title: DAN (NBL1) promotes collective neural crest migration by restraining uncontrolled invasion

status: Public on Aug 07 2017

city: Kansas City

first: Madelaine

last: Gogol

state: MO

address: 1000 E. 50th Street

department: Computational Biology Core

institute: Stowers Institute

summary: Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. Here, we test the function of DAN, a BMP antagonist we detected by analysis of chick cranial mesoderm. Our analysis shows that, prior to neural crest cell exit from the hindbrain, DAN is expressed in the mesoderm, then it becomes absent along cell migratory pathways. Cranial neural crest and metastatic melanoma cells avoid DAN protein stripes in vitro. Addition of DAN reduces the speed of migrating cells, in vivo and in vitro respectively. In vivo loss-of-function of DAN results in enhanced neural crest cell migration by increasing speed and directionality. Computer model simulations support the hypothesis that DAN restrains cell migration by regulating cell speed. Taken together, our results identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion and promotes collective migration in a manner consistent with inhibition of BMP signaling.

overall_design: Neural tube cultures were prepared and plated. DAN was added to the media at 0, 1 and 10 ug/ml. After overnight incubation, neural tubes and any ectoderm were removed from in vitro cultures using a tungsten needle and pipette. Media was then removed from neural tube cultures and the adherent neural crest cells were gently rinsed once with 0.1% DEPC PBS. Three biological replicates were harvested for each of the three conditions (untreated, 1 ug/ml and 10 ug/ml). cDNA was synthesized using SMART-seq v4 Ultra Low Input RNA kit (634888, Clontech) with subsequent library preparation by Nextera XT DNA Sample Prep (FC-131-1096, Illumina) and Index kits (FC-131-1002, Illumina). cDNA samples and libraries were both confirmed on a Bioanalyzer 2100 prior to RNAseq. Libraries were sequenced as 75bp, high output, paired reads on an Illumina NextSeq. Each sample generated in excess of 20 million fastq counts.

first: Rebecca

last: McLennan

first: Caleb

last: Bailey

first: Linus

middle: J

last: Schumacher

first: Jessica

last: Teddy

first: Jason

last: Morrison

last: Kasemeier-Kulesa

first: Lauren

last: Wolfe

first: Madeline

first: Ruth

last: Baker

first: Philip

last: Maini

first: Paul

last: Kulesa


title: Whole tumor RNA-sequencing and deconvolution reveal a clinically-prognostic PTEN/PI3K-regulated glioma transcriptional signature

city: New York

first: Peter

middle: A

last: Sims

state: NY

address: 3960 Broadway, Lasker 203AC

country: USA

institute: Columbia University

type: Expression profiling by high throughput sequencing

summary: The concept that solid tumors are maintained by a productive interplay between neoplastic and non-neoplastic elements has gained traction with the demonstration that stromal fibroblasts and immune system cells dictate cancer development and progression. While less studied, brain tumor (glioma) biology is likewise influenced by non-neoplastic immune system cells (macrophages and microglia) which interact with neoplastic glioma cells to create a unique physiological state (glioma ecosystem) distinct from that found in the normal tissue. To explore this neoplastic ground state, we leveraged several preclinical mouse models of neurofibromatosis type 1 (NF1) optic glioma, a low-grade astrocytoma whose formation and maintenance requires productive interactions between non-neoplastic and neoplastic cells, and employed whole tumor RNA-sequencing and mathematical deconvolution strategies to characterize this low-grade glioma ecosystem as an aggregate of cellular and acellular elements. Using this approach, we demonstrate that optic gliomas generated by altering the germline Nf1 gene mutation, the glioma cell of origin, or the presence of co-existing genetic alterations represent molecularly-distinct tumors. However, these optic glioma tumors share a 25-gene core signature, not found in normal optic nerve, that is normalized by microglia inhibition (minocycline), but not conventional (carboplatin) or molecularly-targeted (rapamycin) chemotherapy. Lastly, we identify a genetic signature conferred by Pten reduction and corrected by PI3K inhibition. This signature predicts progression-free survival in patients with either low-grade or high-grade glioma. Collectively, these findings support the concept that gliomas are composite ecological systems whose biology and response to therapy may be best defined by examining the tumor as a whole.

overall_design: We profiled homogenized optic nerve, optic glioma tissue, and cell lines from mouse models and used a template-switching-based library construction protocol to faciliate low-input RNA-Seq.

first: P


title: Interactions between effector-triggered immunity (ETI) and pattern-triggered immunity (PTI) in an Arabidopsis dde2 ein2 pad4 sid2 mutant

city: St Paul

first: Fumiaki

last: Katagiri

state: Minnesota

address: 1500 Gortner Ave

department: Dept of Plant and Microbial Biology

institute: Univ of Minnesota

summary:
We observed inhibition of the hypersensitive response (a typical ETI response) by PTI signaling in an Arabidopsis quadruple mutant dde2 ein2 pad4 sid2 (quad).
Thus, we designed an experiment to see the interaction between ETI and PTI signaling at the transcriptome level.
The Arabidopsis line dde2 ein2 pad4 sid2 Ed-AvrRpt2 (quadAvrRpt2) was used (Ed-AvrRpt2, estradiol-inducible AvrRpt2 transgene).
In this plant line, ETI can be elicited by estradiol (Ed) treatment via in planta expression of AvrRpt2.
Transcriptome responses to PTI only, ETI only, and PTI+ETI together were recorded.

overall_design:
quadAvrRpt2 was treated with one of (1) Mock, (2) flg22 to elicit PTI response only, (3) Ed to elicit ETI response only via AvrRpt2, (4) flg22+Ed to elicit both PTI and ETI responses together.
At 0 (untouched), 60, 120, 180, and 300 minutes after treatment, treated plant leaves were harvested for RNA-seq analysis.
Three independent experiments were performed (rep1-rep3).

first: Keisuke

last: Mase


title: RNA-Sequencing of RCAS(A) and RCAS(A)-Wnt9a infected basilar papillas to identify genes acting downstream of Wnt9a

city: West Lafayette

first: Donna

middle: M

last: Fekete

state: IN

address: 915 West State St

department: Biological Sciences

institute: Purdue University

summary: This study compared Wnt9a overexpressing basilar papillas to control basilar papillas (BP) from embryonic day 6 (E6) chicken embryos. The goal was to identify genes acting downstream of Wnt9a in the E6 BP. We used Illumina HiSeq with paired-ends and at more than 60 million reads per sample. Differentially expressed genes between Wnt9a overexpressing and control BPs include, but not limited to, genes previously shown to be involved in axon guidance, cell pluripotency, and EMT.  pathway,

overall_design: mRNA profiles were generated from embryonic day 6 basilar papillas infected with RCAS(A) retrovirus (control) or RCAS(A)-Wnt9a retrovirus (experimental). Three biological replicates were collected for both control and experiemental groups. Each biological replicate consisted of RNA from 24 basilar papillas.

first: Mary

middle: K

last: Scott

first: Ankita

last: Thawani

first: Nadia

last: Atallah

first: Vidhya

last: Munnamalai


title: Genome-wide analysis of SRm160 18603 mutat larvae

status: Public on Aug 09 2017

city: Buenos Aires

first: Marcelo

last: Yanovsky

state: Ciudad Autonoma de Buenos Aires

address: Av. Patricias Argentinas 435

country: Argentina

institute: Fundación Instituto Leloir. IIBBA- CONICET

summary: The goal of this study was to compare the transcriptional profile (RNA-seq) of wild type Drosophila melanogaster larvae (w1118) and mutants for the SRm160 gene in the same genetic background.

overall_design: 150 larvae of each genotype were collected 24h after egg laying (AEL) and transferred to a plate with standard food. 3 groups of 100 larvae from each genotype were collected 12h after transferring to standard food. Larvae of each group were collected, rinsed in PBS  and transferred to TRIzol.

first: Esteban

last: Beckwith

first: Carlos

middle: E

last: Hernando

first: Sofía

last: Polcowñuk

first: Agustina

middle: P

last: Bertolin

first: Estefania

last: Mancini

first: M

middle: F

last: Ceriani


title: Impact of Tcf1 and Lef1 deficiency on HSCs

status: Public on Aug 08 2017

city: Nutley

first: Hai-Hui

last: Xue

state: NJ

address: 111 Ideation Way

department: Center for Discovery and Innovation

institute: Hackensack University Medical Center

summary: Comparison of transcriptome between control and Tcf1/Lef1-deficient hematopoietic stem cells (HSCs).

overall_design: Flt3-negative, lineage-negative, Sca1+ and cKit+ cells (Flt3-LSKs) were sorted from bone marrow cells from control mice or those are deficient for Tcf1 and Lef1 transcription factors. Both genes were conditionally deleted using Vav-Cre

first: Shuyang

last: Yu


title: Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions

city: Göttingen

first: Carsten

last: Lueder

address: Kreuzbergring 57

country: Germany

institute: Institut für Medizinische Mikrobiologie

summary: The apicomplexan parasite Toxoplasma gondii infects a broad range of different cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist for the hosts life predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and at 24 hours after infection with T. gondii. However, only 157 to 492 of these DEGs were regulated within the different cell types by infection. Intriguingly, largely diverse sets of genes were regulated in neurons, SkMCs, astrocytes and fibroblasts following infection indicating host cell type-specific transcriptional responses. Only a few genes were identified that were commonly regulated in two or three host cell types after infection. The heterogeneous transcriptomes of the host cells before and during infection coincided with the differential expression of ~5,400 genes in T. gondii residing in the different host cells. Expression of these DEGs mostly differed quantitatively but within neurons, T. gondii also expressed 121 genes in a strictly host cell-type specific manner. Interestingly, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites’ ability to adapt to them may govern the parasite-host cell interaction during toxoplasmosis.

overall_design: Comparison of mRNA profiles of murine neurons, skeletal muscle cells, astrocytes and fibroblasts infected or not with Toxoplasma gondii for 24 hours.

