLPs and their counterparts in vivo, by NextGenSeq. CLPs were sorted from bone marrow of 6-10 weeks old C57BL/6 mice (n=4). Cells were sorted for Lin-, IL7R+, Flk2+, CD27+, Ly6d- marker expressing CLPs by Flow Cytometry for gene expression analysis. In parallel the same cell population was sorted and cultured under cCLP culture conditions. After two months culture the cells sorted again for Lin-, CD27+, Ly6d- cells by Flow Cytometry for gene expression analysis (n=4). The analysis shows that cCLPs maintain a very similar transcription profile to ex vivo sorted common lymphoid progenitors.

overall_design: Comparative gene expression of 2 cell types.

first: Yohei

last: Kawano

first: Georg

last: Petkau

first: Christina

last: Stehle

first: Gitta

last: Heinz

first: Kousuke

last: Tanimoto

first: Hajime

last: Karasuyama

first: Mir-Farzin

last: Mashreghi

first: Chiara

last: Romagnani

first: Fritz

last: Melchers


title: Development and Verification of an RNA-Seq Assay for the Detection of Gene Fusions in Tumors

city: Rochester

first: Numrah

middle: Mohammadali

last: Fadra

state: Minnesota

address: 200 2nd Street SW

department: Health Sciences Research

institute: Mayo Clinic

summary:
Purpose: Assessment of the performance characteristics of an RNA-Seq assay designed to detect gene fusions in 573 genes to aid in the management of cancer patients.
Methods: Polyadenylated RNA was converted to cDNA which was then used to prepare NGS libraries that were sequenced on a HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline.
Results: The assay identified 38 of 41 (93%) gene fusions previously detected by a different laboratory using FISH, RT-PCR, or RNA-Seq for a sensitivity of 93%. No false positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA-Seq in a different laboratory (100% specificity).  The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods.  Nineteen of the 22 fusions had not previously been described. Good intra- and inter-assay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion positive cases with fusion negative RNA.  Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions.
The assay identified 38 of 41 (93%) gene fusions previously detected by a different laboratory using FISH, RT-PCR, or RNA-Seq for a sensitivity of 93%. No false positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA-Seq in a different laboratory (100% specificity).  The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods.  Nineteen of the 22 fusions had not previously been described. Good intra- and inter-assay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion positive cases with fusion negative RNA.  Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions.
This assay should be useful for identifying cancer patients that may benefit from both FDA-approved and investigational targeted therapies.

overall_design: Sequencing data was generated using Hiseq 2500 with a library of 101 paired end reads in the rapid run mode

first: Kevin

last: Halling

first: Jaime

last: Davila

first: Eric

last: Klee

first: Asha

last: Nair

first: Becky

last: Wehrs

first: Jennifer

last: Winters

first: Rory

last: Jackson

first: Lisa

last: Peterson

first: Barb

last: Crusan

first: Amber

last: McDonald


title: Macrophage microRNA-150 promotes pathological angiogenesis as seen in age-related macular degeneration

first: Jonathan

last: Lin

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

platform_organism:
Mus musculus
synthetic construct

overall_design: Refer to individual Series


title: Gene expression signature of brown and inguinal white fat of mice kept at 30°C vs. 5°C

status: Public on Apr 02 2018

city: Neuherberg

first: Yiming

last: Cheng

address: Ingolstädter Landstraße 1

institute: Helmholtz Munich

summary: Activation and recruitment of thermogenic cells in human white adipose tissues (“browning”) can counteract obesity and associated metabolic disorders. However, quantifying the effects of therapeutic interventions on browning remains enigmatic. Here, we devise a computational approach, profiling of fat tissue types (ProFAT), for the quantification of thermogenic potential of heterogeneous fat biopsies based on the prediction of white and brown adipocytes content from raw gene expression profiles. ProFat systematically integrates 103 mouse fat-derived transcriptomes to identify unbiased and robust gene signatures of brown and white adipocytes. Application of ProFAT to 80 mouse and 97 human transcriptional profiles from 14 independent studies correctly predicts browning capacity upon various physiological and pharmacological stimuli. Our study represents the most exhaustive comparative analysis of public data on adipose biology towards quantification of browning after personalized medical intervention. ProFat is freely available and should become increasingly powerful with the growing wealth of transcriptomics data.

