title: Identification and Profiling of microRNAs from Hypothalamus of Estrous Kazakh Sheep Induced by High Nutrition Treatment in Non-breeding Season

city: beijing

first: zhengqin

last: rong

address: Chaoqian Rd,Changping Science Park of Zhongguancun Science Park

institute: hornortech

summary: The identification of significantly differently expressed miRNAs expanded the repertoire of sheep miRNAs and could contribute to further studies on the molecular mechanism of estrous sheep in the non-breeding season under the different levels of nutrition.

overall_design: Total RNA from the hypothalamus of Kazakh sheep in the estrous group (HEN) under the high-nutrition treated and those in the anestrous group (HAN) under the low-nutrition treated in the non-breeding season were used to construct small RNA libraries by Illumina HiSeq sequencing technology.

first: Heng

last: Yang

first: Shan

last: Lin


title: Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics

status: Public on May 18 2017

city: San Francisco

first: Arun

middle: P.

last: Wiita

state: CA

address: 185 Berry St., Suite 290

department: Laboratory Medicine

country: USA

institute: University of California, San Francisco

type: Expression profiling by high throughput sequencing

summary: Ribosome profiling is a widespread tool for studying translational dynamics in human cells.  Its central assumption is that ribosome footprint density on a transcript quantitatively reflects protein synthesis.  Here, we test this assumption using pulsed-SILAC (pSILAC) high-accuracy targeted proteomics.  We focus on multiple myeloma cells exposed to bortezomib, a first-line chemotherapy and proteasome inhibitor. In the absence of drug effects, we found that direct measurement of protein synthesis by pSILAC correlated well with indirect measurement of synthesis from ribosome footprint density.  This correlation, however, broke down under bortezomib-induced stress. By developing a statistical model integrating longitudinal proteomic and mRNA-seq measurements, we found that proteomics could directly detect global alterations in translational rate caused by bortezomib; these changes are not detectable by ribosomal profiling alone.  Further, by incorporating pSILAC data into a gene expression model, we predict cell-stress specific proteome remodeling events.  These results demonstrate that pSILAC provides an important complement to ribosome profiling in measuring proteome dynamics.

overall_design: Timecourse experiment with six points over 48hr after bortezomib exposure in MM.1S myeloma cells.  mRNA-seq and ribosome profiling data at each time point.

first: T-Y

last: Liu

first: H

middle: H

last: Huang

first: D

last: Wheeler

first: Y

last: Xu

first: J

middle: A

last: Wells

middle: S

last: Song

first: A

middle: P


title: RNA binding by the histone methyltransferases Set1 and Set2

status: Public on May 17 2017

city: Grenoble

first: Camille

last: Sayou

address: 17 avenue de Martyrs

department: BGE UA13

country: France

institute: CEA, IRIG

summary: Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo. High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional post-transcriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity.

overall_design: RNA binding by Set1 and Set2 was analysed by CRAC in S. cerevisiae. Set1 full length protein carried either a N-terminal (PTH) or C-terminal (HTP) tag. We also analysed a mutant where Set1 RRM2 was deleted (PTH-Set1dRRM2).  Set2 full length protein carried a HTP tag. All protein genes were tagged at their endogenous locus and were expressed under the control of their endogenous promoter. We have included triplicates or quadruplicates of each sample as well as duplicates of the untagged BY4741 negative control.

first: Gonzalo

last: Millán-Zambrano

first: Helena

last: Santos-Rosa

first: Samuel

last: Robson

first: Elisabeth

last: Petfalski

first: Jonathan

last: Houseley

first: Tony

last: Kouzarides

first: David

last: Tollervey


title: RNA fate determination through co-transcriptional methylation of newely synthesized transcripts

city: Basel

first: Fabio

last: Mohn

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Using murine embryonic stem (mES) cells, here we show that two RNAi factors, Dgcr8 and the RNAseIII Drosha, physically associate with chromatin. We found that these known microRNA-processing factors associate with a subset of actively transcribed genes, as well as non-coding genes, including snoRNA, and lncRNA genes. Dgcr8 recruitment to chromatin was dependent on Methyltransferase-like 3 (Mettl3), which catalyzes N6-methyladenosine (m6A) of RNAs. Chemical inhibition of RNA polymerase II (RNAPII) disrupted the association of Dgcr8 and Mettl3 with chromatin, strongly suggesting RNA methylation and processing events occur co-transcriptionally. We also found that temperature stress causes a radical relocalization of Dgcr8 and Mettl3 to stress-induced genes including Hsp70. Genetic ablation of Dgcr8 or Mettl3 led to the accumulation of Hsp70 mRNA, elongation of its half-life, and increased protein levels only in cells subjected to acute heat stress. This indicates that acute heat-stress co-transcriptionally marks Hsp70 mRNAs by Mettl3 and Dgcr8 for subsequent RNA degradation to control the timing and magnitude of the heat shock response.

overall_design:
CRISPR/Cas9 genome editing was used to endogenously tag the Mettl3, Dicer, Dgcr8, and Drosha genes with FLAG/Avi tags (to identify DNA interactions by immunoprecipitation) in mES cells that express E. coli biotin holoenzyme synthetase (BirA). We performed ChIP coupled to massive parallel DNA sequencing (ChIP-seq) to determine potential interactions of Mettl3, Dicer, Drosha, and Dgcr8 with the mouse genome. We introduced an additional crosslinking step using disuccinimidyl glutarate (DSG), which enables crosslinks over longer distances than formaldehyde alone and hence also captures proteins bound to nascent transcripts.  We also profiled Ribosomal depleted RNA from DGCR8 and Drosha conditional KO mES cells as well as non-4OHT-treated controls by RNA-seq. Finally we also profiled by RNA-seq WT cells mES cells or cells exposed to 42C for 1 hour to identify genes up-regulated upon heat stress.

Please note that there were no antibodies used for the ChIP-seq studies. The proteins were biotin-tagged, and thus Streptavidin magnetic beads were used for all IPs,

first: Philip

last: Knuckles

first: Sarah

last: Carl

first: Alice

last: Wenger

first: Michael

last: Musheev

first: Christof

last: Niehrs

first: Marc

last: Bühler


title: Epigenetic analysis of tumor-specific CD8 T cells in murine and solid tumors

city: New York

first: Andrea

last: Schietinger

state: NY

address: 1275 York Avenue

department: Immunology

institute: Memorial Sloan Kettering Cancer Center

summary: ATACseq analysis of dysfunctional CD8 T cells in progressing tumors. The overall goal of this study was to elucidate chromatin states associated with functional unresponsiveness in tumor-specific CD8 T cells in the context of mouse and human tumors. As comparison we also investigated chromatin state dynamics associated with CD8 T cells differentiation to functional effector and memory T cells during an acute listeria infection.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: CD8 T cells were sorted by flow cytometry and ATAC-seq was performed.

first: Mary

last: Philip

first: Christina

last: Leslie

first: Lauren

last: Fairchild


title: Transcriptome analysis of tumor-specific CD8 T cells in murine solid tumors

summary: RNAseq analysis of CD8 T cells becoming dysfunctional in progressing tumors. The overall goal of this study was to elucidate the molecular program that mediates functional unresponsiveness in tumor-specific CD8 T cells. In comparison, we also investigated CD8 T cells differentiating to functional effector and memory T cells during an acute listeria infection.

overall_design: T cells were sorted by flow cytometry and RNA-seq was performed.


title: The midgut transcriptome of Aedes aegypti fed with saline or protein meals containing chikungunya virus reveals genes potentially involved in viral midgut escape

city: Columbia

first: Alexander

last: Franz

state: MO

address: 303 Connaway Hall

department: Department of Veterinary Pathobiology

institute: University of Missouri

summary: This study seeks to understand the mechanism of midgut escape and identify candidate genes and potential biochemical pathways involved in the midgut infection of chikungunya virus (CHIKV) in vector mosquito Aedes aegypti. We conducted a comparative transcriptomic analysis of midgut samples of female mosquitoes,after feeding a saline meal (SM) or a protein meal (PM) containing CHIKV. Our results allow the conclusion that midgut-expressed genes not involved in blood or protein digestion can be identified by substituting either a bloodmeal or PM for a SM. In presence of orally acquired CHIKV in midguts of SM fed mosquitoes, the majority of the upregulated DE genes belonged to the categories immune system and catalytic activity. These genes included several serine-type endopeptidases, trypsins, collagenases, and M1 zinc metalloproteases, which potentially could be involved in the midgut escape mechanism of CHIKV. One of the serine metalloproteinase genes, AeLT, was further analyzed showing strong (MMP) collagenase activity in vitro. Our results present a set of candidate genes potentially responsible for overcoming the arbovirus midgut escape barrier (MEB) in Ae. aegypti.

overall_design: We used genome-wide transcriptome analyses of midguts to reveal gene expression patterns in midguts of protein meal and saline meal (containing/not containing CHIKV) fed mosquitoes in comparison to a sugarfed control.

first: Shengzhang

last: Dong

first: Susanta

middle: K

last: Behura

middle: W


title: Transcriptome profiling of hematopoietic cells derived from human pluripotent stem cells

city: Boston

first: Areum

last: Han

state: MA

address: Karp Family Building, 7th Floor. 300 Longwood Ave.

institute: Boston Children's Hospital

summary: We performed morphogen-directed differentiation of human PSCs into HE followed by combinatorial screening of 26 candidate HSC-specifying TFs for the potential to promote hematopoietic engraftment in irradiated immune deficient murine hosts. We recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that together were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft primary and secondary murine recipients.

overall_design: Hematopoietic cells derived from human pluripotent stem cells

first: Ryohichi

last: Sugimura

first: George

last: Daley


title: Transcriptome analysis of CNS leukemia

city: Kyoto

first: Akira

last: Watanabe

state: Kyoto

address: Rm504, Miyako Bldg, 233, Isa-cho, kamigyo-ku

country: Japan

institute: CyberomiX Inc

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

overall_design: Refer to individual Series


title: KRAB-ZFP/KAP1 is essential to restrict excessive IGF2 expression during mouse embryo development

city: Bethesda

first: Peng

last: Yang

state: Maryland

address: 6 Center Drive 6B-211

department: DDB

institute: NICHD/NIH

summary: Insulin-like growth factor 2 (Igf2) is the major fetal growth hormone in mammals. Here we identify the Kruppel-associated-box (KRAB) zinc finger protein 568 (ZFP568) as a direct repressor of a placental specific Igf2 transcript (designated Igf2-P0) in early mouse development.  Loss of Zfp568, which leads to gastrulation failure, causes inappropriate Igf2-P0 activation.  Strikingly, deletion of paternal Igf2 can completely rescue Zfp568 knockout-induced gastrulation phenotypes. Mechanistically, ZFP568 and its binding site are required to maintain H3K9me3 and CpG methylation of the Igf2-P0 region. The ZFP568 binding site upstream of the Igf2-P0 transcript is highly conserved throughout Eutheria, along with the DNA binding domain of ZFP568 orthologs, with the exception of human, whose three Znf568 alleles previously linked to human head size at birth have rapidly evolved and mostly lost the ability to bind and suppress Igf2, coincident with the loss of Igf2-P0 transcript activity.  We speculate that the Igf2-P0 transcript and its repressor ZFP568 appeared in a common ancestor of placental mammals as a critical adaptation to boost Igf2 expression specifically in the placenta to regulate maternal supply and fetal demand for nutrients. These data also highlight the exquisite specificity and selectivity by which KRAB-ZFPs have been naturally selected to recognize their genomic targets for pinpoint heterochromatin establishment during mammalian evolution.