middle: G

last: Lüder

first: Izabela

last: Swierzy

first: Ulrike

last: Händel

first: Alexander

last: Kaever

first: Michael

last: Jarek

first: Maren

last: Scharfe

first: Dirk

last: Schlüter


title: Developmentally regulated higher-order chromatin interactions orchestrate B cell fate commitment [Hi-C]

city: Hyderabad

first: Jagan

last: Pongubala

state: Telangana

address: Gachibowli

department: Department of Animal Biology

country: India

institute: University of Hyderabad

summary: Organization of the genome in 3D nuclear-space is known to play a crucial role in regulation of gene expression. However, the chromatin architecture that impinges on the B cell-fate choice of multi-potent progenitors remains unclear. By employing in situ Hi-C, we have identified distinct sets of genomic loci that undergo a developmental switch between permissive and repressive compartments during B-cell fate commitment. Intriguingly, we show that topologically associating domains (TADs) represent co-regulated subunits of chromatin and display considerable structural alterations as a result of changes in the cis-regulatory interaction landscape. The extensive rewiring of cis-regulatory interactions is closely associated with differential gene expression programs. Further, we demonstrate the regulatory role of Ebf1 and its downstream factor, Pax5, in chromatin reorganization and transcription regulation. Together, our studies reveal that alterations in promoter and cis-regulatory interactions underlie changes in higher-order chromatin architecture, which in turn determines cell-identity and cell-type specific gene expression patterns.

overall_design: In situ Hi-C was employed to capture the changes in chromatin organization that underlie the lineage specific gene expression pattern during developmental transition from pre-pro-B to pro-B cell stage.

first: Ravi

last: Boya

first: Anurupa

middle: D

last: Yadavalli

first: Sameena

last: Nikhat

first: Sreenivasulu

last: Kurukuti

first: Dasaradhi

last: Palakodeti


title: Developmentally regulated higher-order chromatin interactions orchestrate B cell fate commitment

type:
Expression profiling by array
Other

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

overall_design: Refer to individual Series


title: Gene expression patterns after chronic clozapine in wild type mice and HDAC2 conditional knock-outs

city: Richmond

first: Mario

last: de la Fuente Revenga

address: 1220 E Broad St.

department: Physiology and Biophysics

institute: School of Medicine, Virginia Commonwealth University

summary: Effect of chronic clozapine in the gene expression in the prefrontal cortex of wild type mice and mice in which the HDAC2 has been knocked-out in CamKIIa-positive pyramidal neurons.

overall_design: An mRNA library was built with samples from four different conditions: wild-type mice treated with chronic clozapine (n=3) or vehicle (n=3), HDAC2 conditional knock-outs treated with chronic clozapine (n=3) or vehicle (n=3)

first: Javier

last: González-Maeso

first: Daisuke

last: Ibi

first: Jeremy

last: Seto

first: Yongchao

last: Ge


title: Time course profiling of IEC and total colonic tissue transcriptome in T.mu colonized animals

first: El-ad David

last: Amir

address: 1470 Madison Avenue

department: Oncological Sciences

institute: Mount Sinai Hospital, New York

summary: In this experiment we profiled the transcriptome of intestinal colonic epithelium and total colonic tissue of animals colonized with T.mu  versus naïve littermate controls at different time points

overall_design: Groups of three mice were inoculated with 2x106 highly purified Tritrichomonas musculis. Total tissue and epithelial cells were collected at 3 days, 14 days and 48 days after inoculation from three different mice. RNA was isolated and sequenced

first: Aleksey

last: Chudnovskiy

first: Arthur

last: Mortha


title: The Transcriptome of the Didelphys virginiana Opossum Kidney OK Proximal Tubule Cell Line

city: Pittsburgh

first: Uma

last: Chandran

state: PA

address: 5150 Center Ave

department: BioMedical Informatics

institute: University of Pittsburgh

summary: The OK cell line derived from kidney of a female opossum Didelphys virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, genomic sequence for Didelphys virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, its relatedness and similarity of the transcriptome to the Didelphys virginiana species is not known. The Monodelphis domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the Didelphys virginiana OK cell line we characterized its transcriptome using de novo transcriptome assembly and alignment to the Monodelphis domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the Monodelphis domestica genome, were compared to publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT specific ion transporters and other key proteins relevant for rodent and human PT function. The sequence and expression data reported here provide a new and important resource for studies on the regulation of PT mRNA and protein expression.

overall_design: de novo and Monodelphis Domestica reference genome based transcriptome assembly and gene expression analysis of 60M paired end reads of one OK cell line sample. Replicate analysis was completed from additional 60M reads from same sample.

first: Megan

middle: L

last: Eshbach

first: Rahil

last: Sethi

first: Raghunandan

last: Avula

first: Janette

last: Lamb

first: Deborah

last: Hollingshead

first: David

middle: N

last: Finegold

first: Joseph

last: Locker

middle: R

first: Ora

last: Weisz


title: Caloric restriction impacts plasma microRNAs in rhesus monkeys

status: Public on Aug 04 2017

city: Orlando

first: Augusto

last: Schneider

state: FL

address: 6900 Lake Nona Blvd

department: Burnett School of Biomedical Sciences

institute: University of Central Florida

type: Non-coding RNA profiling by high throughput sequencing

summary: Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Identification of factors involved in CR brings a promise of translatability to human health and aging. Here, we show that CR induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR’s mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approxi- mately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR-regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identify a role in signaling downstream of the insulin receptor. The highly abundant miR-125a-5p correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR. Putative target pathways of CR- associated miRNAs were highly enriched for growth and insulin signaling that have previously been implicated in delayed aging. Clustering analysis further pointed to CR-induced miRNA regula- tion of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA- based homeostatic mechanisms to coordinate a program of delayed aging.

overall_design: Serum miRNA profile of rhesus monkeys on calorie restriction or control diet was analyzed. For each group 5 samples were processed. Serum was collected, total RNA was isolated using miRNeasy kit (Qiagen). One fourth (5 μL) of the RNA isolated from each plasma sample was used to construct sequencing libraries with the Illumina TruSeq Small RNA Sample Prep Kit (Illumina). The libraries were pooled in equimolar amounts and submitted to sequencing.

last: Dhahbi

first: Hani

last: Atamna

first: Josef

last: Clark

first: Ricki

last: Colman

first: Rozalyn

last: Anderson


title: Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [inDrops]

city: Boston

first: Sowmya

last: Iyer

state: MA

department: Pathology

institute: Massachusetts General Hospital

summary: Recent advances in single-cell transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. Here, we utilized massively parallel single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally-distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune cell deficiencies within prkdcD3612fs, il2rgaY91fs and double homozygous mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants.  Our analysis also uncovered novel cell types including two classes of natural killer immune cells, classically-defined and erythroid-primed hematopoietic stem and progenitor cells, mucin secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first comprehensive single cell transcriptomic analysis of kidney and marrow cells in the adult zebrafish.

overall_design: The goal of our study is to establish the transcriptional profiles of hematopoietic and kidney cell lineages residing in the zebrafish whole kidney marrow. Firstly, we performed single-cell RNA sequencing by a modified Smart-seq2 protocol on sorted single cells from fluorescent transgenic zebrafish lines, which label distinct blood cell types (n = 246 cells total). Secondly, we utilized droplet-based single-cell RNA sequencing (inDrop) to investigate unmarked, comprehensive hematopoietic lineage structure within wild-type, casper-strain zebrafish (N=3 animals, n=3,782 cells total). From this, we identified ten distinct hematopoietic groups of blood and immune identities. Thirdly, we confirmed blood lineage interpretations by comparing hematopoietic lineages within wild-type fish with mutant zebrafish with known immunodeficiencies, including prkdc(D3612fs) (N=3 animals, n=3,201 cells), il2rga(Y91fs) (N=2 animals, n=2,068 cells) and prkdc(D3612fs), il2rga(Y91fs) double compound mutant fish (N=2 animals, n=2,276 cells). Lastly, we identified seven structural and functional cell lineages of kidney identities in the whole kidney marrow (n=1,699 kidney cells).

first: Qin

last: Tang

last: Langenau


title: Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [Smart-seq]


title: Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing [bulk RNA-seq]


title: Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single cell resolution using RNA sequencing


title: Repression by PRDM13 is critical for generating precise neuronal identity (RNA-Seq)

city: Dallas

first: Jane

last: Johnson

state: TX

address: 5323 Harry Hines Blvd

department: Neuroscience

institute: UT Southwestern Medical Center

summary: The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the wide variety of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for these processes during specification of dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for the excitatory neuronal lineages in the dorsal neural tube while also suppressing a battery of genes that determine ventral neural tube fates including Olig1, Olig2 and Prdm12. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing bHLH transcriptional activators that together are required to achieve precise neuronal specification during development.

overall_design: RNA-seq analysis performed on GFP+ cells sorted by FACS from Prdm13GFP/+ or Prdm13GFP/GFP mouse E11.5 neural tubes to identify gene expression in the presence and absence of PRDM13.

first: Ana

last: Uruena

first: Bishakha

last: Mona

first: Rahul

last: Kollipara

first: Zhenzhong

last: Ma

first: Mark

last: Borromeo


title: Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors [RNA-seq]

city: London

first: Emily

last: Saunderson

address: Charterhouse Square

country: United Kingdom

institute: Barts Cancer Institute

summary: Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation in the absence of genetic mutations is not clear. To investigate this question, we use a CRISPR/dCas9 based epigenetic editing tool, where an inactive form of Cas9 is fused to DNMT3A and its regulator DNMT3L. Using this system, we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary myoepithelial cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct and results in sustained repression of CDKN2A and RASSF1 transcripts which prevents cells from entering senescence. The phenotype is associated with retuned expression of a subset of genes to levels in early passage cells; however, the outgrowing myoepithelial cells are not immortal but proliferate for 18-20 population doublings before cell cycle arrest. Finally, we show that the key driver of this phenotype is repression of CDKN2A transcript p16, but prolonged proliferation is enhanced by combined hypermethylation and repression of both CDKN2A transcripts p16 and p14. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, a potential first step in the disease process.

overall_design: RNA-seq experiment with n=3 biological replicates of primary myoepithelial transfection with 26x gRNAs targeting DNA methylation as described.

first: Gabriella

last: Ficz


title: Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics

city: East Lansing

first: Robert

middle: B.

