title: microRNAs are negative and positive post-transcriptional regulators of BRAF-X1 mRNA [miR-CATCH]

first: Romina

last: D'Aurizio

summary: X1 is the most abundant transcript variant of BRAF mRNA and has a long 3’UTR, but its involvement in post-transcriptional regulatory circuits has not been investigated yet.   Here, we describe 20 microRNAs that bind directly to the X1 3’UTR. They were identified in BRAFV600E mutant A375 melanoma cells using miR-CATCHv2.0, an implemented experimental method that combines RNA affinity purification with small RNA sequencing and an ad hoc analytical workflow. X1-targeting microRNAs fall into 4 classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and on the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). In many cases, the microRNA-induced variations in BRAFV600E protein levels are coupled to consistent variations in pMEK levels and, in turn, to melanoma cell proliferation and sensitivity to the BRAF inhibitor vemurafenib. However, examples exist of microRNAs that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein.   Our study describes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.

overall_design: microRNAs that specifically bind the X1 isoform of BRAF were identified by small RNA sequencing, after the X1 3'UTR pulldown was obtained using miR-CATCHv2.0 protocol. Three independent captures performed with ODD and EVEN probes were analyzed.

first: Andrea

last: Marranci

first: Milena

last: Rizzo

first: Laura

last: Poliseno


title: RNA-seq profiling of neural tissue from mouse embryos with combined loss of p53 and Bim

status: Public on Mar 26 2019

city: Parkville

first: Gordon

middle: K

last: Smyth

state: Vic

address: 1G Royal Pde

department: Bioinformatics

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

type: Expression profiling by high throughput sequencing

summary: Neural tube defects (NTDs) are common birth defects in humans and show an unexplained female bias. Female mice lacking the tumor suppressor p53 display NTDs with incomplete penetrance. We found that the combined loss of pro-apoptotic BIM and p53 caused 100% penetrant, female-exclusive NTDs, which allowed us to investigate the female-specific functions of p53. We report that female p53–/– neural tube samples show fewer cells with inactive X markers Xist and H3K27me3, and a concomitant increase in biallelic expression of the X-linked genes, Huwe1 and Usp9x. The depletion of Xist and increase X-linked gene expression was confirmed by RNA sequencing.

overall_design: Compound mutant mice were generated by inter-crossing single mutant strains previously described (PMID: 7922305, PMID: 10576740). RNA was extracted from neural tissue from E9.5 Bim-/+ mouse embryos. RNA-seq profiling was undertaken for n=4 affected female embryos (p53 knockout), n=4 control male mice (p53 knockout) and n=3 control female embryos (p53 wildtype).


title: Genome-wide maps of chromatin state and mRNA expression patterns in leukemic cell lines

status: Public on Mar 30 2019

city: MEMPHIS

first: John

last: Diedrich

state: Tennessee

address: 262 Danny Thomas Place

country: USA

institute: St. Jude Children's Research Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ATACseq and RNAseq were performed on a panel of leukemia cell lines of both B and T cell origin

overall_design: Examination of open chromatin and gene expression in 7 different cell lines

first: Jonathan

first: Daniel

last: Savic


title: microRNAs are negative and positive post-transcriptional regulators of BRAF-X1 mRNA [miRNA]

status: Public on Mar 31 2019

city: Pisa

state: PI

address: via Moruzzi 1

department: IIT

country: Italy

institute: CNR

type: Non-coding RNA profiling by high throughput sequencing

overall_design: The expression levels of microRNAs in A375 cells were measured by small RNA sequencing upon 48h of treatment with DMSO. Two independent replicates were analyzed.

first: Marianna

last: Vitiello


title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors

city: New York

first: Caleb

last: Lareau

state: New York

address: 417 E 68th St, Zuckerman - ZRC 1132

institute: Memorial Sloan Kettering

summary:
CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: PolyA-sequencing in IMR-32 neuroblastoma cells with shRNA mediated depletion of CDK12, CDK13 or GFP.

city: Boston

first: Ruben

last: Dries

address: 360 Longwood Ave

department: Computational Biology

institute: DFCI

summary: We performed RNA-seq analysis of polA transcripts in IMR-32 cells with shRNA-mediated depletion of CDK12 and CDK13 and GFP as a control

overall_design:
Expression of polA transcripts in IMR-32 cells with shRNA-mediated depletion of CDK12 and CDK13 using  the RNA-seq library kit (QuantSeq 3’ mRNA Sequencing REV, Lexogen)  using 2 different shRNA constructs for each target in duplicate, for a total of 10 individual samples
Please note that processed data files were generated from the merged replicates, as indicated in the corresponding sample description field.

first: Malgorzata

last: Krajewska

first: Rani

middle: E

last: George


title: CAR T cell trogocytosis and cooperative killing   regulate tumour antigen escape

status: Public on Mar 27 2019

first: Yu-Jui

last: Ho

address: 417 E 68th St

department: Cancer Biology & Genetics Program

institute: Memorial Sloan Kettering Cancer Center

summary: Chimeric antigen receptors (CARs) are synthetic receptors for antigen that reprogram T cell specificity, function and persistence. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies, and early trial results suggest activity in multiple myeloma. Despite high complete response rates, relapses occur in a large fraction of patients, some of which are antigen-negative and others antigen-low. Unlike mechanisms resulting in complete and permanent antigen loss, those leading to antigen-low tumour escape remain obscure. In murine leukaemia models, we show that CARs provoke reversible antigen loss through trogocytosis, an active process whereby target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide killing and exhaustion. These mechanisms affect both CD28 and 4-1BB-based CARs, albeit differentially, depending on antigen density. They can be offset by cooperative killing and combinatorial targeting.