overall_design: ChIP-seq using WT and KO ESCs/TSCs for ZFP568 and H3K9me3. RNA-seq using WT and KO ESCs/TSCs and hybrid ESCs. ChIP-seq using IGF2 promoter crispr cell lines for ZFP568 and H3K9me3. Two platforms, Illumina Hiseq2500 and Solid 5500xL were used for sequencing. For Solid platforms, raw data was aligned to mm9 by LifeScope™ Genomic Analysis Software v2.5.1 to generate bam files. All data from Illumina platform was deposited in fastq format.


title: INTS8 mutations cause severe neurodevelopmental syndrome

city: Portland

first: Zheng

last: Xia

state: Oregon

address: 3181 SW Sam Jackson Park Rd

department: Department of Biomedical Engineering

institute: Oregon Health & Science University

summary: Integrator (INT) is an RNA polymerase II (RNAPII)-associated complex that was recently identified to have a broad role in both RNA processing and transcription regulation. INT has at least 14 subunits, but INT germline mutations causing human disease have not been reported. We identified mutations in the Integrator Complex Subunit 8 gene (INTS8) causing a rare neurodevelopmental syndrome. In patient cells we identified significant disturbance of gene expression and RNA processing. Also, we show that injection of ints8 oligonucleotide morpholinos into zebrafish embryos leads to prominent underdevelopment of the head demonstrating the evolutionary conserved requirement of INTS8 in brain development.

overall_design: RNA sequencing was carried out using RNA samples from fibroblasts from two individuals with germline bi-allelic INTS8 mutations and from two healthy individuals

first: David

last: Baillat

first: Wei

last: Li

first: Eric

last: Wagner


title: mRNAseq of Huntington's disease and control patient iPSC-derived brain microvascular endothelial cells

status: Public on May 16 2017

city: Irvine

first: Ryan

middle: Gar-Lok

last: Lim

state: California

address: 845 Health Sciences Rd.

department: MIND

institute: UC Irvine

summary:
Purpose: The goals of this study are to compare the transcriptomic profile (mRNA-seq) of HD and control patient iPSC-derived brain microvascular endotheial cells to identify alterations in gene expression.

Methods: RNA were isolated from HD and control iPSC-derived brain microvascular endothelial cells. mRNAseq using Illumina TruSeq mRNA PolyA+ v2 lib prep and HiSeq 2500. Statistical difference in mRNA levels were calculated with subsequent GO and pathway analysis.

Results: mRNAseq and statistical analysis revealed differentially expressed genes between HD and control iPSC-derived brain microvascular endothelial cells.

Conclusions: Our study shows differentially expressed genes between HD and control iPSC-derived brain microvascular endothelial cells, and reveals gene networks that are relevant to the mechanism of HD pathogenesis.

overall_design: mRNA profiles of HD and control iPSC-derived brain microvascular endothelial cells were generated by mRNAseq, in duplicate, using Illumina TruSeq mRNA PolyA+ v2 and Illumina HiSeq 2500.

middle: G

first: Jie

last: Wu

first: Leslie

middle: M

last: Thompson


title: Mov10 in developing brain suppresses retroelements and regulates neuronal development and function

city: Urbana

first: Joseph

last: Seimetz

state: Illinois

address: 506 South Mathews Ave.

department: Biochemistry

institute: University of Illinois Urbana-Champaign

summary: RNA helicase MOV10 participates in RNA-induced gene silencing, nonsense-mediated decay and suppression of retrotransposition in cultured cells. We describe a critical role for MOV10 in early postnatal brain, where there is an ~40 fold increase in MOV10 protein levels along with a nucleo-cytoplasmic localization pattern. The isolation of MOV10-associated RNAs from this stage showed a preponderance of retrotransposon encoded RNAs along with messenger RNAs.  Furthermore, reduced MOV10 expression led to an increase in genomic L1 levels in P2 brain. We show that MOV10 likely suppresses retrotransposition by binding to the LINE1 reverse transcriptase and blocking cDNA synthesis. MOV10 also binds cytoskeletal mRNAs and is required for normal neurite outgrowth in both Neuro2A and cultured hippocampal neurons. Finally, standard MOV10 levels in brain are required for normal mouse behavior. We provide the first evidence of a role for MOV10 in retrotransposon control in brain and in neuronal development and function.

overall_design: The analysis includes 8 samples. The wildtype Neuro2a cells have 2 replicates each of differentiated and undifferentiated cells.The remaining 4 are CRISPR-Cas Knockout lines for MOV10 that are have also been  differentiated or left undifferentiated. These samples are also in replicates.

first: Stephanie

last: Ceman


title: Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells via Transcription Factor Conversion of Hemogenic Endothelium


title: Identification of the RNAs that bind MOV10 in postnatal murine brain

state: IL

address: 601 S. Goodwin Ave

department: Cell and Developmental Biology

institute: University of Illinois-Urbana Champaign

summary: These are the repetitve element RNAs and mRNAs found associated with MOV10 by RNA IP (RIP)

overall_design: RNAs were isolated from 28 C57bl/6 mice aged P2 brains. The RNAs immunoprecipitated with MOV10 were compared for enrichment over input RNA by RIPSeeker

first: Geena

last: Skariah


title: Analysis of paired primary and metastatic cell lines identifies common DNA methylation changes in melanoma metastasis.

city: Dunedin

first: Aniruddha

last: Chatterjee

state: Otago

address: 270 Great king Street

department: Pathology

country: New Zealand

institute: University of Otago

type: Methylation profiling by high throughput sequencing

summary: Using 1 melanocyte and 6 melanoma cell line (3 pair of primary and metastatic), we generated base-resolution DNA methylation maps to document DNA methylation drivers of melanoma metastasis.

overall_design: Here we generated single-nucleotide resoultion DNA methylation map of a total of 7 cell lines using Reduced Representation Bisulfite Sequencing (RRBS)

first: Peter

last: Stockwell

first: Micheal

middle: R

last: Eccles

first: Antonio

last: Ahn

first: Euan

middle: J

last: Rodger


title: Chromatin states of tumor-specific T cells

first: Andrea

last: Schietinger


title: Impact of Runx3 deficiency on mature CD8 T cell responses [RNA-seq]

city: Washington

first: Weiqun

last: Peng

state: DC

address: 1918 F Street, NW , DC

institute: The George Washington University

summary: Comparison of transcriptome between control and Runx3-deficient CD8 effector T cells

overall_design: Control mice or those are deficient for Runx3 transcription factors (deleted by CD2-Cre) were crossed to P14 TCR transgene, and naive P14 CD8 T cells were adoptively tranferred into congenic recipient mice followed by infection with LCMV armstrong. Four days later, the donor-derived CD8 effector T cells were sorted for the KLRG1 high and IL-7Ra low phenotype subset. The sorted cells were used for RNAseq analysis.

first: Hai-Hui

last: Xue


title: inDrop single cell RNA-seq of hematopoietic cells derived from human pluripotent stem cells

first: Areum

last: Han

summary: We performed morphogen-directed differentiation of human PSCs into HE followed by combinatorial screening of 26 candidate HSC-specifying TFs for the potential to promote hematopoietic engraftment in irradiated immune deficient murine hosts. We recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that together were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft primary and secondary murine recipients

overall_design: Examination of  expression pattern in hematopoietic cells.

first: Ryohichi

last: Sugimura

first: George

last: Daley


title: Impact of Runx3 deficiency on the epigenome of early effector CD8 T cells

summary: Define how ablating Runx3 affects histone marks and active enhancers in early effector CD8 T cells

overall_design: Wild-type or Runx3-deficient (RKO) naive P14 CD8 T cells were adoptively tranferred into congenic recipient mice followed by infection with LCMV armstrong. Four days later, the donor-derived CD8 effector T cells were sorted for the KLRG1 high and IL-7Ra low phenotype subset. The sorted cells were used for ChIPseq analysis of various histone marks. The genomic occupancy of the Runx-CBF complex was determined by ChIP-Seq of CBF in naive CD8+ T cells and CD8+ effectors, sorted on day 8 post-infection.


title: Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB

city: Norwich

first: Tung

middle: Ba  Khanh

last: Le

state: Norfolk

address: Colney Lane

department: Department of Molecular Microbiology

country: United Kingdom

institute: John Innes Centre

summary: In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.

overall_design: Whole genome sequencing experiments were performed on (i) untreated swarmer cells and double-strand break induced swarmer cellsof Caulobacter crescentus CB15N; (ii) on induced swarmer cells that lack addAB or recA or express a non-cleavable mutant of lexA or recA(K83A); (iii) on induced swarmer cells harboring chi insertions 30kb or 100kb from the break-site; and (iv) on induced swarmer cells treated with mung bean nuclease.     RNA-seq experiment were performed on RNA extracted from (i) wild-type CB15N swarmer cells ; (ii) swarmer cells induced for double-strand break; and (iii) swarmer cells lacking addAB or recA induced for double-strand break

first: A

last: Badrinarayanan

first: T

middle: B

first: J

last: Spille

first: M

middle: T

last: Laub


title: RNA-SEQ assay for wild type and CRISPR induced endoglin knockout human pulmonary artery smooth muscle cells (PASMC)

city: Durham

first: Hongyu

last: Tian

address: B354 LSRC Research Drive

department: Department of Medicine

institute: Duke University

summary: The goal of this study is to investigate the differential transcripted genes affected by CRISPR induced endoglin knockout in PASMC cells.

overall_design: Total RNA was purified from NTC or ENG-/- PASMC cells using RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quality and concentration were assessed with Agilent Tapestation 200 (Agilent Technologies) and Qubit 2.0 (ThermoFisher Scientific).  ~250-500 ng RNA were used for library construction. The NGS libraries were constructed using the KAPA Stranded mRNA-Seq Kits (KapaBioSystems). mRNA was captured using magnetic oligo-dT beads and 1st strand cDNA was synthesized using random priming. In order to preserve strand-specificity, 2nd strand synthesis, which converts the cDNA:RNA hybrid to double-stranded cDNA (dscDNA), was marked by dUTP incorporation. cDNA framents were A-tailed by adding dAMP to the 3'-ends of the dscDNA library fragments. dsDNA Illumina TruSeq "forked”  adapters 3'-dTMP overhangs were then ligated to A-tailed library insert fragments. Each of the six libraries were ligated with a unique Truseq 6bp barcode. Library fragments were amplified using the KAPA HiFi HotStart polymerase. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Fragment length and library quality was assessed on a 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies).  Libraries were diluted to 10nM and pooled at equimolar ratios. The pool was then diluted to 2nM and denatured in NaOH following Illumina recommendations. 10pM of denatured library pool was loaded in one HiSeq lane and flowcell was clustered on the Illumina C-bot. 5% PhiX control was spiked-in. The flowcell was sequenced on a HiSeq 2500 V4 chemistry with 50bp Single read protocol. Data was demultiplexed and Fastq files were generated using BcptoFastq 1.8.4 script provided by Illumina.