last: Abramovitch

state: MI

address: 567 Wilson Road

department: Microbiology and Molecular Genetics

institute: Michigan State University

summary: The purpose of this study was to characterize the mechanism of action of the small molecule AC2P36

overall_design: Mtb strain CDC1551 was grown at 37ºC in T-25 vented, standing tissue culture flasks in 8 mL of buffered 7H9 medium (pH 7.0 or 5.7) lacking catalase to an OD600 of 0.5. Cultures were subsequently treated with 10 µM AC2P36. For the control, cultures treated with an equal amount of DMSO.  Following a 4 h incubation period, total bacterial RNA was extracted, quality controlled and sequenced. Each treatment was repeated with two independent (biological) replicates.

first: Gary

middle: B

last: Coulson

first: Benjamin

first: Christopher

last: Colvin

last: Fillinger

first: Huiqing

last: Zheng

first: Elizabeth

last: Haiderer

first: Neal

last: Hammer


title: Early fruit abscission is associated with the IAA14/SLR-ARF7 signaling cascade in pedicels of self-abscising apple

status: Public on Aug 05 2017

city: Seoul

first: Seong

last: Heo

address: Sinlim-dong 151-921

department: Department of Plant Science

country: South Korea

institute: Seoul National University

summary: The self-abscising characteristic of fruit is a prominent trait for labor-saving cultivation in apple (Malus × domestica). Up to 30 days after full bloom, early abscission leaves only central fruit in a cluster, while four lateral fruits are abscised. Since fruit abscission is possibly induced during earlier phases of fruit development, samples were collected at full bloom and 10 days after full bloom. Comparative analysis between central and lateral pedicels in self-abscising apple can be used to identify the genes that trigger the abscission mechanism, as the destinies of the pedicels in a cluster are obvious. Transcriptome analysis was performed using RNA-Seq to compare expression profiles between the surviving central pedicel to be survived and abscised lateral pedicel to be abscised from self-abscising apple. A total of 797,647 ESTs were assembled into 65,876 contigs which were annotated and analyzed with using Blast2GO. A total of 1,585 differentially expressed genes in central and lateral pedicels were identified using the NOISeq software. , and these genes were were characterized using the MapMan ontology software. Transcription factors involved in vascular bundle differentiation functioned in the central pedicel, while the signaling cascade of IAA14/SLR-ARF7, which progressed during lateral root emergence from primary roots, operated in lateral pedicels. Future studies should explore associations between the mechanisms of lateral root emergence and pedicel abscission during early phases of fruit development, as well as the interconnection among hormones.

overall_design: mRNA profile of 2 different pedicel tissue of apple using 454 GS-FLX

first: Hee

last: Lee


title: Mutual potentiation of Dnmt3a loss-of-function and Idh2 neomorphic mutations in malignant hematopoiesis

city: Houston

first: Margaret

last: Goodell

address: One Baylor Plaza, N1030

institute: Baylor College of Medicine

type: Methylation profiling by high throughput sequencing


title: Global methylation alteration in Dnmt3aKO-Idh2 mutant double mutant HSPCs

summary: We investigated DNA methylation alterations in Dnmt3aKO-Idh2 mutant HSPCs in comparison with the megarkaryocyte erythroid progenitor (MEP) and Granulocyte Macrophage Progenitor(GMP) of Dnmt3a knockout, Idh2 mutant, WT genotypes. We generated an average of 17.42 million reads per sample (12.17 - 24.53 million) of which an average of 16.39 million could be mapped to the mm9 genome (11.74 - 23.73 million). We achieved an average CpG coverage depth of 27.88-fold (19.52 - 35.89-fold) and average bisulfite conversion efficiency of 99.61% (98.43 - 99.94%).

overall_design: Reduced representation bisulfite sequencing of megarkaryocyte erythroid progenitor (MEP) and Granulocyte Macrophage Progenitor(GMP) of Dnmt3a knockout, Idh2 mutant, WT and Dnmt3aKO-Idh2 mutant HSPC using Illumina HiSeq 2000

last: Rodriguez

first: Wei

last: Li


title: Gene expression alteration in Dnmt3aKO-Idh2 mutant double mutant HSPCs

summary: We assessed global gene expression changes in Dnmt3aKO-Idh2 mutant HSPCs in comparison with the megarkaryocyte erythroid progenitor (MEP) and Granulocyte Macrophage Progenitor(GMP) of Dnmt3a knockout, Idh2 mutant, WT genotypes.

overall_design: Mouse HSPC mRNA profiles of megarkaryocyte erythroid progenitor (MEP) and Granulocyte Macrophage Progenitor(GMP) of Dnmt3a knockout, Idh2 mutant, WT and Dnmt3aKO-Idh2 mutant HSPC were obtained by sequencing on Illumina HiSeq 2000


title: Integrative analysis of translatome and transcriptome of adipocytes

city: Durham

first: David

last: Reid

state: NC

address: 436 Nanaline Duke Bldg., Box 3709 DUMC

department: Biochemistry

institute: Duke University

summary: To systemically determine the translational control of gene expression in adipose, we performed ribosome profiling and RNA-seq in parallel to depict the translatome and transcriptome changes during primary brown and white adipogenesis, and between brown and white adipose tissue.

overall_design: ribosome profiling and mRNA profiling were performed using the same biological material for interscapular brown fat, epididymal white fat, in vitro cultured white and brown preadipocytes and differentiated adipocytes

middle: W

first: Lei

last: Sun


title: RNA-seq of mitotic and meiotic cells from Drosophila testis

city: Beijing

first: Zhaohui

last: Wang

address: No 1 Westbeichen Road

country: China

institute: Institute of genetics and developmental biology

summary: we observed distinct profiles of transcriptome-wide 3’UTR in the accurately dissected mitotic and meiotic cells of wildtype testes, also in the over-amplifying mitotic germ cells of mutant testes

overall_design: Triplicate sampling for laser-dissected SG (mitotic germ cells) and SC (meiotic germ cells); duplicate sampling for tut, bam, or bgcn mutant testes(mainly mitotic germ cells)

first: Lingjuan

last: Shan

first: Chan

last: Wu

first: Di

last: Chen


title: RNA sequencing of Hordeum vulgare near-isogenic lines

status: Public on Aug 03 2017

city: Cologne

first: Kurt

last: Stueber

address: Carl von Linne-Weg 10

department: Genome Centre

institute: Max Planck Institute for Plant Breeding Research

summary:
RNA sequencing of ILs of vrs1, vrs3, vrs4 and int-c.5 in cv. Bowman
Purpose: Expression analysis and variant calling

overall_design: Enriched apical meristem tissue was isolated at Waddington stage (W) 3.5 and W5.0. The samples were harvested at the middle of the day, 6 to 8 h before the end of the light period.

first: Agatha

last: Walla

first: Wilma

last: van Esse

first: Maria

last: von Korff


title: Transcriptome analysis of CD133 overexpressed U87MG glioblastoma cells

first: Seon Yong

address: 145 Anam-ro, Seungbuk-gu

institute: Korea University

summary: This study investigated the biological function of CD133 by ectopic expression of CD133 in U87MG cell line. Although CD133 is widely used as a cancer stem cell marker, there are a few studies that examined its own biological functions. While a number of loss-of-function studies about CD133 have shown that CD133 have effects on cancer progression, there are few gain-of-function studies about functions of CD133. Thus, we identified the potential function of CD133 by its overexpression in U87MG glioblastoma cell line. Though there were no significant changes in cell growth and sphere forming ability, elevated IL-1β and its downstream chemokines (CCL3, CXCL3, CXCL5) may function as chemoattractants which affect recruitment of Ly6G+ neutrophils surrounding necrotic regions in vivo and migration of neutrophil-like dHL-60 cells. Taken together, this results imply that CD133 can regulate IL-1β signaling, and promotes the environmental change.

overall_design: A Glioblastoma cell line, U87MG, was transfected with pCDH-CMV-MCS-EF1-Puro (U87MG-control) or pCDH-CMV-CD133-EF1-Puro (U87MG-CD133). Plasmids for CD133 overexpression (NM_001145847) were gifted by Dr. Young-Gyu Ko (Korea University). The 3 biological replicate of RNA extracts in these cells was used for transcriptome analysis.

first: Xiong

last: Jin

first: Hyunggee

last: Kim


title: RNA-Seq analysis of Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f primary chondrocytes

status: Public on Aug 06 2017

city: HangZhou

first: JUN

last: DAI

state: ZheJiang

address: #866 Yu Hang Tang Rd

department: School of Medicine

institute: Zhe Jiang University

summary: Purpose: The aims of this study were to identify differentially expressed genes between Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f primary chondrocytes. Methods: RNA samples of primary chondrocytes were prepared from Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f mice and were sequenced and analyzed by Shanghai Novel Bioinformatics Co, Ltd. Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences, reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20 percent of bases with qualities of <13. The clean reads were then aligned to the mouse genome (version: hg19_GRCh37) using the MapSplice program (v2.1.6). We applied EBseq algorithm to filter the differentially expressed genes (DEGs), after the significant analysis and FDR analysis under the following criteria: i) Fold Change>2 or <0.5; ii) FDR<0.05. DEGs were used for gene ontology (GO) and pathway analysis. We downloaded the GO annotations from NCBI, UniProt and the Gene Ontology. Fisher’s exact test was applied to identify the significant GO categories and FDR was used to correct the p-values. Pathway analysis was used to determine the significant pathway of the differentially expressed genes according to the KEGG database. The Fisher’s exact test was utilized to select the significant pathway, and the threshold of significance was defined by P-value and FDR. Conclusions: Our study represents the first detailed analysis of the role of Kdm6b in joint cartilage development and homeostasis, which provided a new therapeutic modalities for OA treatment.

overall_design: Kdm6bf/f and Col2a1-CreERT2;Kdm6bf/f primary chondrocytes were treated with 4-hydroxytamoxifen (4OH-TM) (1 μM) for 48 hours, and then RNA samples were prepared from these cells for RNA-Seq.

first: Jun

last: Dai


title: Age-dependent comparison of transcriptional signature of lamina propria anti-inflammatory macrophages of Il10rb-/- (KO) and Il10rb+/- (het) mice

first: Naresh

middle: Singh

last: Redhu

state: Massachusetts

address: 300 Longwood Ave, Enders 670

department: GI/Nutrition

institute: Boston Children's hospital

summary: We report that colonic lamina propria anti-inflammatory macrophages from Il10rb-/- (KO) mice at 12 weeks possessed significantly high levels of proinflammatory transcripts compared to the ones from Il10rb+/- (het) controls. While the transcriptional profile between macrophages from KO and het mice was comparable at 1 weeks and did not possess significant levels of proinflammatory transcripts, we show that the macrophages from KO mice were enriched for proinflammatory transcripts at 3 weeks compared to the ones from 3 week old het controls. These data indicate that the anti-inflammatory signature of colonic lamina propria macrophages from Il10rb-/- mice is compromized at 3 weeks.