overall_design: The experiment was performed with female, 16 weeks old WT mice. All mice were bred, born and weaned at 30°C. At the age of 10-12 weeks, half of the mice were transferred to a temperature-controlled (+/- 0.5°C) climate chamber and kept for two weeks at 18°C followed by 3 weeks at 5°C. Afterwards, mice kept at 30°C and 5°C were euthanized after 2-3 hours food withdrawal, and serum and tissue samples were collected.

first: Martin

last: Jastroch

first: Fabiana

last: Perocchi

first: Susanne

last: Keipert


title: Quiescent tissue stem cells evade immune surveillance

city: New York

first: Brian

last: Brown

state: NY

address: 1470 Madison Avenue

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

summary: Stem cells are critical for the maintenance of many tissues, and thus their integrity is crucial, but whether this integrity is maintained in the face of an immune response is not well defined. Here we set out to determine the outcome of T cell interactions with adult stem cells in their physiological niche. We find that fast cycling epithelial stem cells, including Lgr5+ intestinal stem cells, as well as ovary and mammary stem cells, are eliminated by activated T-cells, but slow cycling stem cells, specifically hair follicle and muscle stem cells, are resistant to T-cell killing. This is an intrinsic property of the quiescent stem cells resulting from systemic downregulation of the antigen presentation machinery, including MHC class I, Tap proteins, and the transactivator Nlrc5, which is reversed upon stem cell entry into the cell cycle.  These findings identify a link between tissue stem cell quiescence, antigen presentation, and immune evasion, and may help explain why mutations in long-lived stem cells would not lead to immune editing. They also suggest how cancer-initiating cells may evade immune surveillance

overall_design: Lgr5-positive cells were flow-cytometry sorted from the back epidermis of mice that were either 45 days old (Telogen) or 35 days old (Anagen)

first: Judith

last: Agudo


title: Expression data from SUM149 inflammatory breast cancer cells cultured in monolayer or Matrigel

city: HOUSTON

first: Kazuharu

last: Kai

state: TX

address: 2130 West Holcombe Blvd. Life Science Plaza

department: Translational Molecular Pathology

institute: MD Anderson Cancer Center

summary:
Inflammatory breast cancer (IBC) is a rare type of breast cancer but accounts for up to 10% of breast cancer-related deaths. Plasticity between epithelial and mesenchymal feature is reported to be crucial in metastasis of IBC.
Using Matigel culture, we induced epithelial to mesenchymal transition (EMT) in epithelial-like SUM149 IBC cells and identified overexpressed genes in this EMT process.

overall_design: SUM149 cells were plated on monolayer or Matrigel culture (triplicates each) on day 0. On day 2, SUM149 cells on monolayer culutre were collected by tripsinizing them and SUM149 cells on Matrigel culture were recovered by using Cell Recovery Solution for RNA extraction and hybridization on Affymetrix microarrays.

first: Dongwei

last: Zhang


title: Epigenome analysis of alcohol consumption in whole blood (WB) samples

city: Iowa City

first: Robert

middle: Alan

last: Philibert

state: IA

address: 500 Newton Rd

institute: University of Iowa

type: Methylation profiling by genome tiling array

summary: Genome wide DNA methylation profiling of drinkers and non-drinkers in WB samples. The Illumina Infinium EPIC Human DNA methylation Beadchip was used to obtain DNA methylation profiles across  485,577 CpGs in WB samples that oevrlapped with CpGs from Illumina Infinium450k Human DNA methylation Beadchip. Samples included 47 drinkers (cases) and 47 non-drinkers (controls).