first: Gerard

middle: C

last: Blobe


title: Genome-wide maps of Gata6 in primary mouse keratinocytes

city: London

first: Giacomo

last: Donati

address: Guy's Hospital, Great Maze Pond

institute: King's College London

summary: In adult K14ΔNLef1 mouse, the overexpression of ∆NLef1, a ß-catenin dominat negative, in basal keratinocytes leads to the conversion of hair follicles into multilayered epithelial cysts and ectopic sebaceous gland. ∆NLef1 transcriptional activity led to Gata6 overexpression in the pilosebaceous unit in transgenic mice. To uncover direct target genes of Gata6 we performed ChIP-Seq experiments in primary mouse keratinocytes.

overall_design: Examination of Gata6 genomic targets in mouse primary keratinocytes

first: Fiona

last: Watt


title: Gata6 network regulates epidermal junctional zone cell fate.


title: Evolutionary re-wiring of p63 regulatory landscape has both epigenetic and transcriptomic implications and is the underlying cause for epidermal differences between mouse and human [ChIP-seq]

city: Buffalo

first: Satrajit

last: Sinha

address: 701 Ellicott Street

department: Department of Biochemistry

institute: State University of New York at Buffalo

summary: Mapping p63 regulatory and epigenetic landscape in mouse keratinocytes using ChIP-Seq techniques

overall_design: p63 ChIP DNA, histone ChIP DNA were extracted from 2 similar mouse keratinocyte cell-lines (MK1, MK2) and sequenced

first: Isha

last: Sethi

first: Christian

last: Gluck

first: Michael

last: Buck


title: Gene expression signature of Gata6 positive epidermal cells.

summary: To characterize gene expression in Gata6 positive epidermal cells we analyzed a Gata6 reporter mouse in which the endogenous Gata6 promoter drives expression of mTomato. We performed flow cytometry followed by transcriptome analysis. We compared four subpopulations of telogen epidermal cells: Gata6+/Itga6+ cells, Gata6+/itga6- cells, CD34+/Itga6+ cells (which are Gata6-) and all remaining Itga6+ cells (Gata6-/CD34-). The RNA was isolated from age and sex matched mice.

overall_design: We compared four subpopulations of telogen epidermal cells: Gata6+/Itga6+ cells, Gata6+/itga6- cells, CD34+/Itga6+ cells (which are Gata6-) and all remaining Itga6+ cells (Gata6-/CD34-). The RNA was isolated from age and sex matched mice. Three biological replicates for each cell population were analyzed


title: Gene expression signature of the three major epidermal differentiation compartments: interfollicular epidermis (IFE), hair follicle (HF) and sebaceous gland (SG).

summary: There are multiple stem cells in adult mammalian epidermis, but the mechanisms controlling lineage specification are poorly understood. To identify gene expression signatures of the three major epidermal differentiation compartments we micro-dissected individual SG, IFE and HF from adult epidermis. The RNA was isolated from age and sex matched wild-type mice and performed transcriptome analysis with Affymetrix Exon microarrays

overall_design: We micro-dissected individual interfollicular epidermis (IFE), hair follicle (HF) and sebaceous gland (SG) from adult tail epidermis. The RNA was isolated from age and sex matched wild-type mice. Three biological replicates for each epidermal differentiation compartment were analyzed.


title: RNA-seq analysis of primary patient samples to characterize the CNS leukemia

first: Akira

last: Watanabe

summary: We present a detailed genetic, transcriptional and physiological study of leukemic cells isolated from the CNS and bone marrows (BM) of both affected children and xenograft model of B-ALL which recapitulate the key features of CNS involvement. We reveal that leukemic cells in CNS possess distinct hypoxic signature such as proliferation suppression, decrease of mitochondrial oxidative phosphorylation and increase of glycolysis compared with leukemic cells in BM.

overall_design: We compared the gene expression profiles of leukemic cells isolated from CSF and BM of primary 3 patients (ALL#6-8) with RNA-seq.

first: Itaru

last: Kato

first: Hiroki

last: Ozawa

first: Dapeng

last: Wang

first: Masahiro

last: Nakamura


title: Fam60a defines a variant Sin3a-Hdac complex in embryonic stem cells required for self-renewal

city: Dublin

first: Bruce

last: Moran

address: Nutley Lane, Dublin 4

department: Dept. of Pathology

country: Ireland

institute: St. Vincent's University Hospital

summary: RNA-Sequencing (RNA-seq). The aim of this RNA-seq experiment was to monitor the genome-wide transcriptional changes in mouse embryonic stem cells depleted of either Fam60a or Sin3a.

overall_design: RNA-Seq of mRNA level of mESCs depleted for Sin3a and Fam60a.

first: Gundula

last: Streubel

first: Sudipto

last: Das

first: Adrian

last: Bracken


title: The novel quantitative trait locus qTGW3 encodes a GSK3-like kinase and negatively regulates grain size and weight in rice

status: Public on May 15 2017

city: Shanghai

first: Zejun

last: Hu

address: Yangpu District Songhu Road No. 2005

country: China

institute: Fudan University

summary: qTGW3 encodes a GLYCOGEN SYNTHASE KINASE 3 (GSK3)/SHAGGY-like family protein kinase GSK5. We demonstrate that GSK5 interacts with and phosphorylates the AUXIN RESPONSE FACTOR (ARF) transcription factor ARF4. Both GSK5 and ARF4 are mainly expressed in reproductive tissues, and loss-of-function mutation of ARF4 also increases rice grain length and weight. ARF4 functions as a transcriptional repressor in plants and RNA-Seq analysis reveals that, compared to the wild-type plants,  totally 887 and 1179 genes were up-regulated in the GSK5 and ARF4 mutants, respectively. Among them, 373 genes were commonly up-regulated in both mutants. In contrast, there were 681 and 669 genes were down-regulated in the GSK5 and ARF4 mutants, respectively. Among them, 288 genes were commonly up-regulated in both mutants. These results showed that GSK5 and ARF4 have overlapping roles and regulate a common set of genes during rice grain development.

overall_design: Rice grain (7-8 days after flowering) mRNA profiles of wild type (WT), GSK5-edited and ARF4-edited plants were generated by deep sequencing, in triplicate, using Illumina GAIIx.

first: Sun-Jie

last: Lu


title: Epidermal overexpression of ∆NLef1 leads to deregulation of epidermal stem cell fate.

first: Giacomo

last: Donati

summary: In adult K14ΔNLef1 mouse, the overexpression of ∆NLef1, a B-catenin dominat negative, in basal keratinocytes leads to the conversion of hair follicles into multilayered epithelial cysts and ectopic sebaceous gland. To uncover in vivo changes in gene expression associated to ∆NLef1 activity, we compared the expression profiles of unfractionated  keratinocytes  in wild type and K14ΔNLef1 transgenic mice. Cells were collected from 9.5 week old mice, when the hair follicle are in the resting (telogen) phase of the hair growth cycle. At this stage the ∆NLef1 phenotype is not yet evident enabling us to detect early molecular events.

overall_design: We compared the expression profiles of unfractionated keratinocytes in wild type and K14ΔNLef1 transgenic 9.5 week old mice. Three biological replicates for each cell population were analyzed.


title: A Novel Perivascular Cell Population in the Zebrafish Brain

first: Marina

last: Venero Galanternik

address: 6 Center Drive Building 6B - Room 4B-412

department: NICHD

institute: NIH

summary:
Purpose: RNA-seq analysis from isolated zebrafish fluorescent granular perithelial (FGP) cells in adult brains compared to endothelial cells or entire fish.
Method: FGPs and endothelium were isolated by FACS sorting using transgenic line Tg(kdrl:mCherry);Tg(mrc1a:eGFP).

overall_design: Total RNA from isolated FGPs, endothelial cells and whole fish.

first: Brant

last: Weinstein


title: Genome-wide analysis of STAT3 mediated transcription reveals immune disease associated SNPs in STAT3 binding sites

city: Aalto

first: Kari

last: Nousiainen

country: Finland

institute: Aalto University

summary: The development of therapeutic strategies to combat immune-associated diseases requires the molecular mechanisms of human Th17 cell differentiation to be fully identified and understood. To investigate transcriptional control of Th17 cell differentiation we used primary human CD4+ T cells in siRNA-mediated gene silencing and chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) to identify both the early direct and indirect targets of STAT3. The integrated data set presented in this study confirms that STAT3 is critical for transcriptional regulation of early human Th17 cell differentiation. Importantly, we found that a large number of SNPs from loci associated with immune mediated disorders were located at sites where STAT3 binds to induce Th17 cell specification. Introduction of such SNPs alters STAT3 binding in DNA Affinity Precipitation Assays. Overall, our study provides important new insights for modulating Th17-mediated pathogenic immune responses in humans.

overall_design: Examination of STAT3 binding sites

first: Subhash

last: Tripathi

first: Zhi

last: Chen

first: Antti

last: Larjo

first: Tarmo

last: Äijö

first: Isis

last: Ricaño-Ponce

first: Barbara

last: Hrdlickova

first: Soile

last: Tuomela

first: Essi

last: Laajala

first: Verna

last: Salo

first: Vinod

last: Gopalaiah

first: Cisca

last: Wijmenga

first: Harri

last: Lähdesmäki

first: Riitta

last: Lahesmaa


title: Chd2 knockout effect on chromatin remodelling and the expression of developmental genes in mouse embryonic stem cells (RNA-Seq)

city: Fukuoka

first: Yasuyuki

last: Ohkawa

address: 3-1-1 Maidashi

institute: Medical Institute of Bioregulation

summary: We demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in a proportion of nucleosomes of developmentally regulated genes in Chd2-depleted ESCs to enhance histone variant H3.3 enrichment, leading to elevated trimethylation of histone H3 lysine 27.Chd2 is essential to prevent suppressive chromatin formation in developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state.

overall_design: Evaluation of three histone modifications, H3.3 and total H3 in WT and Chd2mut/mut mESCs (ChIP-Seq), and of mRNA expression (RNA-Seq).

first: Yuichiro

last: Semba

first: Akihito

last: Harada

first: Kazumitsu

last: Maehara

first: Jumpei

last: Nogami


title: Chd2 knockout effect on chromatin remodelling and the expression of developmental genes in mouse embryonic stem cells (ChIP-Seq)


title: Runx3 regulates effector CD8 T cell differentiation and enhancer landscape

first: Weiqun

last: Peng

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

overall_design: Refer to individual Series


title: Chd2 knockout effect on chromatin remodelling and the expression of developmental genes in mouse embryonic stem cells


title: Single cell analysis of atherosclerosis baseline and regression macrophages-surgical model

city: Tarrytown

first: Yurong

last: Xin

address: 777 Old Saw Mill River Road

institute: Regeneron Pharmaceuticals, Inc.