overall_design: Lamina propria macrophages were FACS sort-purified from 1, 3, and 12 weeks old KO and het mice based on gating strategy; CD45+CD11b+CD11cintCD103-CD64+Ly6C-MHCII+. One mouse each for 3, 12 weeks, and a pool of 3-5 mice at 1 weeks was included in RNA-seq analysis.

middle: S

first: Vasudevan

last: Bakthavatchalu

first: Evan

last: Conaway

first: Dror

last: Shouval

first: Amy

last: Tsou

first: Jeremy

last: Goettel

first: Amlan

last: Biswas

first: Chuanwu

first: Michael

last: Field

first: Werner

last: Muller

first: Andre

last: Bleich

first: Ning

first: Georg

last: Gerber

first: Lynn

last: Bry

first: James

last: Fox

first: Scott

last: Snapper

first: Bruce

middle: H

last: Horwitz


title: Lamin B1 regulates somatic mutations and progression of B cell malignancies

first: Andrejs

last: Braun

state: London

address: Charterhouse Square,

department: Barts Cancer Institute

institute: Queen Mary University of London

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Somatic hypermutation (SHM) is a pivotal process in adaptive immunity that occurs in the germinal centre and allows B-cells to change their primary DNA sequence and diversify their antigen receptors. Here, we report that genome binding of Lamin B1, a component of the nuclear envelope involved in epigenetic chromatin regulation, is reduced during B cell activation and formation of lymphoid germinal centres. ChIP-Seq analysis showed that kappa and heavy variable immunoglobulin domains were released from the Lamin B1 suppressive environment when SHM was induced in B cells. RNAi-mediated reduction of Lamin B1 resulted in spontaneous SHM as well as kappa-light chain aberrant surface expression. Finally, Lamin B1 expression level was directly proportional to 5-year survival rate in chronic lymphocytic leukaemia, and was strongly involved in transformation of follicular lymphoma.   In summary, here we report that Lamin B1 is a negative epigenetic regulator of SHM in normal B-cells and a "mutational gatekeeper", suppressing the aberrant mutations that drive lymphoid malignancy.

overall_design: Lamin B1 ChIP sequencing data from two independent biological replicates in control vs somatic hypermutation-induced BL2 cells

first: Tetyana

last: Klymenko

first: Jun

last: Wang


title: RNASeq comparing Thpok and LRF-deficient Tregs to Control

city: Bethesda

first: Margaret

middle: C

last: Cam

state: MD

institute: NIH

summary: The transcription factor Thpok is required for intrathymic CD4+ T cell differentiation and, together with its homolog LRF, supports CD4+ T cell helper effector responses, However, it is not known if these factors are needed for the T regulatory arm of MHC-II responses. We inactivated the genes encoding both factors in differentiated Treg cells, to see whether how they are redundantly required to maintain the size and function of the post-thymic Treg pool and for immune homeostasis.

overall_design: We compared YFP+ LRF and Thpok-deficient Treg cells with two distinct controls: (i) CD25+ YFP– cells (Thpok and LRF-sufficient ‘animal control’ cells, expressing the Foxp3+ allele) obtained from the same female mice as the YFP+ Treg cells, and (ii) YFP+ Treg cells from Foxp3+/ YFP Cre Thpok+/+ Lrf+/+ female mice (‘genetic controls’ which express the Foxp3YFP Cre allele).

first: Remy

last: Bosselut

first: Maggie


title: Epigenome profiling and editing of neural progenitor cells in the developing mouse neocortex [RNA-seq]

city: Dresden

first: Mareike

last: Albert

address: Fetscherstr. 105

department: Center for Regenerative Therapies TU Dresden

institute: TUD Dresden University of Technology

summary: The generation of neocortical neurons from neural progenitor cells (NPCs) is primarily controlled by transcription factors binding to DNA in the context of chromatin. To understand the complex layer of regulation that orchestrates different NPC types from the same DNA sequence, epigenome maps with cell type resolution are required. Here we present genome-wide histone methylation maps for distinct neural cell populations in the developing mouse neocortex. Using different chromatin features, we identify potential novel regulators of cortical NPCs available for future exploration. Moreover, we identify extensive H3K27me3 changes between NPC subtypes coinciding with major developmental and cell biological transitions. Interestingly, we detect dynamic H3K27me3 changes on promoters of several crucial transcription factors, including the basal progenitor regulator Eomes. We used catalytically inactive Cas9 fused with the histone methyltransferase Ezh2 to edit H3K27me3 at the Eomes locus in vivo, which results in reduced Tbr2 expression and lower basal progenitor abundance, underscoring the relevance of dynamic H3K27me3 changes during neocortex development. Taken together, we provide a rich resource of neocortical histone methylation and outline an approach to investigate its contribution to the regulation of selected genes during neocortical development.

overall_design:
Gene expression profile of mouse purified neuroepithelial cells (NECs) was generated by RNA-seq.
---------------
This represents the RNA-Seq component only

first: Marta

last: Florio

first: Wieland

last: Huttner


title: Cdk8 overexpression effects in heart

city: Iowa City

first: Long-Sheng

last: Song

state: IA

address: 285 Newton Rd

department: Internal Medicine

institute: University of Iowa

summary: Analysis of ventricular derived mRNA from cardiac specific Cdk8 overexpressing mice.  Results provide insight into the molecular mechanisms underlying dilated cardiomyopathy and heart failure.

overall_design: Methods: Ventricular samples (5 per group) from 21-day-old cardiac specific Cdk8 overexpressing versus wildtype male mice were used to generate polyA enriched stranded RNA libraries followed by RNAseq using the Illumina HiSeq platform.  Raw sequence reads were analyzed with BaseSpace (www.illumina.com) by trimming to 125bp and aligning reads to the mus musculus mm10 genome using the TopHat Alignment app.  Transcripts were assembled and significant differentially expressed genes were determined with the Cufflinks Assembly and DE app using a false discovery rate <0.05. Genes with >1.5 fold expression changes were used for further analyses.  qRT–PCR validation was performed using SYBR Green assays.

first: Chad

last: Grueter


title: Effect of FGF13 depletion on the H460 cell line

city: Rehovot

first: Moshe

last: Oren

address: Herzl Street

department: Department of Molecular Cell Biology

country: Israel

institute: Weizmann Institute of Science

summary: The purpose of our study was to explore the relevance of the FGF13 protein in a NSCLC cell line

overall_design: RNA seq was performed on H460 cells transiently transfected with siRNA against FGF13 (siFGF13) or control siRNA (siC)

first: Débora

last: Bublik


title: High-throughput RNA sequencing on circular RNA profiles of human bladder cancer tissues and normal bladder tissues

city: Wuhan

first: Yawei

last: Li

state: Hubei

address: Jiefang road

institute: Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary: In order to find out circular RNAs profiles in human bladder cancer tissues and normal bladder tissues, we characterized circuclar RNA transcripts by performing RNA-Seq on ribosomal RNA-depleted total RNA from three pairs of human bladder cancer tissues and paired normal bladder tissues.A computational pipeline based on the anchor alignment of unmapped reads was used to identify circular RNAs. Collectively, we identified16,535 distict circular RNAs, most of them origined from exons (88.96%), others from introns, linc RNA, intergenic region, 3’UTR and 5’UTR. Among all these circRNAs, 571 circRNAs were differentially expressed between bladder cancer tissues and normal bladder tissues, and 524 circRNAs were downregulated in bladder cancer tissues (91.2%), others were upreguluated. These significantly differential expressed circular RNA might have regulatory function in bladder cancer, and worth to be further explored.

overall_design: Circular RNAs profiles of thredd pairs of bladder cancer tissues and paired adjacent normal bladder tissues were generated by RNA deep-sequencing, using HiSeq2000, Illumina.

first: Guosong

last: Jiang


title: Transcriptional profiling identifies differential expression of long non-coding RNAs in Jo-1 associated and inclusion body myositis

status: Public on Aug 02 2017

city: Bath

first: Mark

middle: Andrew

last: Lindsay

address: Claverton Down

department: Pharmacy and Pharmacology

institute: University of Bath

summary: Myositis is characterised by muscle inflammation and weakness. Although generally thought to be driven by a systemic autoimmune response, increasing evidence suggests that intrinsic changes in the muscle might also contribute to the pathogenesis. Long non-coding RNAs (lncRNAs) are a family of novel genes that regulate gene transcription and translation. To determine the potential role of lncRNAs, we employed next generation sequencing to examine the transcriptome in muscle biopsies obtained from two histologically distinct patient populations, inclusion body myositis (IBM) and anti-Jo-1-associated myositis (Jo-1).

overall_design: PolyA+ RNA was sequenced in human muscle biopsies obtained from control individuals or patients with inclusion body myositis or Jo-1 myositis

middle: A


title: Cell type-specific translation profiling reveals a novel strategy for treating fragile X syndrome

city: Edinburgh

first: Emily

last: Osterweil

address: Hugh Robson building

institute: University of Edinburgh

summary: Excessive mRNA translation downstream of group I metabotropic glutamate receptors (mGlu1/5) is a core pathophysiology of fragile X syndrome (FX), however the differentially translating mRNAs that contribute to altered neural function are not known. We used Translating Ribosome Affinity Purification (TRAP) and RNA-seq to identify mistranslating mRNAs in CA1 pyramidal neurons of the FX mouse model (Fmr1-/y) hippocampus, which exhibit exaggerated mGlu1/5-induced long-term synaptic depression (LTD). In these neurons, we find the Chrm4 transcript encoding muscarinic acetylcholine receptor 4 (M4) is excessively translated, and synthesis of M4 downstream of mGlu5 activation is mimicked and occluded. Surprisingly, enhancement rather than inhibition of M4 activity normalizes core phenotypes in the Fmr1-/y, including excessive protein synthesis, exaggerated mGluR-LTD, and audiogenic seizures. These results suggest that not all excessively translated mRNAs in the Fmr1-/y brain are detrimental, and some may be candidates for enhancement to correct pathological changes in the FX brain.