overall_design: Bisulfite converted DNA from the 94 samples were hybridized to the Illumina InfiniumEPIC Human Methylation Beadchip


title: A  New Family of Vertebrate Specific Polycomb Proteins Encoded by the LCOR and LCORL Gene Loci Reveal Antagonism Between PRC2 Subtypes

city: Dublin 2

first: Darren

middle: John

last: Fitzpatrick

state: Ireland

address: College Green

department: Smurfit Institute of Genetics

country: Ireland

institute: Trinity College Dublin

summary: The Polycomb Repressive Complex 2 (PRC2) is composed of core subunits SUZ12, EED, RBBP4/7 and EZH1/2, which together are responsible for all di- and tri- methylation of lysine 27 on Histone H3 (H3K27me2/3) in higher eukaryotes. While two distinct forms, PRC2.1 (containing one Polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2) exist, little is known about their differential functions or interplay. Here we report the discovery of a new family of vertebrate specific PRC2.1 associated proteins; ‘PRC2 associated LCOR isoform 1’ (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. We uncover an antagonistic relationship between the PALI-PRC2.1 and AEBP2-PRC2.2 subtypes and establish that both are required for balanced regulation of Polycomb target genes during differentiation. This discovery links the Polycomb epigenetic system with co-repressors and nuclear receptors in the regulation of cellular identity.

overall_design: ChIP seq analysis of Pali WT, Pali1 KO, Pali1/2 double KO, C129 WT and Aebp2 gene trap mouse embryonic stem cells. Each has 1.67% spike in of Drosophila chromatin (S2 cell line). 5 samples are included.

first: Eric

last: Conway

first: Emilia

last: Jerman

first: Evan

last: Healy

first: Shinsuke

last: Ito

first: Daniel

last: Holoch

first: Orla

last: Deevy

first: Eleanor

last: Glancy

first: Giorgio

last: Oliviero

first: Ariane

last: Watson

first: Alan

middle: M

last: Rice

first: Marlena

last: Mucha

first: Paul

last: Chammas

first: Christine

last: Huang

first: Indigo

last: Pratt-Kelly

first: Yoko

last: Koseki

first: Manabu

last: Nakayama

first: Tomoyuki

last: Ishikura

first: Gundula

last: Streubel

first: Kieran

last: Wynne

first: Karsten

last: Hokamp

first: Aoife

last: McLysaght

first: Claudio

last: Cifferi

first: Luciano

last: Di Croce

first: Raphael

last: Margueron

first: Gerard

last: Cagney

first: Haruhiko

first: Adrian

last: Bracken


title: Bioinformatics analysis of microRNAs related to blood stasis syndrome in diabetes mellitus patients

first: Ruixue

last: Chen

summary: In traditional Chinese medicine (TCM), blood stasis syndrome (BSS) is mainly manifested by the increase of blood viscosity, platelet adhesion rate and aggregation, and the change of microcirculation, resulting in vascular endothelial injury. It is an important factor in the development of diabetes mellitus (DM). The aim of this study was to screen out the potential candidate microRNAs (miRNAs) in DM patients with BSS by high-throughput sequencing (HTS) and bioinformatics analysis. CRL-1730 human umbilical vein endothelial cells (HUVECs) were incubated with 10% human serum to establish models of DM with BSS, DM without BSS (NBS) and normal control (NC). Total RNA of each sample was extracted and sequenced by the Hiseq2000 platform. Differentially expressed miRNAs (DE-miRNAs) and mRNAs (DE-mRNAs) were screened between samples. Target genes of miRNAs were predicted by softwares. Gene Ontology (GO) and pathway enrichment analysis of the target genes were conducted. According to the significantly enriched GO annotations and pathways (P value ≤0.001), we selected the key miRNAs of DM with BSS.

overall_design: Bioinformatics analysis of miRNAs in DM patients with BSS by establishing cell models of DM with BSS.