summary: CD45+ CD11b+ F4/80+ macrophages were isolated using the BD FACS Aria IIu SORP from pre-transplant baseline (Apoe-/- fed 16 weeks westernd diet) and grafted arches from WT recipient mice (regression). Cells were then sorted using the C1 Fluidigm to analyze their transcriptomes at a single cell level.

overall_design: Our study used the transplant model of atherosclerosis regression for these experiments. Donor Apoe-/- mice were fed a high fat diet for 16 weeks (baseline). Then atherosclerotic aortic arches from donor mice were transplanted into WT on chow (regression). The mice were sacrificed 3 days after transplantation, the aortic arches enzymatically digested to create single cell suspension and antibody stained to label surface markers. We sorted macrophages by selecting CD45+CD11b+ F4/80+ cells and these cells were used for single cell RNA seq.

first: Edward

last: Fisher


title: Genomic characterization of murine monocytes reveals C/EBPb dependence of Ly6C-cells [Single Cell RNA sequence]

city: REHOVOT

first: Steffen

last: Jung

address: Herzl

department: Immunology and Regenerative Biology

country: Israel

institute: The Weizmann Institute of Science

summary: Single cell RNA sequencing of murine circulating blood monocytes under steady state conditions. 2 plates of cx3cr1-cre:rosa26YFP monocytes and 4 plates (3 plates total monocytes and 1 plate Ly6Cint monocytes) were pre-enriched by CD115-biotin MACS and afterwards FACS sorted.

overall_design: Indexed FACS sorting in 384well plates followed by MARS-Seq (Jaitin et al., Science 2014).

first: Mildner

last: Alexander

first: Amit

last: Ido

first: Jung

last: Steffen

first: David

last: Eyal


title: Tight Regulation of the Interaction between Brassica napus and Sclerotinia sclerotiorum at the MicroRNA Level

city: Hangzhou

first: cao

last: jiayi

address: 866 Yu Hang Tang Road

institute: Zhejiang University

type: Non-coding RNA profiling by high throughput sequencing


title: Tight Regulation of the Interaction between Brassica napus and Sclerotinia sclerotiorum at the MicroRNA Level [HTS]

summary: MicroRNAs are multifunctional non-coding short nucleotide molecules. Nevertheless, the role of miRNAs in the interactions between plants and necrotrophic pathogens is largely unknown. Here, we report the identification of the miRNA repertoire of the economically important oil crop oilseed rape (Brassica napus) and those involved in interacting with its most devastating necrotrophic pathogen Sclerotinia sclerotiorum. We identified 280 B. napus miRNA candidates, including 53 novel candidates and 227 canonical members or variants of known miRNA families, by high-throughput deep sequencing of small RNAs from both normal and S. sclerotiorum-inoculated leaves. Target genes of 15 novel candidates and 222 known miRNAs were further identified by sequencing of degradomes from the two types of samples. MicroRNA microarray analysis revealed that 68 miRNAs were differentially expressed between S. sclerotiorum-inoculated and uninoculated leaves. A set of these miRNAs target genes involved in plant defense to S. sclerotiorum and/or other pathogens such as NBS-LRR R genes and nitric oxygen and reactive oxygen species related genes. Additionally, three miRNAs target AGO1 and AGO2, key components of post-transcriptional gene silencing (PTGS). Expression of several viral PTGS suppressors reduced resistance to S. sclerotiorum. Arabidopsis mutants of AGO1 and AGO2 exhibited reduced resistance while transgenic lines over-expressing AGO1 displayed increased resistance to S. sclerotiorum in an AGO1 expression level-dependent manner. Moreover, transient over-expression of miRNAs targeting AGO1 and AGO2 decreased resistance to S. sclerotiorum in oilseed rape. Our results demonstrate that the interactions between B. napus and S. sclerotiorum are tightly regulated at miRNA level and probably involve PTGS.

overall_design: To identify the miRNAome in B.napus, especially those involved in S. sclerotiorum response, one small RNA library was constructed from an equivalent mix of total RNA from S. sclerotiorum-inoculated and uninoculated B. napus leaves and was sequenced via the high-throughput Illumina Solexa platform.Target genes of the miRNAs were further identified by sequencing of degradomes.To understand response of oilseed rape miRNA to S. sclerotiorum, miRNA microarray analyses were performed to obtain miRNA expression profiles at both early (3 hpi) and late (12 hpi) stage after inoculation with S. sclerotiorum.

first: Xin-Zhong

last: Cai

first: Jia-Yi

last: Cao


title: Genomic characterization of murine monocytes reveals C/EBPb dependence of Ly6C-cells [ChIP data]

summary: Chromatin status of murine blood Ly6C+ and Ly6C- monocytes indicates that both population share K4me3 marks (promoters) while Ly6C- monocytes establish de novo enhancers (K4me1, K4me2) during differentiation

overall_design: Monocytes were isolated from murine blood with CD115 biotin MACS and subsequently stained. The cells were fixed and afterwards FACS sorted. 20.000-50.000 monocytes were sorted from each population and analyzed for K4me1, K4me2, K4me3 and K27ac in duplicates (except Ly6Cint monocytes)

first: Alexander

last: Mildner


title: Genomic characterization of murine monocytes reveals C/EBPb dependence of Ly6C-cells [ATAC data]

summary: Analysis of open chromatin by ATAC sequencing in murine blood monocyte subsets

overall_design: 20.000 - 50.000 blood monocytes were isolated in duplicates after CD115 biotin MACS enrichment by FACS sorting and open chromatin regions were identified by transposase accessibility


title: Genomic characterization of murine monocytes reveals C/EBPb dependence of Ly6C-cells [RNA sequence]

summary: We show that monocyte development is accompanied by stepwise RNA expression changes and that Ly6C+ monocytes upon transfer develop into Ly6C- monocytes indicated by a comparable expression profile to host Ly6C- monocytes

overall_design: Examination of RNA expression changes in different monocyte populations

first: teffen


title: Nm-seq finds thousands of modified 2’-O-methylation sites in mRNA with base precision

city: Chicago

first: Chuan

last: He

state: ILLINOIS

address: 929 E 57th St., GCIS E319

department: Department of Chemistry

institute: The University of Chicago

summary:
Nm-seq maps 2'-O-methylation sites in human mRNA with base precision
The ribose of rna nucleotides can be 2′-O-methylated (nm). despite advances in high-throughput detection, the inert chemical nature of nm still limits sensitivity and precludes mapping in mrna. We leveraged the differential reactivity of 2′-O-methylated and 2′-hydroxylated nucleosides to periodate oxidation to develop nm-seq, a sensitive method for transcriptome-wide mapping of nm with base precision. nm-seq uncovered thousands of nm sites in human mrna with features suggesting functional roles.

overall_design:
we developed a conceptually distinct approach based on the different chemical properties of nucleosides with 2′-OH and 2′-OMe22–25, combining enrichment with detection of a positive signal (rather than the lack of signal) to produce a sensitive method suited for discovery of Nm sites in rare RNA molecules or at low stoichiometry. Nm-seq leverages oxidative cleavage of ribose 2′,3′-vicinal diols by periodate to expose, enrich and map Nm sites in the transcriptome without bias and with single-nucleotide precision.
Nm-seq first exposes internal Nm sites in RNA fragments by iterative oxidation–elimination–dephosphorylation (OED) cycles that remove 2′-unmodified nucleotides (one per cycle) in the 3′-to-5′ direction.

first: Dan

last: Dominissini

first: Qing

last: Dai

first: Sharon

last: Moshitch-Moshkovitz

first: Dali

last: Han

first: Nitzan

last: Kol

first: Gideon

last: Rechavi


title: Genomic characterization of murine monocytes reveals C/EBPb dependence of Ly6C-cells

first: Steffen


title: Genome wide study of Methylated regions in mouse medial Prefrontal cortex (mPFC)

city: Esch-sur-Alzette

first: Rashi

last: Halder

address: 7, Avenue des Hauts-Fourneaux

country: Luxembourg

institute: Luxembourg Center for Systems Biomedicine

summary: We investigated the methylated regions using MeDIP in naïve mice mPFC

overall_design: 5 replicates were used for MeDIP and 5 input samples were pooled for sequencing; for RNA seq 3 replicates were used

first: Sanaz

last: Javan


title: RNA-seq analysis in RISP WT and KO hematopoietic stem cells

city: Memphis

first: Jian

last: Xu

state: Tennessee

address: 262 Danny Thomas Place, MS 345

institute: St. Jude Children's Research Hospital

summary: Adult and fetal hematopoietic stem cells (HSCs) display a glycolytic phenotype required to maintain stem cell properties; however, whether mitochondrial respiration is required to maintain HSC function is not known. Here we report that loss of the mitochondrial complex III subunit Rieske iron sulfur protein (RISP), encoded by the Uqcrfs1 gene, in fetal mouse HSCs, results in anemia and prenatal death. RISP null fetal HSCs displayed impaired respiration, resulting in a decreased NAD+/NADH ratio and an increase in glycolysis. While they were viable and able to robustly proliferate in vivo, RISP null fetal HSCs had impaired differentiation, resulting in a depletion of the progenitor pool. RNA-seq analysis revealed widespread changes in gene expression due to loss of RISP in fetal HSCs. Furthermore, RISP null fetal HSCs and progenitors exhibited an increase in both DNA and histone methylation concomitant with increases in 2-hydroxyglutarate (2-HG), a metabolite known to inhibit DNA and histone demethylases. RISP inactivation in adult HSCs also impaired respiration, resulting in a decreased NAD+/NADH ratio, increased glycolysis and loss of quiescence, followed by rapid depletion resulting in severe pancytopenia and lethality. The effect of RISP ablation in fetal or adult HSCs was cell-autonomous. Together, these data indicate that respiration is dispensable for HSC proliferation, but essential for HSC differentiation and maintenance of adult HSC quiescence.

overall_design: RNA-seq was performed to determine gene expression changes in RISP (Uqcrfs1) WT and KO fetal liver HSCs

first: Elena

last: Anso

first: Samuel

last: Weinberg

first: Lauren

last: Diebold

first: Benjamin

last: Thompson

first: Sebastien

last: Malinge

first: Paul

last: Schumacker

first: Xin

last: Liu

first: Yuannyu

last: Zhang

first: Zhen

last: Shao

first: Mya

last: Steadman

first: Kelly

last: Marsh

first: John

last: Crispino

first: Navdeep

last: Chandel


title: RNA-seq transcriptonal profiling in human primary adult erythroid progenitor cells (ProEs) after shRNA-mediated depletion of TFAM and PHB2 expression

summary: The developing erythroid cells require highly coordinated gene expression and metabolism. By comparing the proteomic and transcriptomic changes in human hematopoietic stem/progenitor cells (HSPCs) and lineage-committed erythroid progenitors (ProEs), and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Two principal mitochondrial factors TFAM and PHB2 are tightly regulated at the protein level and indispensable for mitochondria and erythropoiesis. To determine the role of TFAM and PHB2 in mitochondrial function during erythroid development, we employed shRNA-mediated depeltion of TFAM and PHB2 expression in differentiating erythroid cells, and performed RNA-seq transcriptional profiling analysis.