overall_design: 6 biological replicates of total hippocampal mRNA (Input) from WT and Fmr1 KO littermate pairs and CA1-TRAP-IP (IP) from the same 6 WT and KO littermate pairs.

first: Sophie

last: Thomson

first: Sang

last: Seo

middle: K


title: Epigenome profiling and editing of neural progenitor cells in the developing mouse neocortex [ChIP-seq]

overall_design: Histone methylations profiles of mouse purified neural progenitor cells and neurons were generated by ChIP-seq.

first: Naharajan

last: Lakshmanaperumal


title: Genomic architecture of biomass heterosis in Arabidopsis

first: Xuncheng

last: Wang

address: 5 Yiheyuan Road

institute: Peiking University

summary: Heterosis is most frequently manifested by the substantially increased vigorous growth of hybrids compared with their parents. Investigating genomic variations in natural populations is essential to understand the initial molecular mechanisms underlying heterosis in plants. Here, we characterized the genomic architecture associated with biomass heterosis in 200 Arabidopsis hybrids. The genome-wide heterozygosity of hybrids makes a limited contribution to biomass heterosis, and no locus shows an obvious overdominance effect in hybrids. However, the accumulation of significant genetic loci identified in genome wide association studies (GWAS) in hybrids strongly correlates with better-parent heterosis (BPH). Candidate genes for biomass BPH fall into diverse biological functions, including cellular, metabolic, and developmental processes and stimulus-responsive pathways. Important heterosis candidates include WUSCHEL, ARGOS, and some genes that encode key factors involved in cell cycle regulation. Interestingly, transcriptomic analyses in representative Arabidopsis hybrid combinations reveal that heterosis candidate genes are functionally enriched in stimulus-responsive pathways, including responses to biotic and abiotic stimuli and immune responses. In addition, stimulus-responsive genes are repressed to low-parent levels in hybrids with high BPH, whereas middle-parent expression patterns are exhibited in hybrids with no BPH. Our study reveals a genomic architecture for understanding the molecular mechanisms of biomass heterosis in Arabidopsis, in which the accumulation of the superior alleles of genes involved in metabolic and cellular processes improve the development and growth of hybrids, whereas the overall repressed expression of stimulus responsive genes prioritizes growth over responding to environmental stimuli in hybrids under normal conditions.

overall_design: First leaf mRNA profiles of 14-day old A.thaliana Col-0, Aa-0, Ler-1, Col-0×Aa-0 and Col-0×Per-1 were generated by deep sequencing, in duplicate, using Illumina Hiseq 2500.

first: Mei

last: Yang

first: Guangming

last: He

first: Xing

last: Deng


title: Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID

city: Aurora

first: Srinivas

last: Ramachandran

state: CO

department: Biochemistry and Molecular Genetics

institute: University of Colorado School of Medicine

summary: Previous studies suggested that expression of most yeast mRNAs is dominated by either transcription factor TFIID or SAGA.  We reexamined this longstanding problem by rapid depletion of TFIID subunits and measurement of changes in nascent transcription.  We find that transcription of nearly all mRNAs is strongly dependent on TFIID function.  Degron-dependent depletion of Tafs 1,2,7,11, and 13 showed similar transcription decreases for genes in the Taf1-depleted, Taf1-enriched, TATA-containing and TATA-less gene classes.  The magnitude of TFIID-dependence varies with growth conditions, although this variation is similar at all gene classes.  Many studies have suggested differences in gene regulatory mechanisms between TATA and TATA-less genes and these differences have been attributed in part to differential dependence on SAGA or TFIID.  Our work indicates that TFIID contributes significantly to expression of nearly all yeast mRNAs and that differences in regulation between these two gene classes is due to other properties.

overall_design: We used the auxin-inducible degron system to rapidly deplete selected TFIID subunits and Med14 subunit of Mediator. We then monitored genome-wide nascent transcription using native Pol II ChIP to analyze the position of Pol II elongation complexes before and after auxin-induced depletion of selected TFIID subunits and Med14 subunit of Mediator.

first: Linda

last: Warfield

first: Steven

last: Hahn


title: RNA-seq analysis of wild type and upf1 knockout strains in the filamentous fungus Neurospora crassa.

city: Guangzhou

first: Yin

last: Zhang

address: No. 135, Xingang Xi Road

institute: SYSU

summary: Analysis of genes differentially expressed in wild type and upf1 knockout strains

overall_design: Total RNA obtained from isolated Neurospora crassa strains (wild type and upf1 knockout).

first: Jinhu

last: Guo


title: Measuring the effect of MYC on transcription during the DNA double-strand break response by RNA-seq of newly synthesized transcripts

first: Eric

last: Batchelor

address: 10 Center Dr

department: LP/CCR/NCI

institute: National Institutes of Health

summary: We sequenced newly synthesized mRNA to determine which genes are differentially transcribed during the DNA double-strand break (DSB) response. Moreover, as the transcription factor MYC is repressed in a p53-dependent manner during the DSB response, we determined the effect of this repression on transcription by maintaining MYC above its basal expression level. To control for differences in cell size, we spiked in S. pombe RNA proportional to cell number, then normalized human transcript counts to S. pombe transcript counts. We found that ~77% of transcripts were produced at a lower level during the time interval 2.5‑3.5 h after treatment with neocarzinostatin (NCS), which causes DSBs, than in a one-hour time interval without NCS treatment. When MYC was maintained above its basal level during the DSB response, only ~59% of transcripts were produced at a lower level in NCS-treated cells. Known p53 target genes were upregulated during the same time interval, and maintaining MYC above its basal level did not change this significantly. Consistent with previous observations, we found that increasing MYC levels increased transcription of most genes, with a larger effect on more highly transcribed genes. We observed the opposite in NCS-treated cells, in which more highly transcribed genes were downregulated to a greater degree. These data support a model that p53, by repressing MYC, redistributes transcription during the DSB response, inhibiting global transcription while activating transcription of specific genes involved in the response.

overall_design: mRNA transcribed over a 1-h interval in cells with/without DNA double-strand breaks and with/without increased MYC levels, in triplicate. RNA from S. pombe was spiked in proportional to cell number.

first: Joshua

last: Porter


title: RNA-sequencing of human liver tissue

city: Huntsville

first: Ryne

last: Ramaker

state: AL

address: 601 Genome Way

department: Genetics

institute: University of Alabama at Birmingham

summary: We performed quadruplicate RNA-sequencing on two human livers

overall_design: RNA-sequencing of 2 fresh frozen human liver tissues

first: Dan

last: Savic

first: Sara

last: Cooper

first: Rick

last: Myers


title: Expression data from AEG-1fl/fl and AEG-1DHEP mice which have conditional hepatocyte-specific knockout of AEG-1 (Astrocyte elevated gene 1)

city: Richmond

first: Mikhail

last: Dozmorov

state: VA

address: 830 E Main St

department: Biostatistics

institute: Virginia Commonwealth University

summary:
AEG-1 is overexpressed in human hepatocellular carcinoma (HCC) and positively regulates development and progression of HCC
A conditional hepatocyte-specific knockout mouse was generated by crossing floxed AEG-1 mouse with Alb/Cre mouse in C57BL/6 background

overall_design: Livers were harvested from chow-fed 12 wks old AEG-1fl/fl and AEG-1DHEP mice. RNA was extracted and subjected to RNA-Seq.

first: Devanand

last: Sarkar


title: The Xenopus Primordial Germ Cell Transcriptome: Unexpected Role for sox7 in Early PGC Development

status: Public on Aug 01 2017

city: Miami

first: Mary Lou

last: King

department: Cell Biology

institute: University of Miami, Miller School of Medicine

summary: Xenopus primordial germ cells (PGCs) are determined by the presence of maternally derived germ plasm. Germ plasm components both protect PGCs from somatic differentiation and begin a unique gene expression program. Segregation of the germline from the endodermal lineage occurs during gastrulation and PGCs subsequently initiate zygotic transcription. However, the gene-networks that operate to both preserve the potential for totipotency and promote germline differentiation are poorly understood. Here, we utilized RNA-sequencing analysis to comprehensively interrogate PGC and neighboring endoderm cell RNAs after lineage segregation. We identified 1,865 transcripts enriched in PGCs compared to endoderm cells. Over 50% of maternal, vegetally-enriched transcripts were enriched in the PGC transcriptome, including sox7. PGC-directed sox7 knockdown and over-expression studies revealed an early requirement for sox7 in proper germ plasm localization, zygotic transcription, and PGC number. We identified oct60 as the most highly expressed and enriched OCT3/4 homologue in PGCs. Lastly, we compared the Xenopus PGC transcriptome with human PGC transcripts and showed that 80% of transcripts are conserved, identifying Xenopus as a relevant model system for understanding the gene-networks necessary for human germline development.

overall_design: Examination of X. laevis primordial germ cell (PGC) and neighboring endoderm cell (Endo) RNAs after lineage segregation to determine PGC-enriched transcripts that may contribute to germline development.

first: Amanda

middle: M

last: Butler

first: Dawn

last: Owens

first: Lingyu

last: Wang

first: Mary


title: RNA-Seq profile of PSGL-1loCD4+ T cells from chronic GVHD mice

city: Duarte

first: Xiwei

last: Wu

state: CA

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

institute: City of Hope National Medical Center

summary: Our studies have shown that CD44hiCD62LloPSGL-1loCD4+ T (PSGL-1loCD4+ T) cells play an important role in chronic GVHD pathogenesis (R. Deng et al: Nature Communications 2017).  Thus, PSGL-1loCD4+ T cells were sorted from spleen of chronic GVHD BALB/c recipient mice transplanted with spleen cells and T cell-depleted bone marrow (TCD-BM) cells from C57BL/6 donor mice. The control no GVHD BALB/c recipient mice were transplanted with C57BL/6 donor TCD-BM only.  21 days after transplantation, PSGL-1loCD4+ T cells from spleen of chronic GVHD and control mice were sorted and subjected to RNA-seq analysis.

overall_design: Spleen cells from 8 chronic GVHD recipients or control recipients were combined. PSGL-1loCD4+ T cells from each group were sorted with flow cytometry. mRNA of sorted PSGL-1lo cells were extracted and RNA-seq was performed with duplicates.