first: Meixia

last: Fang


title: RNA seq_PDX2_SHP099

city: Amsterdam

first: Sara

last: Mainardi

state: -- Please Select --

address: Plesmanlaan 121

country: Netherlands

institute: Netherlands Cancer Institute

summary: RNA seq data of PDX2 treated with vehicle of SHP099 --> to check senescence and SASP gene expression signatures

overall_design: PDX2 KRAS mut NSCLC tumors implanted in 12 different mice, 5 treated with vehicle, 7 with SHP099, during 19 days.

first: Iris

last: de Rink

first: Cor

last: Lieftink


title: Epigenetic profiling of OVA-specific CD8+ TILs from mice treated with control, anti-B7S1, anti-PD-1 or anti-B7S1+anti-PD-1 antibodies

city: Beijing

first: Chen

last: Dong

address: Haidian District

department: Institute for Immunology and School of Medicine

institute: Tsinghua University

summary: Combinational blockade of B7S1 and PD-1 exhibits stronger anti-tumor efficacy than monotherapies. In order to further understand the mechanisms whereby blockade of B7S1 or PD-1 inhibits tumor growth, we conducted ATAC-seq analysis of OVA-specific CD8+ TILs from E.G7-bearing mice treated with control antibodies, anti-B7S1, anti-PD-1 monotherapy or combinational therapy.

overall_design: 1 million E.G7 were inoculated subcutaneously into mice on Day 0. On Day 21, OVA-specific CD8+ TILs from E.G7-bearing mice treated with control, anti-B7S1, anti-PD-1 or anti-B7S1+anti-PD-1 antibodies(6 mice per group) were sorted using BD FACSAria Cell Sorter. The collected cells were then subjected to construction of library for ATAC-seq.

first: Jing

last: Li

first: Yanjian


title: Global gene expression profile of CD8+ TILs from WT and B7S1 KO mice

summary: B7S1 negatively regulates T cells and its expression correlates with poor prognosis of cancer patients. In order to understand how B7S1 signaling contributes to dysfunction of CD8+ T cell in the TME, we conducted transcriptional analysis of OVA-specific CD8+ TILs and different TIL subsets from E.G7-bearing WT and B7S1 KO mice (Day 21).

overall_design: One million E.G7 were inoculated subcutaneously into mice on Day 0. On Day 21, OVA-specific CD8+ TILs from E.G7-bearing WT and B7S1 KO mice (6 mice per group), or PD-1+Tim-3-B7S1R+(B7S1R_SP), PD-1+Tim-3+B7S1R-(TIM-3_SP), PD-1+Tim-3-B7S1R-(DN) CD8+ TILs from E.G7-bearing WT or B7S1 KO mice were sorted into lysis buffer using BD FACSAria Cell Sorter. cDNA was made and library was constructed using SMARTer Ultra Low Input RNA for Illumina Sequencing-HV kit and Creator SMART cDNA Library Construction Kit (Clontech) according to manufacturer’s instructions. The library products were sequenced via Illumina HiSeq 2500/4000 and successfully mapped to mouse genome. Reads counts were normalized based on RPKM/FPKM, fold changes were calculated for all possible comparisons.

first: Huiping

last: Lu

first: Xiaohong

last: Zhao


title: Differential 3’ Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types

first: Saša

last: Jereb

state: New York

address: 1230 York Ave

department: Laboratory of Molecular Neuro-Oncology

institute: The Rockefeller University

summary: Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length.

overall_design: Examination of Pabpc1-GFP bound RNA in cerebellar granule in Purkinje cells and cerebellar granule cells during development using a cTag-PAPERCLIP. RNA-seq of RNA from cerebellar granule cells at two developmental timepoints to compare with cTag-PAPERCLIP data.

first: Hun-Way

last: Hwang

first: John

middle: J

last: Fak

first: Mary

last: Hatten

first: Robert

last: Darnell