overall_design: Human primary adult erythroblasts were generated ex vivo from CD34+ hematopoietic stem/progenitor cells (HSPCs) using a serum-free two-phase liquid culture system. CD34+ HSPCs were transduced with lentiviruses containing shRNAs against TFAM, PHB2, or the non-targeting (shNT) control, selected and differentiated to proerythroblasts (ProEs). Cells were harvested at day 5 of erythroid differentiation and total RNA were extracted for RNA-seq analysis.


title: Quantitative Proteomic and Transcriptomic Analysis Reveals Post-Transcriptional Regulation of Mitochondrial Biogenesis during Erythropoiesis


title: ChIP-seq analysis of H3K27ac in K562 cells with shRNA-mediated depeltion of TFAM expression

summary: The developing erythroid cells require highly coordinated gene expression and metabolism. By comparing the proteomic and transcriptomic changes in human hematopoietic stem/progenitor cells (HSPCs) and lineage-committed erythroid progenitors (ProEs), and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Two principal mitochondrial factors TFAM and PHB2 are tightly regulated at the protein level and indispensable for mitochondria and erythropoiesis. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, defective mitochondria function and erythropoiesis.

overall_design: To examine the global effects of histone hyperactylation in Tfam-deficient erythroid cells, we performed ChIP-seq analysis of H3K27ac in human K562 cells transduced with TFAM-specific or control (shNT) shRNAs.


title: RNA-seq transcriptonal profiling in E13.5 fetal liver erythroid cells from Tfam WT and KO mice

summary: The developing erythroid cells require highly coordinated gene expression and metabolism. By comparing the proteomic and transcriptomic changes in human hematopoietic stem/progenitor cells (HSPCs) and lineage-committed erythroid progenitors (ProEs), and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Two principal mitochondrial factors TFAM and PHB2 are tightly regulated at the protein level and indispensable for mitochondria and erythropoiesis. To determine the role of TFAM in mitochondrial function during erythroid development, we generated Tfam conditional knockout (KO) mice by an erythroid-specific EpoR-Cre allele. We isolated the CD71+Ter119+ embryonic day (E)13.5 fetal liver erythroid cells by FACS sorting, and performed RNA-seq transcriptional profiling analysis.

overall_design: CD71+Ter119+ embryonic day (E)13.5 fetal liver erythroid cells were isolated by FACS sorting. Total RNA were extracted and processed for RNA-seq transcriptional profiling analysis.


title: RNA-seq transcriptional profiling in human primary fetal and adult CD34+ hematopoietic stem/progenitor cells (HSPCs) erythroid progenitor cells (ProEs)

summary: Advances in sequencing-based genomic profiling present a new challenge of explaining how changes in DNA/RNA are translated into proteins linking genotypes to phenotypes. The developing erythroid cells require highly coordinated gene expression and metabolism, and serve as a unique model in dissecting regulatory events in development and disease. Here we compare the proteomic and transcriptomic changes in human hematopoietic stem/progenitor cells and lineage-committed erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Two principal mitochondrial factors TFAM and PHB2 are tightly regulated at the protein level and indispensable for mitochondria and erythropoiesis. mTORC1 signaling is progressively enhanced to promote translation of mitochondrial proteins during erythroid specification. Genetic and pharmacological perturbation of mTORC1 or mitochondria impairs erythropoiesis. Our studies suggest a new mechanism for regulation of mitochondrial biogenesis through mTORC1-mediated protein translation, and may have direct relevance to the hematological defects associated with mitochondrial diseases and aging.

overall_design: Transcriptional profiling in human primary fetal and adult CD34+ hematopoietic stem/progenitor cells (HSPCs) erythroid progenitor cells (ProEs) by RNA-seq analysis.


title: Transcriptional profiling of mouse and hamster oocytes

city: Zagreb

first: Vedran

last: Franke

address: Horvatovac 102a

department: Department of biology

country: Croatia

institute: University of Zagreb

summary: We profiled transcriptomes from mouse GV, MII oocytes and 1 cell embryos and Hamster oocytes.

sample_organism:
Mesocricetus auratus
Mus musculus

platform_organism:
Mesocricetus auratus
Mus musculus

overall_design: Examination of gene expression in different stages of oogenesis and early development

first: Petr

last: Svoboda


title: LSD1 inhibition induces neurodegeneration and provides a common link to Alzheimer's disease and Frontotemporal dementia

status: Public on May 13 2017

city: Atlanta

last: Barwick

state: GA

address: 1365 Clifton Rd. NE WCI-C 4th Floor Benches 36-37

department: Hematology and Medical Oncology

institute: Emory University

summary: Alzheimer’s disease (AD) and Frontotemporal dementia (FTD) are the two most common forms of dementia that occur during aging. Both AD and FTD are associated with the pathological aggregation of proteins. Nevertheless, it remains unclear how these protein aggregates lead to neuronal cell death in these dementias. Here we show that the histone demethylase LSD1 is mislocalized with cytoplasmic aggregates in human cases of AD and FTD. In addition, loss of LSD1 systemically in adult mice is sufficient to recapitulate many aspects of these diseases. From these data, we propose that the aggregation of Tau and TDP-43 lead to neuronal cell death in AD and FTD by interfering with the continuous requirement for LSD1 to repress inappropriate transcription. Furthermore, we observe the inappropriate reactivation of stem cell loci in the hippocampal neurons of LSD1 mutant mice. This suggests that LSD1 may function to maintain the fate of differentiated neurons by repressing stem cell transcription.

overall_design: Hippocampus tissue was compared between Lsd1-sufficient (Lsd1(fl/fl)) and Lsd1-deficient mice (Cre-ER(TM)Lsd1(fl/fl))

first: David

last: Katz

first: Michael

last: Christopher


title: Altered Ca+2 homeostasis induces Calpain-Cathepsin axis activation in sCJD.

city: Göttingen

first: Ashish

last: Rajput

state: Niedersachsen

address: Von-Siebold-Straße 3A

department: Research Group for Computational Systems Biology

country: Germany

institute: German Center for Neurodegenerative Diseases (DZNE)

summary: Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent form of human prion disease and it is characterized by the presence of neuronal loss, spongiform degeneration, chronic inflammation and the accumulation of misfolded and pathogenic prion protein (PrPSc). The molecular mechanisms underlying these alterations are largely unknown, but the presence of intracellular neuronal calcium (Ca+2) overload, a general feature in models of prion diseases, is suggested to play a key role in prion pathogenesis.  Here we describe the presence of massive regulation of Ca+2 responsive genes in sCJD brain tissue, accompanied by two Ca+2-dependent processes: endoplasmic reticulum stress and the activation of the cysteine proteases Calpains 1/2. Pathogenic Calpain activation in sCJD is linked to the cleavage of their cellular substrates, impaired autophagy and lysosomal damage, which is partially reversed by Calpain inhibition in a cellular prion model. Calpain 1 treatment enhances seeding activity of PrPSc in a prion conversion assay. Neuronal lysosomal impairment caused by Calpain over activation leads to the release of the lysosomal protease Cathepsin S that in sCJD mainly localises in axons. Additionally, massive Cathepsin S overexpression is detected in microglial cells. Alterations in Ca+2 homeostasis and activation of Calpain-Cathepsin axis already occur at pre-clinical stages of the disease as detected in a humanized sCJD mouse model.   Altogether our work indicates that unbalanced Calpain-Cathepsin activation is a relevant contributor to the pathogenesis of sCJD at multiple molecular levels and a potential target for therapeutic intervention.

overall_design: To identify differentially expressed genes during development of sCJD pathology we analysed the expression levels in the cortical region of tg340-PRNP129MM mice infected with sCJD MM1 brain homogenates at pre-clinical (120 dpi) and clinical (180 dpi) stages.

first: Franc

last: Llorens

first: Katrin

last: Thüne

first: Beata

last: Sirkoska

first: Matthias

last: Schmitz

first: Maria

last: Cramm

first: Nadine

last: Gotzmann

first: Waqas

last: Tahir

first: Margarita

last: Carmona

first: Olivier

last: Andréoletti

first: Uwe

last: Michel

first: Stefan

last: Bonn

first: Pawel

last: P. Liberski

first: Juan

last: Espinosa

last: Torres

first: Isidre

last: Ferrer

first: Inga

last: Zerr


title: Androgen receptor agonists increase muscle mass and function in preclinical models of DMD

status: Public on May 12 2017

first: Daniel

middle: Lee

last: Johnson

address: 71 S Manassas

department: Molecular Informatics Core

institute: UTHSC

summary: Mdx mice were castrated and treated with selective androgen receptor modulator (SARM) GTx-026. Ten weeks after treatment, gastrocnemious muscle were isolated, RNA extracted, and sequenced using ion torrent sequencer.

overall_design: n=3/group. Group-1 wildtype mice, group-2 mdx mice treated with vehicle, and group-3 mdx mice treated with 10 mg/kg s.c. GTx-026

first: Ramesh

last: Narayanan


title: Analysis of gene expression changes in the cranial base (chondrocranium) of Memo1 mutant embryos versus controls

city: Iowa City

first: Eric

last: Van Otterloo

state: Iowa

address: 801 Newton Rd

institute: University of Iowa

summary: We report the gene expression changes in the embryonic day (E) 15.5 mouse cranial base associated with loss of Memo1, as assessed by next-generation RNA-sequencing

overall_design: E15.5 cranial base samples dissected from 3 control and 3 Memo1 mutant mouse embryos - total RNA extracted using Norgen Biotek Total RNA Purification Kit

first: Trevor

last: Williams


title: Transcriptome analysis in HT29 and SW480 cells depleted of Prdx2

city: Daejeon

first: Yukyung

last: Jun

address: 245 Daehak-ro, Yuseong-gu

department: Center for Supercomputing Applications

country: South Korea

institute: Korea Institute of Science and Technology Information

summary: Prdx2 is the thioredoxin-dependent peroxidase that reduces H2O2 using reducing power NADPH in the presence of thioredoxin and thioredoxin reductase. Prdx2 plays an important role in growth. factor signaling in mammlian cells. Therefore, we examined the gene expression in colon adenocarcinoma cell line HT29 after Prdx2 depletion. Prdx2 depletion resulted in a significant alteration on gene expression, including protein synthesis, metabolisms, and cell cycle.

overall_design: Control-siRNA-transfected versus PRDX2-siRNA-transfected HT29 and SW480 cells

first: Dong

last: Kang

first: Eunkyung

last: Lee

first: Sang


title: Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia

city: Oxford

first: Claus

last: Nerlov

address: John Radcliffe Hospital, University of Oxford

department: MRC Molecular Haematology Unit

institute: Weatheral Institute of Molecular Medicine

summary: Single-cell whole transcriptome analysis of  chronic myeloid leukemia stem cells, defined as Lin-CD34+CD38-.