first: Ruishu

last: Deng

first: Defu

last: Zeng


title: Bacterial dispersal on fungal hyphae

city: Medford

first: Benjamin

middle: E

last: Wolfe

address: 200 Boston Ave

department: Microbiology

institute: Tufts University

summary: The rate, timing, and mode of species dispersal is recognized as a key driver of the structure and function of communities of macroorganisms, and may be one ecological process that determines the diversity of microbiomes. Many previous studies have quantified the modes and mechanisms of bacterial motility using monocultures of a few model bacterial species. But most microbes live in multispecies microbial communities, where direct interactions between microbes may inhibit or facilitate dispersal through a number of physical (e.g., hydrodynamic) and biological (e.g., chemotaxis) mechanisms, which remain largely unexplored. Using cheese rinds as a model microbiome, we demonstrate that physical networks created by filamentous fungi can impact the extent of small-scale bacterial dispersal and can shape the composition of microbiomes. From the cheese rind of Saint Nectaire, we serendipitously observed the bacterium Serratia proteamaculans actively spreads on networks formed by the fungus Mucor. By experimentally recreating these pairwise interactions in the lab, we show that Serratia spreads on actively growing and previously established fungal networks. The extent of symbiotic dispersal is dependent on the fungal network: diffuse and fast-growing Mucor networks provide the greatest dispersal facilitation of the Serratia species, while dense and slow-growing Penicillium networks provide limited dispersal facilitation. Fungal-mediated dispersal occurs in closely related Serratia species isolated from other environments, suggesting that this bacterial-fungal interaction is widespread in nature. Both RNA-seq and transposon mutagenesis point to specific molecular mechanisms that play key roles in this bacterial-fungal interaction, including chitin utilization and flagellin biosynthesis. By manipulating the presence and type of fungal networks in multispecies communities, we provide the first evidence that fungal networks shape the composition of bacterial communities, with Mucor networks shifting experimental bacterial communities to complete dominance by motile Proteobacteria. Collectively, our work demonstrates that these strong biophysical interactions between bacterial and fungi can have community-level consequences and may be operating in many other microbiomes.

overall_design: Serratia spp. was grown with or without Mucor spp. Present

first: Erik

last: Kastman


title: The Dynamic Epigenetic Landscape of the Retina During Development, Reprogramming, and Tumorigenesis [ATAC-Seq_Mm]

city: Memphis

first: Beisi

last: Xu

state: Tennessee

address: 262 Danny Thomas Pl

department: Center for Applied Bioinformatics

institute: St Jude Children's Research Hosipital

summary: In the developing retina, as in many other regions of the central nervous system, multipotent neural progenitor cells undergo unidirectional changes to produce differentiated cells in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinal development in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell type–specific differentiation programs. We identified developmental stage–specific superenhancers and showed that the majority of epigenetic changes during murine retinal development are conserved in the human retina. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed the same integrated epigenetic analysis of murine and human retinoblastomas and iPSCs derived from murine rod photoreceptors.  The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from a neurogenic to a terminal pattern of cell division and murine retinoblastomas initiate earlier in development than human tumors. The epigenome of retinoblastomas was far more similar to that of normal retina than was the epigenome of retinal-derived iPSCs but we were able to identify retinal specific epigenetic memory. Together, these data provide an in depth view of the dynamic epigenome during neurogenesis and how that relates to developmental tumors and epigenetic memory in iPSCs produced from neurons.

overall_design: Examination of 8 different histone modifications and 3 transcription factors, transcriptome, DNA methylation in 23 cell types

first: Issam

last: Aldiri

first: Lu

first: Xiang

last: Chen

first: Daniel

last: Hiler

first: Lyra

last: Griffiths

first: Marc

last: Valentine

first: Abbas

last: Shirinifard

first: Marie-Elizabeth

last: Barabas

first: Jiakun

last: Zhang

first: Dianna

last: Johnson

first: Sharon

last: Frase

first: John

last: Easton

first: Jinghui

first: Elaine

last: Mardis

first: Richard

last: Wilson

first: James

last: Downing

first: Michael

last: Dyer


title: Epigenome profiling and editing of neural progenitor cells in the developing mouse neocortex

first: Mareike

last: Albert

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

overall_design: Refer to individual Series


title: Synergistic activity and heterogeneous acquired resistance of combined MDM2 and MEK inhibition in KRAS mutant cancers

city: Charlestown

first: Aaron

last: Hata

address: 149 13th Street

department: Cancer Center

institute: Massachusetts General Hospital

summary: There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) + SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and CRC.

overall_design: A427, DV-90, H1944, H358, LU-99A, SW1573 cells treated with 10 nM, 100 nM, 1000 nM pimasertib, SAR405838 or combination (3 replicates each)

first: Steve

last: Rowley

first: Fei

last: Ji

first: Ruslan

middle: I

last: Sadreyev


title: RNA-sequencing profiles of five different pancreas cancer cell lines that were engineered to express a form of the basic helix-loop-helix transcription factor, E47, that localizes to the nucleus in response to tamoxifen.

city: La Jolla

first: Roman

last: Sasik

address: 9500 Gilman Dr.

department: Center for Computational Biology and Bioinformatics

institute: UCSD

summary: E47 had been shown previously to inhibit cell cycle progression in pancreas cancer cell lines. This study was designed to identify global changes in gene expression that occurred in response to E47 with the goal of identifying molecular mechanisms involved inE47-mediated inhibition of the cell cycle.

overall_design: Total RNA was isolated from No Tamoxifen (NT) and With Tamoxifen (WT) treated samples 48-72 hrs after induction of nuclear E47, sequenced and analyzed to identify candidate regulators of cell cycle inhibition that were common to all five cell lines examined.

first: Kathleen

last: Scully

first: Reyhaneh

last: Lahmy

first: Pamela

last: Itkin-Ansari


title: Rho dependent transcription termination is a dominant mechanism of transcription termination in Mycobacterium tuberculosis

city: Bangalore

first: Valakunja

last: Nagaraja

state: Karnataka

address: C V Raman Avenue

department: Department of Microbiology and Cell Biology

institute: Indian Institute of Science (IISc)

overall_design: anti-Rho ChIP-seq of Mycobacterium tuberculosis H37Rv

first: A

last: Mitra

first: W

last: Ahmed

first: C

last: Sala

last: Ezaz

first: S

last: Hegde

middle: T

last: Cole

first: V


title: YTHDC2 regulates spermatogenesis through promoting the translation of N6-methyladenosine-modified RNA

city: Chicago

first: zhike

last: lu

state: IL

address: 5801 South Ellis Avenue

department: department of chemistry

institute: University of Chicago

summary: N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as an essential layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to promote the translation of m6A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the splicing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6A along its consensus motif GGACU. YTHDC2 promotes translation of its targets, and associates with cellular fractions involved in translation initiation. Ythdc2 knockout mice are infertile and have significantly smaller testes compared to those of littermates. In the testes, Ythdc2 is temporally expressed as meiosis begins, and germ cells of Ythdc2 knockout mice do not develop past the spermatogonium stage. Thus, YTHDC2 is an m6A binding protein that plays essential roles in spermatogenesis.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We used CLIP-seq replicates and RIP-seq replicates to identify the binding targets and sites of YTHDC2. We used m6A-seq to identify changes in m6A and RNA expression in Ythdc2-/- mice

first: Phillip

middle: J

last: Hsu

first: Yunfei

last: Zhu

first: Honghui

last: Ma

first: Yiqiang

last: Cui

first: Xiaodan

last: Shi

first: Zhike

last: Lu

first: Hailing

first: Guanzheng

last: Luo

first: Qing

last: Dai

first: Bin

last: Shen

first: Chuan

last: He


title: RNA-seq analysis of gene expression levels duing callus formation on CIM using WT and lbd16-2

city: Shanghai

first: Lin

last: XU

address: 300 Fenglin Road

department: Chinese Academy of Sciences

institute: Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences

summary: To study the molecular mechanism of how LBD16 contributes to pluripotency acquisition in callus cells, we performed RNA-seq to analyze its potential downstream genes, using leaf explants from wild-type Col-0 and lbd16-2 before culture (0 d) and at 4 days on CIM. Our study suggests that gain of the root primordium-like identity is the key event for callus to acquire pluripotency, and LBD16 gene is a specific root primordium gene that ensures the callus to be pluripotent.

overall_design: Total RNA obtained from Arabidopsis wild-type Col-0 or lbd16-2 mutant leaf explants at time 0 and 4 days on callus inducing medium (CIM).

last: Xu

first: Jie

last: Liu


title: Identification of splice variants and RNA binding partners of STAT3-induced long noncoding RNAs (STAiRs) in multiple myeloma

city: Leipzig

first: Stefanie

last: Binder

address: Perlickstr. 1

department: Diagnostics

institute: Fraunhofer Institute for Cell Therapy and Immunology

summary: Interleukin 6 (IL-6) activates the transcription factor signal transducer and activator of transcription 3 (STAT3). We previously identified several STAT3-induced long noncoding RNAs (STAiRs) in INA-6 multiple myeloma cells (Hackermüller et al. 2014). Here, we analyze five STAiRs by CAPTURE- and ChIRP-RNA-sequencing, a pulldown of a target RNA by transcript tiling oligonucleotides. The CAPTURE technique allows the identification of different STAiR splice variants and shall give rise to the transcript architecture. ChIRP, a method first published by Chu et al 2011, is the original method on which the CAPTURE approach is based on and enables the identification of RNAs interacting with our STAiRs.

overall_design: Identification of (1) the transcript architecture analyzed by Capture-seq and (2) the RNA interaction partners analyzed by ChIRP-seq of five different STAiRs in INA-6 multiple myeloma cells

first: Diana

last: Riedel

first: Tilo

last: Buschmann

first: Kristin

last: Reiche


title: RNA-seq on developing mouse heart

city: Bunkyo-ku

first: Yutaro

last: Hori

state: Tokyo

department: Department of Scinece

country: Japan

institute: The University of Tokyo

summary: In order to compehensively determine long non-coding RNAs (lncRNAs) expressed in the mouse heart, we conducted RNA-seq on heart ventricles at three stages. We found that although lncRNAs are generally at a distance from protein-coding genes, cardiac transcription factor genes have lncRNAs in their proximity. Detailed examination revealed that many of these ln cRNAs were transcribed from the neighboring genes' promoter in a head-to-head divergent manner.

overall_design: Paired-end RNA-seq on mouse heart ventricles. One sample for each stage.


title: Diverse Repetitive Element RNA Expression Define Epigenetic and Immunologic Features of Colon Cancer

city: Boston

first: David

last: Ting

state: MA

address: 149 E 13th street

summary: There is tremendous excitement for the potential of epigenetic therapies in cancer, but the ability to predict and monitor response to these drugs remains elusive. This is in part due to the inability to differentiate the direct cytotoxic and the immunomodulatory effects of these drugs on cancers. The DNA hypomethylating agent 5-azacytdine (AZA) has shown these distinct effects in a variety of cancers, and appears to be linked to the de-repression of repetitive element RNA. Although AZA generally induces global de-repression of repeats, we have found that there is significant diversity of repeat behavior that has not been well characterized in cancer. LINE and HERV are two of the largest classes of repeat elements in the human genome, and despite many commonalities, we find there is heterogeneity in behavior amongst different subtypes of repeats. Specifically, the LINE-1 and HERV-H subtypes were found to be abundant across colon cancers by RNA-seq and RNA in situ hybridization, but had distinct expression patterns, which suggested that these repeats are correlated to transcriptional programs marking different biological states in cancer cells. We find that low LINE-1 expression correlates with global DNA hypermethylation, wildtype TP53 status, and responsiveness to AZA. HERV-H repeats were not concordant with LINE-1 expression, but were found to be linked with differences in FOXP3+ regulatory T-cell tumor infiltrates. Together, distinct repeat RNA expression patterns define new molecular classifications of colon cancer and provide biomarkers that better distinguish cytotoxic from immunomodulatory effects by epigenetic drugs.

overall_design: There are a total of 15 bisulfite genome sequencing samples from 3 colon cancer cell lines (HCT116, HCT8, and DLD1).  Each cell line has been treated at different concentrations of 5-azacitidine (0, 500, 1000 nM) with technical replicates for the 0 and 1000 nM concentrations


title: DNA Methylation of Synaptic Genes in the Prefrontal Cortex is Associated with Aging and Age-Related Cognitive Impairment

city: Gainesville

first: Thomas

last: Foster

state: Florida

address: 1149 Newell Drive Bldg 59

department: Neuroscience

institute: University of Florida-McKnight Brain Institute

summary:
This study investigated the epigenetic mark of DNA methylation in the medial prefrontal cortex (mPFC) as a function of age and cognition. Young and aged F344 rats were characterized in a cognitive flexibility task, set shifting, and whole-genome bisulfite sequencing was performed in an Illumina system.
The results indicate that differential methylation was linked to the expression of genes within functional categories which may mediate impaired cognition in aging. Differences in aging included hypermethylation of genes linked to synaptic function and GTPase activity. Further, age-related cognitive flexibility impairment was correlated to hypermethylation of synaptic, postsynaptic density and ion channel activity genes.

overall_design: The study contains 10 young and 20 aged rats utilized to investigate the epigenetic alterations in the mPFC associated to aging and executive function.

first: Lara

last: Ianov

first: Alberto

last: Riva

first: Ashok

last: Kumar


title: Replication defective viral products exploit a cellular pro-survival mechanism to establish persistent infections

city: St Louis

first: Carolina

last: Lopez

state: MO

address: 425 S Euclid Ave

institute: Washington University in St. Louis

summary: Replication defective viral genomes (DVGs) generated during virus replication are the primary triggers of antiviral immunity in many infections. However, DVGs also provide viruses with an evolutionary advantage by facilitating virus persistence. Why and how DVGs and the host interact to achieve these two divergent functions remains unknown. We report that DVGs engage a MAVS-mediated antiviral response that promotes apoptosis on highly infected cells while protecting cells dominated by DVGs from death. We find that MAVS signaling leads to the expression of TNF and of the pro-survival TNFR2/TRAF1 proteins. TNF induces apoptosis of infected cells lacking DVGs, while its signaling in cells enriched in DVGs promotes their survival. These results identify DVGs as pivotal for virus-host co-existence and reveal a MAVS-mediated axis that while promoting the death of infected cells, protects those cells producing toxic cytokines from apoptosis, thereby facilitating viral persistence.

overall_design: Four sub-populations of cells (ND, FL-high, DVG-high and INT) were sorted from single viral infection previously by FISH FACS sorting. RNA-seq was performed in two technical repeats with the RNA sample of each repeats pooled from 3 independent FISH-FACS sortings (6 independent sortings in total).

first: Daniel

last: Beiting

first: Yan

last: Sun

last: Xue


title: Blimp1 induces transient metastatic heterogeneity in pancreatic cancer [688M_ATAC-seq]

city: Stanford

first: Shin-Heng

last: Chiou

address: 450 Serra Mall

institute: Stanford University

summary:
Purpose: Due to its high metastatic proclivity, pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly types of cancer.  Therefore, it is imperative to better understand how the disease spreads as it progresses. Using a novel genetically engineered mouse model that allows us to isolate a subpopulation of cancer cells with superior metastatic capacity, we show that this aggressive phenotype correlates exclusively with a strong hypoxia signature. We subsequently identified the novel hypoxia-inducible gene Blimp1, which appears to play a critical role in regulating the hypoxic response upon its induction. Furthermore, genetic ablation of Blimp1 greatly reduces the level of metastasis in a PDAC mouse model. The nature of this Blimp1-regulated hypoxia signature is very unstable, since the seeded metastatic lesions mostly re-adopt similar transcriptomic profiles as the primary tumors. In conclusion, our results offer a potential mechanistic insight into how hypoxia drives metastasis in PDAC.
Methods: The liver metastasis cell line 688M was subjected to control or knockdown of Blimp1 before assay. Cells ere validated for knockdown efficiency and then cultured under normoxia and hypoxia (0.5% O2) for 24 hours before preparation for ATAC-Seq (total of 4 groups with 2 technical replicates per group, overall 8 samples). Reference for ATACSeq: Buenrostro et al. 2013. Nat Methods 12:1213-8.
Results: For the control knockdown group, 0.5% O2 culture (hypoxia) for 24 hours induced dramatic changes in global genome accessibility, and Blimp1 knockdown appeared to induce minimum changes in chromatin accessibility under hypoxia or normoxia (20% O2).
Conclusions: Compared to our RNASeq profiles of the same liver met PDAC cell line under identical conditions, Blimp1 appeared to impact a global gene expression changes under hypoxia that is not associated with a corresponding changes of chromatin accessibility.

overall_design: Transcriptomes of derivatives of liver metastasis cell line 688M (control and Blimp1 knockdown) derived from the KPCT (Kras-LSL-G12D;p53-LSL-R172H;Pdx1-cre;R26-LSL-Tomato) mice were cultured under 0.5% or 20% O2 for 24 hours before ATAC-Seq preparation (Illumina NextSeq).

first: Monte

last: Winslow

first: Shin-Hen


title: NAFLD in IL-4 and IL-10/4 KO Mice

city: Bethesda

first: Timothy

middle: G

last: Myers

address: 50 South Drive, Room 5509

department: Research Technologies Branch

institute: National Institute of Allergy and Infectious Diseases

summary: Whole liver RNA isolated from wild type, IL-4 knockout and IL-10/4 double knockout mice on normal or high fat diet for 15 weeks.

overall_design: 23 samples, 4 mice from each genotype and diet except IL-4 knockout HFD, three mice.

last: Wynn

first: Kevin

last: Hart


title: RNA-seq on Tbx5ua Knockdown mice

summary: By comprehensive screening of long non-coding RNAs (lncRNAs) over mouse heart development, we have identified Tbx5 upstream antisense RNA (Tbx5ua). In order to understand its function, we produced Tbx5ua knock down ES cells by inserting triple bovine polyadenylation signal to the second exon of Tbx5ua. From the ES cells we made chimeric mouse embryos via tetraploid complementation assay. We conducted RNA-seq analysis on the WT and KD heart ventricles at E9.5 to further elucidate the lncRNA's molecular functions.

overall_design: Single-end RNA-seq of total RNAs extracted from the ventricles of tetraploid chimeric mice generated from wildtype or Tbx5ua knockdown B6J ES cells


title: Blimp1 induces transient metastatic heterogeneity in pancreatic cancer [RNA-seq]

summary:
Purpose: Due to its high metastatic proclivity, pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly types of cancer.  Therefore, it is imperative to better understand how the disease spreads as it progresses. Using a novel genetically engineered mouse model that allows us to isolate a subpopulation of cancer cells with superior metastatic capacity, we show that this aggressive phenotype correlates exclusively with a strong hypoxia signature. We subsequently identified the novel hypoxia-inducible gene Blimp1, which appears to play a critical role in regulating the hypoxic response upon its induction. Furthermore, genetic ablation of Blimp1 greatly reduces the level of metastasis in a PDAC mouse model. The nature of this Blimp1-regulated hypoxia signature is very unstable, since the seeded metastatic lesions mostly re-adopt similar transcriptomic profiles as the primary tumors. In conclusion, our results offer a potential mechanistic insight into how hypoxia drives metastasis in PDAC.
Methods: Pure, paired GFP-negative/Tomato-positive and GFP-positive/Tomato-positive cancer cells or pure Tomato-positive cancer cells were sorted from primary PDAC samples from 6 KPC-colors mice or KPCT mice, respectively, with the following criteria: single cell based on FSC-A/H; CD45-negative; CD31-negative; Ter119-negative; F4/80-negative; DAPI-negative; and Tomato-positive. RNA were extracted from 10^4 to 5x10^4 freshly sorted cancer cells using AllPrep DNA/RNA Micro Kit (Qiagen). RNA quality was assessed with the RNA6000 PicoAssay kit by using the Bioanalyzer 2100 (Agilent).  All ex vivo RNA samples used for RNA-seq analyses had an RIN > 8.0. Total RNA (15 ng/sample) was used for cDNA synthesis and amplification with the Ovation RNA-Seq system (NuGEN Technologies, Inc.). Subsequently, the amplified DNA samples were fragmented through sonication (Covaris model S1) and subjected to library preparation using the Illumina TruSeqTM DNA sample preparation kit (Low-Throughput protocol) according to manufacturer's protocol. The quality of purified cDNA library products was confirmed by bioanalyzer and prepared for cluster generation on HiSeq paired-end flow cells using the CBot automated cluster generation system followed by sequencing on HiSeq 2000 machines. We obtained 101bp, paired-end reads from fragments of an average length of 250bp. Subsequently, RNA-Seq reads were aligned to the mouse genome (mm10) using the STAR aligner with standard input parameters (Dobin et al., 2013). The number of reads uniquely aligned to exons of individual genes were counted with HTSeq against the UCSC KnownGene (mm10) transcriptome (Anders et al., 2015).
Results: Compared to the GFP-negative counterparts, GFP-positive pure PDAC cancer cells express higher levels of genes that are highly enriched with hypoxia signature. Additionally, compared to the GFP-negative counterparts, GFP-positive pure PDAC cancer cells express lower levels of cell cycle-related genes. In contrast, pure cancer cells isolated based on locations reveal few consistent differentially expressed genes between primary tumor and liver metastases; no consistent differentially expressed gene between primary tumor and lymph node metastases.
Conclusions: Transcriptome profiles of both GFP-negative/positive PDAC cancer cells suggest that Hmga2/GFP-expressing cancer cells are highly enriched for signatures that correspond to cells residing within hypoxic enrivonment.