overall_design: We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 single cells from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML stem cells, including the identification of a subgroup of CML stem cells with a distinct molecular signature that selectively persisted during prolonged therapy. We performed differential gene expression and the gene set enrichment analyses of BCR-ABL+ vs BCR-ABL- single cells from either patients or normal donors, analyzed at different stages of the disease.

first: Supat

last: Thongjuea

first: Alice

last: Giustacchini

first: Sten

middle: E

last: Jacobsen

first: Adam

last: Mead


title: Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes in PB- and PB-ID2-transfected 46C mouse embryonic stem cells

status: Public on May 11 2017

city: Hefei

first: Mengmeng

last: Guo

address: Anhui University No.111 Jiulong Road  Hefei City Anhui Prov China

institute: Anhui University

summary: Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes in 46C mouse embryonic stem cells overexpressing PB-ID2 or PB empty vector at gene expression level. Results provide important information of the response of up-regulating ID2, such as specific mechano-responsive genes, up- or down-regulated genes that were mainly enriched in signaling pathways regulating pluripotency of stem cells

overall_design: Total RNA obtained from ID2-overexpressed 46C ESCs compared to empty vector-transfected control 46C ESCs. Cells were cultured in serum medium with LIF

first: Shoudong

last: Ye


title: RNA-seq of HEK293T cells overexpressing TET1-FL or TET1-ALT

city: Camden

first: Jaroslav

last: Jelinek

state: NJ

address: 403 Haddon Avenue

department: Research Labs

institute: Coriell Institute for Medical Research

summary: TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine, resulting in regulation of DNA methylation and gene expression.  Full length TET1 (TET1-FL) has a CXXC domain that binds to unmethylated stretches of CpG dinucleotides known as CpG islands (CGIs).  This CXXC domain allows TET1 to protect CGIs from aberrant methylation but it also limits its ability to regulate genes outside of CGIs.  Here we report a novel isoform of TET1 (TET1-ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site.  Importantly, TET1-ALT lacks the CXXC domain but retains the catalytic domain. To identify the gene expression targets of TET1-ALT  and to compare them to the gene expression targets of TET1-FL, we performed RNA-seq on HEK293T cells that overexpress either Empty Vector (Control), TET1-FL or TET1-ALT.

overall_design: Biological triplicates were performed for a total of 9 samples. Fold change of each gene was calculated by comparing changes in expression after overexpression compared to the empty vector control samples

first: Charly

last: Good

first: Jozef

last: Madzo

first: Bela

last: Patel

first: Nora

last: Engel

first: Jean-Pierre

last: Issa

first: Shinji

last: Maegawa


title: Lineage-specific functions of TET1 in the post-implantation mouse embryo

city: Leuven

first: Rekin's

last: Janky

address: Herestraat 49 Box 816

department: Nucleomics Core

country: Belgium

institute: VIB

summary: The TET dioxygenases erase mediate DNA dedemethylation in pre-implantation embryos and in primordial germ cells, yet limited studies address their contribution to the global gain of DNA methylation following implantation. Here, we show that Tet1 is expressed and non-redundantly contributes to 5-hydroxymethylctyosine (5hmC) non-redundantly in the pre-gastrulation mouse epiblast. Ablation of Tet1 in primed epiblast cells results in widespread loss of 5hmC associated with gain of 5-methylcytosine at CpG islands and promoters.  Moreover, Tet1 is expressed, albeit at lower levels, in the extra-embryonic ectoderm. Tet1-deficiency in the pre-streak mouse embryos causes dysregulation of early lineage regulators in the epiblast and increased expression of metabolic genes in the extra-embryonic ectoderm. Our studies reveal a distinct role of Tet1 in regulating the methylome landscape of the post-implantation mammalian epiblast and a hitherto unknown gene repressive effect in the extra-embryonic lineage, providing insights into the early developmental origins of epigenetic-based basis of imprinting and developmental disorders.

overall_design: Lysates of E6.25 epiblast (E) and extra-embryonic (ExE) tissues were pooled, based on retrospective genotyping, from at least 6 embryos (collected from 2-3 litters). Four pooled biological replicates were collected for each tissue compartment and each genotype, i.e. wild type (wt), Tet1gt/wt (Zwt), Tet1gt/gt(ZZ); in 3 of 4 pooled replicates, the E and ExE samples were obtained from the same litters and given the same numbering. One ExE sample of ZZ was lost.  RNA was purified using the RNeasy micro Kit (Qiagen) to obtain 1-10 ng of RNA per pool. RNA quality was assessed using a Bioanalyzer 6000 Pico chip and only samples with RNA integrity number (RIN) > 8.0 were processed further. First-strand cDNA synthesis and amplification was performed by using the SMARTer Ultra Low Input RNA amplification kit v3 (Clontech). Libraries were prepared using NEBNext Ultra DNA library prep for Illumina and sequenced on NextSeq 500 in high-output to generate 75-bp single-end reads.  Low quality ends and adapter sequences were trimmed off from the Illumina reads with FastX 0.0.13 and cutadapt 1.7.1. Using FastX and ShortRead 1.16.3, we filtered subsequently small reads (length < 35 bp), polyA-reads (>90% of the bases equal A), ambiguous reads (containing N) and low quality reads (>50% of the bases < Q25).

first: Kian

last: Koh

first: Rita

last: Khoueiry

first: Abhishek

last: Sohni


title: RNAseq analysis of murine brainstem gliomas with and without H3.3K27M

city: New York

first: Oren

middle: Josh

last: Becher

state: NY

address: 1468 Madison Avenue, A21-02

department: Pediatrics

institute: Mount Sinai

summary: Using the RCAS/tv-a system, we induced murine brainstem gliomas (PDGF-B; p53 loss using RCAS-Cre with and without H3.3K27M) in Nestin tv-a; p53 floxed mice

overall_design: Three independent tumors with H3.3K27M (PKC) and three independent tumors without H3.3K27M (PDC) were snap frozen, RNA extracted, and RNAseq analysis was performed (PDGF-B; H3.3K27M; p53 loss vs. PDGF-B; p53 loss)

first: Francisco

last: Cordero


title: Circadian networks in human embryonic stem cell-derived cardiomyocytes

city: Utrecht

first: Pieterjan

last: Dierickx

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: Cell-autonomous circadian oscillations strongly influence tissue physiology and pathophysiology of peripheral organs. Recent in vivo findings in the heart demonstrate that the circadian clock controls oscillatory gene expression programs in the adult myocardium. However, whether in vitro human embryonic stem (ES) cell-derived cardiomyocytes can establish circadian rhythmicity is unknown. Here we report that while undifferentiated human ES cells do not possess a functional clock, oscillatory expression of known core clock genes emerges during directed cardiac differentiation, with robust rhythms in day 30 cardiomyocytes. Our data reveal a stress related oscillatory network of genes that underlies a time-dependent response to doxorubicin, a frequently used anti-cancer drug with cardiotoxic side effects. These results provide a set of oscillatory genes that is relevant to functional cardiac studies and that can be deployed to uncover the potential contribution of the clock to other processes such as cardiac regeneration.

overall_design: Human embryonic stem cells (ES cells) were differentiated via a directed differentiation protocol in vitro towards cardiomyocytes for a period of 30 days. Cardiomyocytes were synchronized with dexamethasone and triplicate samples for RNA extraction and sequencing were taken every 4 hours for 48 hours in total. RNA was then extracted using TRIzol, barcoded and amplified following the CEL-Seq protocol.

first: Marit

last: Vermunt

first: Mauro

last: Muraro

first: Menno

last: Creyghton

first: Pieter

middle: A

last: Doevendans

first: Alexander

last: van Oudenaarden

first: Niels

last: Geijsen

first: Linda

last: Van Laake


title: Expression Profiling of Term Placenta in Viviparous Mammals by RNA-Seq

city: Urbana

first: Don

middle: Leigh

last: Armstrong

address: 1206 W Gregory Drive

department: Institute for Genomic Biology

institute: University of Illinois at Urbana Champaign

summary: The placenta is arguably the most anatomically variable organ in mammals. Understanding gene expression patterns among placentas from multiple species can illuminate the molecular basis of placenta function as well as potentially enrich our knowledge of the biology of obstetrical syndromes. We measured the expression profiles of the term placenta of multiple therian mammals using RNA-Seq. We identified multiple genes and transcripts which are differentially expressed across lineages including prolactin, relaxin, and galectins, as well as identifying the non-housekeeping genes which are core to placenta function (such as components of annexin complexes) in all species studied.

sample_organism:
Dasypus novemcinctus
Ateles fusciceps
Pan paniscus
Canis lupus familiaris
Loxodonta africana
Bos taurus
Mus musculus
Monodelphis domestica

overall_design: RNAseq data from term placentas of 8 species with one sample per species

middle: L

first: Derek

last: Wildman


title: Identifying Novel Therapeutic Targets by Combining Transcriptional Data with Ordinal Clinical Measurements

city: Cambridge

first: Brook

last: Wassie

state: MA

address: 77 Massachusetts Ave.

institute: Massachusetts Institute of Technology

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

overall_design: Refer to individual Series


title: The role of PCNA ubiquitylation during unperturbed genome replication in S.pombe

city: Tokyo

first: Yasukazu

last: Daigaku

address: 3-8-31 Ariake Koto-ku

department: Cancer institute

institute: Japanese Foundation for Cancer Research

summary: To establish if PCNA ubiquitylation affects the DNA replication kinetics of specific loci, we compared rates of  DNA replication across the genome between rad18+ and rad18-delta cells using the BrdU-IP sequencing method.

overall_design: We mapped BrdU-incorporation at 300-bp resolution and subsequent informatics analysis was performed to generate datasets of replication kinetics.

first: Antony

last: Carr


title: RNA-seq Analysis of Tcf1-regulated Genes in Human ESCs

first: shoudong

last: ye

address: No 111, Jiu Long Road

department: Cell Biology

summary: Purpose:The goal of this study is to analyze the candidate genes motified by Tcf1 to regulate the human embryonic stem cell (ESC) fate through RNA-seq approach.

overall_design: HES2 human ESCs expressing PB or PB-Tcf1 were cultured in 10% KSR basal media supplemented with 10 ng/ml FGF2, 10 ng/ml Activin A and 4 µM IWR1 for two passages and then collected by using Trizol . These samples were sent to BGI (China) company to do deep sequencing by using BGISEQ-500.