overall_design: Freshly sorted GFP/Hmga2-positive and GFP/Hmga2-negative PDAC cancer cells derived from tumors of 6 KPCT;Hmga2-CK/+ (KPC-colors) mice were subjected to transcriptome profiling by paired-end RNA-Seq (total of 6 pairs of samples with overall 12 samples). Additionally, pure Tomato-positive PDAC cancer cells isolated from different anatomical locations were also subjected to transcriptome profiling by paired-end RNA-Seq (n = 23, not including technical replicates).


title: Blimp1 induces transient metastatic heterogeneity in pancreatic cancer


title: Single cell sequencing of primary dorsal root ganglia from CAST/Ei or C57BL/6 mouse highlights strain differences in cellular populations.

city: Santa Barbara

first: Kenneth

middle: S.

last: Kosik

state: California

address: UC Santa Barbara

department: NRI

institute: UCSB

summary: Peripheral nerve regeneration after injury is a complex process involving a large number of transcriptional changes. How these changes impact the regenerative outcome is though, poorly understood. Here, we take advantage of the genetically based differences in the peripheral and central regenerative capacity of CAST/Ei and C57BL/6j inbred mice to better understand the molecular bases driving superior regeneration in the CAST/Ei mouse strain. Single cell RNA sequencing highlighted the existence of three populations of cells, one of which expressed genes enriched for mature neuronal function, another one had a gene expression profile enriched for an immature dedifferentiated state while the third one showed an intermediate profile between these two extremes. The immature expression state was observed after injury in both strains but a larger proportion of the CAST/Ei neurons retained an expression pattern consistent with neuronal identity whereas a larger proportion of C57BL/6 neurons acquired an immature gene expression state accompanied by expression of stress markers. This finding suggests that unexpectedly, maintenance of a mature differentiated state in injured neurons increased regenerative success.

overall_design: Mouse DRG from two strains (CAST/Ei or C57BL/6) were dissected and processed into single cell suspension. The naive cells were loaded into a fluidigm C1 for cell capture, lysis and cDNA preamplification. IVA (in vitro axotomy) cells were plated for 48h before being resuspended and captured as the naive cells. Overall, 516 individual cells were analyzed.

first: Véronique

last: Lisi

first: Bhagat

last: Singh

first: Michel

last: Giroux

first: Elmer

last: Guzman

first: Michio

last: Painter

first: Yung-Chih

last: Cheng

first: Eric

last: Huebner

first: Giovanni

last: Coppola

first: Michael

last: Costigan

first: Clifford

last: Woolf


title: Loss of Apela peptide in mice causes low penetrance embryonic lethality and defects in early mesodermal derivatives

city: Paris

first: Laina

last: Freyer

address: 25-28 rue du Dr Roux

department: Developmental and Stem Cell Biology

country: France

institute: Institut Pasteur

summary: Apela (also referred to as Elabela, Ende and Toddler) is a small signaling peptide that activates the G protein-coupled receptor Aplnr. We used CRISPR/Cas9 to generate a null, reporter-expressing allele, in order to study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low penetrance cardiovascular defects that manifest after the onset of circulation. Targeted Apela null alleles exhibited different transcriptional activity depending on the presence or absence of a Neomycin selection cassette. These are referred to as Apela KO NEO-IN and Apela KO NEO-OUT strains, respectively. Despite subtle phenotypic characteristics that were unique to the NEO-OUT mutants, both Apela null strains shared the same variable expressivity of cardiovascular defects and the same penetrance of embryonic lethality. To investigate the earliest regulatory events leading to physical abnormalities in Apela mutants, we performed RNA-Seq on whole stage-matched and morphologically normal E7.5 embryos (3 wild-type, 6 Apela KO NEO-IN, and 6 Apela KO NEO-OUT individuals). We chose this stage because Apela is initially expressed in the embryo at late gastrulation, shortly after the emergence of extraembryonic mesoderm progenitors. Since modification of the Apela locus may influence the expression of neighboring genes, we examined the expression of upstream and downstream sequences and found no significant difference in their expression. Downregulated genes of interest included several mitochondrial genes, Ceacam2, Ulk4, and Mov10l1. Upregulated genes included the vascular endothelial growth factor Vegfc. Principal component analysis identified outliers (KO1 and KO9), both of which expressed lower levels of mesoderm markers. KO9 was further characterized by aberrant upregulation of erythroid and myeloid markers. This finding was confirmed in our study by qRT-PCR analysis of additional Apela null individuals.

overall_design: 15 individual embryos were analyzed at E7.5. Embryos were stage-matched according to morphological landmarks. Control samples were wild-type (n=3), and Apela KO samples were null embryos from the NEO-IN (n=6, ‘KO1-6’) and NEO-OUT (n=6, ‘KO7-12) mutant strains. Whole embryos (including embryonic and extraembryonic tissues) were used for the analysis. Apela KO samples were isolated from homozygous KO intercrosses and therefore did not require genotyping.

first: Anna-Katerina

last: Hadjantonakis


title: PGK cleavage mapping

first: Hani

last: Zaher

address: 1 Brookings Drive

department: Biology

institute: Washington University in St Louis

summary: Mapping 3'ends of cleaved mRNAs derived from a reporter that is subject to no-go decay for the purpose of characterizing the cleavage reaction.

overall_design: Libraries constructed from mRNA collected from S. cerevisiae expressing a PGK-stall seqeunce reporter gene. Three different libraries were seqeunced, each expressing a different stalling reporter: a stem loop, (CGA)12 or (AAA)12.

first: Carrie

middle: L

last: Simms


title: Blimp1 induces transient metastatic heterogeneity in pancreatic cancer [688M_RNA-seq]

first: Shin-Heng

last: Chiou

department: Genetics

summary:
Purpose: Due to its high metastatic proclivity, pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly types of cancer.  Therefore, it is imperative to better understand how the disease spreads as it progresses. Using a novel genetically engineered mouse model that allows us to isolate a subpopulation of cancer cells with superior metastatic capacity, we show that this aggressive phenotype correlates exclusively with a strong hypoxia signature. We subsequently identified the novel hypoxia-inducible gene Blimp1, which appears to play a critical role in regulating the hypoxic response upon its induction. Furthermore, genetic ablation of Blimp1 greatly reduces the level of metastasis in a PDAC mouse model. The nature of this Blimp1-regulated hypoxia signature is very unstable, since the seeded metastatic lesions mostly re-adopt similar transcriptomic profiles as the primary tumors. In conclusion, our results offer a potential mechanistic insight into how hypoxia drives metastasis in PDAC.
Methods: The liver metastasis cell line 688M was subjected to control or knockdown of Blimp1 before assay. Cells ere validated for knockdown efficiency and then cultured under normoxia and hypoxia (0.5% O2) for 24 hours before preparation for RNA-Seq (total of 4 groups with duplicates, overall 8 samples).
Results: For the control knockdown group, 0.5% O2 culture(hypoxia)  for 24 hours induced dramatic changes in global gene expression, and Blimp1 knockdown potently mitigated changes of significant amount of genes induced under hypoxia. In brief, cell cycle-related genes that are normally suppressed by hypoxia (which contributes to hypoxia-induced cell cycle arrest) are not suppressed under hypoxia in the Blimp1 knockdown cells. In contrast, ~35% of genes that are induced under hypoxia in the control knockdown cells are instead induced less than 50% (under hypoxia) upon Blimp1 knockdown.
Conclusions: Blimp1 is a critical regulator of hypoxic response in pancreatic cancer.

overall_design: Transcriptomes of derivatives of liver metastasis cell line 688M (control and Blimp1 knockdown) derived from the KPCT (Kras-LSL-G12D;p53-LSL-R172H;Pdx1-cre;R26-LSL-Tomato) mice were cultured under 0.5% or 20% O2 for 24 hours before RNA-Seq preparation (Illumina TruSeq).

first: Monte

last: Winslow

first: Shin-Hen


title: Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs (MethylC-seq)

city: Columbia

first: Md Shakhawat

last: Hossain

state: Missouri

address: 201 Life Science Center 1201 Rollins Road

department: Plant Sciences

institute: University of Missouri

summary:
Chromatin modifications, such as cytosine methylation of DNA, play a significant role in mediating gene expression in plants, which affects growth, development, and cell differentiation. Since root hairs are single cell extensions of the root epidermis and the primary organs for water uptake and nutrients, we sought to use root hairs as a single cell model system to measure the impact of environmental stress.
We measured changes in cytosine DNA methylation in single cell root hairs as compared to multi-cellular stripped roots, as well as in response to heat stress.
Differentially methylated regions (DMRs) in each methylation context showed very distinct methylation patterns between cell types and in response to heat stress. Intriguingly, at normal temperature, root hairs were more hypermethylated as compared to stripped roots. However, in response to heat stress, both root hairs and stripped roots showed more hypomethylation in each context, especially in the mCHH context. Moreover, expression analysis of mRNA from similar tissues and treatments identified some associations between DMRs, genes and transposons.
Taken together, the data indicate that changes of dynamic DNA methylation directly or indirectly is associated with expression of genes and transposons either within the context of specific tissues/cells or stress (heat).

overall_design: MethylC-seq from soybean root hair cells and stripped root cells under normal and heat stress condition

first: Gary

last: Stacey