first: Shoudong

last: Ye


title: ChIP-seq dataset for H3K9ac in Identifying Novel Therapeutic Targets by Combining Transcriptional Data with Ordinal Clinical Measurements

summary: The widespread adoption of transcriptional profiling has led to immense repositories of data that may contain critical insights for developing new therapies. Current approaches to mining these data largely rely on binary classifications of disease versus control.  These methods are not able to incorporate measures of disease severity, which are often available. We report an analytical approach to integrate ordinal clinical information with transcriptomics. We have applied this method to public data for a large cohort of Huntington’s disease patients and controls, identifying and prioritizing phenotype-associated genes. To validate the approach, we have performed viability, siRNA knockdown, mRNA-seq, and ChIP-seq on striatal precursor cells expressing either full-length wild type (STHdh Q7) or mutant huntingtin (STHdh Q111). We have verified the role of a high-ranked gene in dysregulation of sphingolipid metabolism in the disease and demonstrated that inhibiting the enzyme, SPL, has neuroprotective effects in HD models. We have shown that one consequence of inhibiting SPL is the intracellular inhibition of HDACs, thus linking our observations in sphingolipid metabolism to a well-characterized HD pathway. Our approach is easily applied to any data with ordinal clinical measurements, and may deepen our understanding of disease processes.

overall_design: Wild type cells were treated with serum-free DMEM for 24 hours (Q7 SST). Cells expressing mutant huntingtin were treated with either serum-free DMEM with vehicle (Q111 SST) or serum-free DMEM supplemented with 4mM 4-deoxypyridoxine hydrochloride (Q111 DOP). H3K9ac ChIP was performed on two replicates for each condition and sequenced with Illumina HiSeq 2000 platform.

first: Leila

last: Pirhaji

first: Pamela

last: Milani

first: Ernest

last: Fraenkel


title: Comparative transcriptome profiling of transgenic Arabidopsis thaliana lines expressing the Hyaloperonospora arabidopsidis candidate effector HaRxL106 and knock-out mutants of HaRxL106-interacting host proteins

city: Norwich

first: Ghanasyam

last: Rallapalli

address: Norwich Research Park

institute: The Sainsbury Laboratory

summary: To prevent activation of plant innate immunity the oomycete pathogen Hyaloperonospora arabidopsidis translocates effector proteins into infected cells of its host Arabidopsis thaliana. We noticed that some H. arabidopsidis effectors, when over-expressed in A. thaliana, render the plant more susceptible to infection by biotrophic pathogens (Fabro et al., 2011, PubMed PMID: 22072967). Here we performed transcriptome profiling of a representative transgenic line constitutively expressing H. arabidopsidis effector HaRxL106. We compared the transcriptomes of A. thaliana wild-type (Col-0) plants and an isogenic line expressing HaRxL106 before pathogen challenge and 24 h after infection with the compatible bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000. HaRxL106 interacts with several Arabidopsis proteins (Mukhtar et al., 2011, PubMed PMID: 21798943; Wirthmueller et al., 2015, PubMed PMID: 25284001). To test whether the HaRxL106-interacting A. thaliana proteins MODIFIER OF SNC1, 6 (MOS6), 6B-INTERACTING PROTEIN 1-LIKE 1 (ASIL1) or RADICAL-INDUCED CELL DEATH1 (RCD1) are altered in their transcriptional response to a biotrophic pathogen we performed transcriptome profiling of mos6-1, asil1-1 and rcd1-1 mutants before and 24 h after infection with P. syringae pv. tomato DC3000.

overall_design: Five weeks old Arabidopsis plants (Col-0, HaRxL106-overexpressing Col-0 plants, mos6-1, asil1-1 and rcd1-1 mutants) were either left untreated (untreated), infiltrated with P. syringae pv. tomato DC3000 (DC3000) or infiltrated with 10 mM MgCl2 buffer (Mock) at 12:00 (4h after lights on). All samples were harvested 24 hours later for total RNA extraction. mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol. Each biological replicate set of samples were sequenced as one batch (biorep1, biorep2 and biorep3 in filenames).

first: Lennart

last: Wirthmueller

first: Shuta

last: Asai

first: Georgina

last: Fabro

first: Jonathan

middle: D

last: Jones


title: RNA-seq dataset for Identifying Novel Therapeutic Targets by Combining Transcriptional Data with Ordinal Clinical Measurements

overall_design: Wild type cells were treated with serum-free DMEM for 24 hours (Q7 DOP). Cells expressing mutant huntingtin  were treated with either serum-free DMEM with vehicle (Q111 SST) or serum-free DMEM supplemented with 4mM 4-deoxypyridoxine hydrochloride (Q111 DOP).


title: Macrophage function in tissue repair and remodeling requires IL-4/IL-13 with apoptotic cells [Run2]

city: New Haven

first: Lidia

last: Bosurgi

state: Connecticut

address: 300 Cedar street

department: Immunobiology

institute: Yale University

summary: Tissue repair is a subset of a broad repertoire of IL-4/IL-13-dependent host responses during helminth infections. Here, we show that IL-4/IL-13 alone were not sufficient, but IL-4/IL-13 together with apoptotic cells induced the tissue repair program in macrophages. Genetic ablation of sensors of apoptotic cells impaired the proliferation of tissue-resident macrophages and induction of anti-inflammatory/tissue repair genes in the lung following helminth infection or the damage caused by induction of colitis in the gut. In contrast, recognition of apoptotic cells was dispensable for cytokine-dependent induction of pattern recognition receptor, cell adhesion or chemotaxis genes in macrophages. Detection of apoptotic cells can therefore spatially compartmentalize or prevent premature or ectopic activity of pleiotropic, soluble cytokines, such as IL-4/IL-13.

overall_design: RNA-seq of bone marrow-derived macrophages stimulated with apoptotic neutrophils, IL-4, or both

first: Y

last: Cao

first: Mar

last: Cabeza-Cabrerizo

first: Andrea

last: Tucci

first: Lindsey

last: Hughes

first: Yong

last: Kong

first: Jason

middle: S

last: Weinstein

first: Paula

last: Licona-Limon

first: Edward

last: Schmid

first: Facundo

last: Pelorosso

first: Nicola

last: Gagliani

first: Joseph

last: Craft

first: Richard

last: Flavell

first: Sourav

last: Ghosh

first: Carla

middle: V

last: Rothlin


title: Macrophage function in tissue repair and remodeling requires IL-4/IL-13 with apoptotic cells [Run1]

overall_design: RNA sequencing of lung resident macrophages from WT and Axl-/-Mertk-/- mice upon infection with N. brasiliensis

middle: G


title: A practical solution for preserving single cells for RNA sequencing

first: Eshita

last: Sharma

state: Oxfordshire

address: Roosevelt Drive, Headington

department: Bioinformatics

institute: Wellcome Trust Centre for Human Genetics

summary: We present a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomant's Reagent, to stabilise cell samples for single-cell transcriptomic applications.

overall_design: Single-cell RNAseq from 2,3 and 7 day old DSP fixed cells were compared with RNAseq from fresh cells

first: Moustafa

last: Attar

first: Rory

last: Bowden


title: Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures

city: South San Francisco

first: Christopher

middle: John

last: Bohlen

state: CA

address: Room 15-2006, Genentech Building 15

department: Neuroscience

institute: Genentech

summary: Microglia, the resident macrophages of the central nervous system (CNS), engage in various CNS-specific functions that are critical for development and health. To better study the properties that distinguish microglia from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly purified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components (CSF-1/IL-34, TGF-b2, and cholesterol). Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic change in intrinsic phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression profiles after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia require continuous instructive signaling from the intact CNS.  This RNA-seq dataset describes changes in rat microglial transcriptome in culture with or without serum exposure.

overall_design: Three to six biological replicates per group were collected: Freshly isolated P21 rat microglia, P21 rat microglia 8 days in culture in serum-free TIC medium, P21 rat microglia 7 days in culture in serum-free TIC medium and 1 day supplemented with 10% fetal calf serum (FCS), P21 rat microglia 5 days in culture in serum-free TIC medium and 3 days supplemented with 10%  FCS, or P21 rat microglia 3 days in culture in serum-free TIC medium and 5 days supplemented with 10%  FCS.

first: Hannah

middle: Y

last: Collins

first: Ben

last: Barres


title: A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer

first: Jaroslav

last: Jelinek

summary: TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine, resulting in regulation of DNA methylation and gene expression.  Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated stretches of CpG dinucleotides known as CpG islands (CGIs).  This CXXC domain allows TET1 to protect CGIs from aberrant methylation but it also limits its ability to regulate genes outside of CGIs.  Here we report a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site.  Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is silenced in ESCs but becomes activated in embryonic and adult tissues while TET1FL is expressed in ESCs, but repressed in adult tissues.  Overexpression of TET1ALT shows production of 5-hydroxymethylcytosine with distinct (and weaker) effects on DNA methylation or gene expression when compared to TET1FL. TET1ALT (but not TET1FL) is aberrantly activated in multiple cancer types including breast cancer, lung cancer and lymphomas. Our data suggest that the predominantly expressed isoform of TET1 in cancer cells does not protect from CGI methylation and likely mediates dynamic site specific demethylation outside of CGIs.

overall_design: Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of human genomic DNA from HEK293T cell with empty vector, TET1-FL and TET1-ALT overexpression. Briefly, genomic DNA was sequentially digested by SmaI and XmaI, which both recognize the sequence CCCGGG. SmaI is methylation sensitive, where XmaI is methylation insensitive. Distinct signatures, 5’-GGG at unmethylated sites or 5’-CCGGG at methylated sites were created by enzyme digestion and ultimately high througput sequencing was used to map these sites to the genome. Methylation ratios for each individual CCCGGG site were calculated as a proportion of methylated counts to the sum of unmethylated and methylated counts, and subsequently adjusted for differences in restriction enzyme efficiency using values obtained from spiked in standards.

first: Charly

middle: R

last: Good

first: Jozef

last: Madzo

first: Bela

last: Patel

first: Nora

last: Engel

first: Jean-Pierre

middle: J

last: Issa

first: Shinji

last: Maegawa


title: ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator

city: Mainz

first: Sergi

last: Sayols

address: Ackermannweg 4

institute: Institute of Molecular Biology, Mainz


title: The INO80 complex removes H2A.Z to promote presynaptic filament formation during homologous recombination

status: Public on May 14 2017

city: Martinsried

first: Claudio

middle: Amadeo

last: Lademann

address: Am Klopferspitz 18

institute: Max Plack Institute of Biochemistry

summary: INO80 is an evolutionary conserved nucleosome remodelling complex that acts in transcription, replication and genome stability. It is required for resistance against genotoxic agents and involved in the repair of DNA double-strand breaks (DSB) by homologous recombination (HR). However, causes underlying the HR defect of INO80-C mutant cells have been controversially discussed. We here unite previous findings using a high-resolution system to study HR in the yeast Saccharomyces cerevisiae. We find that INO80-C has at least two distinct functions during HR, which promote DNA end resection and presynaptic filament formation, respectively. Importantly, specifically the second function is linked to the histone variant H2A.Z. In the absence of H2A.Z, presynaptic filament formation and HR are restored in INO80-C-deficient mutants, suggesting that presynaptic filament formation is the crucial INO80-C function during HR.

overall_design: (ChIP) DNA was analyzed following different time points after the induction of a site-specific DSB in the genome of Saccharomyces cerevisiae.

first: Boris

last: Pfander


title: ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator [RNA-seq]

summary: Here we show that ZBTB48 binds directly both to telomeric as well as to subtelomeric variant repeat sequences. ZBTB48 is found at telomeres of human cancer cells regardless of the mode of telomere maintenance and it acts as a negative regulator of telomere length. In addition to its telomeric function, we demonstrate through a combination of RNAseq, ChIPseq and expression proteomics experiments that ZBTB48 acts as a transcriptional activator on a small set of target genes, including mitochondrial fission process 1 (MTFP1). This discovery places ZBTB48 at the interface of telomere length regulation, transcriptional control and mitochondrial metabolism.

overall_design: ZBTB48 WT clones were generated by single sorting parental cells (HeLa & U2OS pools) into 96 well plates. ZBTB48 KO clones were generated by transiently transfecting a ZBTB48 TALEN pair (H12250; from TALEN library Resource, Seoul National University) into HeLa and U2OS cells, followed by single sorting into 96 well plates. Clones were first phenotypically screened for absence of ZBTB48 IF staining and subsequently verified by sequencing the exact genomic alterations across the TALEN cut site. Counts provided are not normalized.

first: Dennis

last: Kappei

first: Falk

last: Butter


title: ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator [ChIP-seq]

overall_design: ZBTB48 WT clones were generated by single sorting parental cells (HeLa & U2OS pools) into 96 well plates. ZBTB48 KO clones were generated by transiently transfecting a ZBTB48 TALEN pair (H12250; from TALEN library Resource, Seoul National University) into HeLa and U2OS cells, followed by single sorting into 96 well plates. Clones were first phenotypically screened for absence of ZBTB48 IF staining and subsequently verified by sequencing the exact genomic alterations across the TALEN cut site. Anti-ZBTB48 antibodies from HPA030417 (Atlas Antibodies) & GTX118671 (Genetex) were used in our ChIP-seq experiments.


title: Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Osr2−/− Tooth Mesenchyme Transcriptomes

city: Cincinnati

first: Rulang

last: Jiang

state: Ohio

address: 3333 Burnet Avenue

department: Developmental Biology

institute: Cincinnati Children's Hospital Medical Center

summary: Mutations in MSX1 cause craniofacial developmental defects, including tooth agenesis, in humans and mice. Previous studies suggest that Msx1 activates Bmp4 expression in the developing tooth mesenchyme to drive early tooth organogenesis. Whereas Msx1−/− mice exhibit developmental arrest of all tooth germs at the bud stage, however, mice with neural crest-specific inactivation of Bmp4 (Bmp4ncko/ncko), which lack Bmp4 expression in the developing tooth mesenchyme, showed developmental arrest of only mandibular molars. We recently demonstrated that deletion of Osr2, which encodes a zinc finger transcription factor expressed in a lingual-to-buccal gradient in the developing tooth bud mesenchyme, rescued molar tooth morphogenesis in both Msx1−/− and Bmp4ncko/ncko mice. In this study, through RNA-seq analyses of the developing tooth mesenchyme in mutant and wildtype embryos, we found that Msx1 and Osr2 have opposite effects on expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Remarkably, both Dkk2 and Sfrp2 exhibit Osr2-dependent preferential expression on the lingual side of the tooth bud mesenchyme and expression of both genes was up-regulated and expanded into the tooth bud mesenchyme in Msx1−/− and Bmp4ncko/ncko mutant embryos. We show that pharmacological activation of canonical Wnt signaling by either lithium chloride (LiCl) treatment or by inhibition of Dkk in utero was sufficient to rescue mandibular molar tooth morphogenesis in Bmp4ncko/ncko mice. Furthermore, whereas inhibition of Dkk alone was insufficient to rescue tooth morphogenesis in Msx1−/− mice, pharmacological inhibition of Dkk in combination with genetic inactivation of Sfrp2 and Sfrp3 rescued maxillary molar morphogenesis in Msx1−/− mice. Together, these data reveal a novel mechanism that the Bmp4-Msx1 pathway drives tooth organogenesis by activating Wnt signaling via regulation of the secreted Wnt antagonists.

overall_design: E14.5 mouse embryos tooth germs were micro-dissected by LCM, mandibular molar and maxillary molar were separated, 3 pairs of control and mutant samples were pooled for the RNA extraction. Osr2+/− lower molar and Osr2-/− lower molar.

first: Shihai

last: Jia

first: Hyuk-Jae

last: Kwon


title: CRISPR/Cas9 Screens Reveal Epstein-Barr virus Synthetic Lethal Targets

city: Boston

first: Benjamin

last: Gewurz

state: Massachusetts

address: 181 Longwood Ave

department: Medicine

institute: Brigham and Women's Hospital

summary: Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma and immunosuppression-related lymphomas.  These B-cell malignancies arise by distinct transformation pathways and utilize divergent viral and host expression programs.  To identify host dependency factors elicited by EBV latent-infection states, we performed parallel genome-wide CRISPR/Cas9 screens in Burkitt lymphoma (BL) and lymphoblasotid cell lines (LCL). Our results highlighted 57 BL and 87 LCL genes selectively critical for their growth and survival.  LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets and multiple kinases.  We uncovered key CD19/CD81 roles in EBV membrane protein-driven PI3K/AKT activation and mechanisms by which EBV evades tumor suppressor responses to its growth program. LMP1-induced cFLIP was critical for LCL defense against TNFa-mediated programmed cell death, while EBV-induced BATF/IRF4 were critical for LCL BIM suppression and MYC induction.  EBV super-enhancer targeted IRF2 protected LCLs against BLIMP1 responses.  Collectively, our results identify host/pathogen interaction-driven synthetic lethal targets for therapeutic intervention.

overall_design: CRISPR was employed to knock out IRF2, IRF4 and BATF in LCLs. Control sgRNA was included as a control. RNA-seq was performed on these samples to conduct transcriptome profiling.

first: Yijie

last: Ma


title: Transcriptional profile of Krt15- and Krt15+ basal cells in mouse esophageal epithelium

city: Philadelphia

first: Veronique

last: Giroux

state: PA

address: 421 Curie Blvd, BRB II/III Room 941

institute: University of Pennsylvania

summary: We report different transcriptional profiles of Krt15+ and Krt15- basal cells in the mouse eosphageal epithelium.

overall_design: Isolation of Krt15- (CD29highGFP-) and Krt15+(CD29highGFP+) basal cells through FACS using Krt15-CrePR1;R26mTomato/mGFP mice

first: Anil

middle: K

last: Rustgi


title: Diversification of Root Hair Development Genes in Vascular Plants

city: Ann Arbor

first: John

last: Schiefelbein

state: Michigan

address: 830 North University Avenue

department: Molecular, Cellular, and Developmental Biology

institute: University of Michigan

summary:
Purpose: The root hair is a model for understanding evolution of individual cell differentiation programs in plants. We compare the expression of the genes that participate in root hair development between Arabidopsis and other vascular plants to assess the conservation/diversification of the root hair development programs in vascular plants.
Methods: We used RNA-Seq, in triplicates, to measure the genome-wide transcription activity of the root-hair cells isolated by Fluorescence-activated cell sorting (FACS) in Arabidopsis (COBL9::GFP transgeneic line, AtRH) and rice (EXPA30::GFP transgenic line, OsRH). We also generated RNA-Seq data, in triplicates, on the Arabidopsis rhd6 WER::GFP and WT WER::GFP by FACS to identify the RHD6-regulating root hair morphogenesis genes (AtRHM). For Arabidopsis, rice, tomato, soybean, cucumber and maize, we used RNA-seq, in triplicates, to measure genome-wide transcription activity of root hair cells filtered by sieves after stirred in liquid nitrogen (HAIR genes). Each sample was trimmed to retain high-quality reads, mapped to the reference genome by TopHat, and quantified by Cufflinks. The number of raw reads of Arabidopsis rhd6 WER::GFP and WT WER::GFP sample was counted by HTSeq and analyzed by edgeR to identify the differentially expressed genes.
Results: We defined the root-hair transcriptome in diverse vascular plant species and analyzed the relative conservation/divergence in the expression of a large set of gene families.

sample_organism:
Cucumis sativus
Arabidopsis thaliana
Glycine max
Solanum lycopersicum
Oryza sativa
Zea mays

overall_design: RNA-Seq on the FACS isolated root hair-cells of Arabidopsis COBL9::GFP and rice EXPA30::GFP transgenic lines to get the gene expression of the root-hair cells of Arabidopsis and rice. RNA-Seq on the FACS isolated epidermal cells of Arabidopsis rhd6 WER::GFP and WT WER::GFP transgenic lines to identify the RHD6-regulating root hair genes. RNA-Seq on sieve-filtered root hair cells from Arabidopsis, rice, tomato, soybean, cucumber, and maize to define the root-hair transcriptome in diverse angiosperms. Each measurement includes 3 biological replicates.

first: Ling

last: Huang

first: Kook Hui

last: Ryu

first: Wenjia

last: Wang

first: Xinhui

last: Shi


title: Methyl-Spec-seq control data sets

status: Public on May 10 2017

city: Palo Alto

first: Zheng

last: Zuo

address: Stanford

department: Genetics

institute: Stanford university

summary: We developed a novel in-vitro experimental method to characterize the protein-DNA interaction specificity and methylation sensitivity, we called Methyl-Spec-seq. In this data set, mouse ZFP57 (F1-F3) was used as a positive control example to show that Methyl-Spec-seq can determine the relative binding energy for variants with different methylation property, i.e., unmethylated, top hemimethylated, bottom hemimethylated, duplex methylated by either chemical synthesis or enzymatic treatment.

overall_design: Except different methylation types of DNA, the overall design is the same as conventional Spec-seq, which we have published work before.


title: Transgenic Expression Of Mitochondrial Chaperone Trap1 Accelerates Prostate Cancer Development

first: Priyankara

last: Wickramasinghe

address: 3601 Spruce Street

institute: The Wistar Institute

summary: Protein homeostasis, or proteostasis is critical for organelle function, including mitochondria, but its role in cancer is controversial. Here, we show that transgenic mice expressing the mitochondrial chaperone, TRAP1 in the prostate develop prostatic hyperplasia and cellular atypia. When examined on a Pten+/- background, a common alteration in prostate cancer patients, TRAP1 transgenic mice showed accelerated incidence of invasive prostatic adenocarcinoma, characterized by increased cell proliferation and reduced apoptosis, in situ. Conversely, homozygous deletion of TRAP1 delays prostatic tumorigenesis in Pten+/- mice, without affecting hyperplasia or prostatic intraepithelial neoplasia (PIN). Global RNA sequencing and reverse phase protein array profiling of Pten+/--TRAP1 transgenic tumors reveals modulation of oncogenic networks of cell proliferation, apoptosis, cell motility, DNA damage and metabolism. Mechanistically, reconstitution of Pten+/- prostatic epithelial cells with TRAP1 results in increased cell proliferation, reduced apoptosis, heightened cell invasion, and no changes in mitochondrial bioenergetics. Therefore, TRAP1 promotes invasive prostate cancer, and provides an “actionable” therapeutic target in patients with advanced disease.

overall_design: RNA-seq for Pten +/- mice with and without transgenic Trap1

first: Sofia

last: Lisanti

first: Dario

last: Altieri