overall_design: Examination of mRNA CD19 expression in  NALM6 retrieved from relapsed mice treated with 19-BBζ CAR T cells (D0),  retrieved NALM6 after ex-vivo culture (D6), and  NALM6 culture in vitro (ctrl).

first: Mohamad

last: Hamieh

first: Michel

last: Sadelain


title: RNAseq in BAP1 KO primary mouse mesothelial cells

city: South San Francisco

first: Rohit

last: Reja

state: CA

address: 1 DNA Way

institute: Genentech

summary: Malignancies arising from mutation of tumor suppressor genes display an unexplained tissue proclivity. For example, tumor suppressor BAP1 encodes a ubiquitously expressed deubiquitinase for histone H2A but germline mutations predominantly cause uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver and pancreas, whereas melanocytes and mesothelial cells remain viable. E3 ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing the pro-survival genes Bcl-2 and Mcl-1. Our data argue that BAP1 modulates gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program. We propose that intolerance of BAP1 loss, and perhaps the loss of other tumor suppressors, restricts the mutant tumor spectrum.

overall_design: RNA was extracted from following genotypes - BAP1 wildtype (WT) and BAP1 knockout (BAP1 KO).

first: Meng

last: He


title: Combination of high-dimensional approaches for skeletal muscle tissue cartography

city: Hong Kong

first: Tom

last: Cheung

address: Clear Water Bay, Kowloon

department: Division of Life Science

country: Hong Kong

institute: Hong Kong University of Science and Technology

summary:
The regenerative capacity of skeletal muscle relies on muscle stem cells (MuSCs, or satellite cells) and its niche interactions with different neighboring cells. To understand the cellular diversity within adult skeletal muscle tissue, we harvested mononuclear cells from hindlimb skeletal muscles, sorted into single cells and profiled them by single-cell RNA-seq.
To further understand and compare the expression profile between MuSCs and a novel smooth-muscle/mesenchymal-like cells (SMMCs) population,  we isolated the two cell types by FACS and profiled them respectively by bulk RNA-seq.

overall_design:
(scRNA-seq) Mononuclear cells from hindlimb skeletal muscle from wild type mouse were profiled by taking advantage of 10X Chromium platform.

(bulk RNA-seq) MuSCs and SMMCs cell population were isolated from hindlimb skeletal muscle from wild type mouse. Their polyA+ RNA were profiled using Smart-seq2.

first: Gary

middle: J

first: Justin

last: Law

first: Raymond

last: Wan

middle: H


title: Tau drives translational selectivity by interacting with ribosomal proteins

city: Gainesville

first: Jose

middle: F

last: Abisambra

state: FL

address: 1275 Center Dr

department: Department of Neuroscience & Center for Translational Research in Neurodegenerative Research

institute: University of Florida

summary: There is a fundamental gap in understanding the consequences of tau-ribosome interactions. Tau oligomers and filaments hinder protein synthesis in vitro, and they associate strongly with ribosomes in vivo. Here, we investigated the consequences of tau interactions with ribosomes in vivo and in human brain tissues to identify tau as a direct modulator of ribosomal selectivity. We performed microarrays and nascent proteomics to measure changes in protein synthesis using rTg4510 tau transgenic mice. We determined that tau expression differentially shifts the transcriptome and the proteome and that the synthesis of ribosomal proteins is reversibly dependent on tau levels. We further extended these results to human brains and show that tau pathologically interacts with ribosomal protein S6 (rpS6 or S6). Consequently, synthesis of ribosomal proteins coded by 5’TOP-mRNAs was reduced under tauopathic conditions in Alzheimer’s disease brains. Our data establish tau as a driver of RNA translation selectivity. Moreover, considering that regulation of protein synthesis is critical to learning and memory, aberrant tau-ribosome interactions in disease could explain the linkage between virtually every tauopathy and cognitive impairment and memory decline.

overall_design: Nontransgenic mice and rTg4510 tau transgenic mice were fed either normal feed or doxycycline to suppress transgenic tau expression. Biological replicates: Non + veh = 3, Non + dox = 2, Tg + veh = 3, Tg + dox = 3

first: Koren

middle: A

last: Shon

first: Matthew

last: Hamm

first: Meier

last: Shelby

first: Weiss

last: Blaine

first: Grant

last: Nation

first: Chishti

last: Emad

first: Chen

last: Jing

first: Arango

last: Juan Pablo

first: Zhu

last: Haining

first: Blalock

middle: M

last: Eric

first: Abisambra

last: Jose


title: A role for ZNF598 in post-transcriptional gene regulation

city: Copenhagen

first: Christopher

last: Tiedje

address: Blegdamsvej 3B

department: Department of Molecular and Cellular Medicine (ICMM)

country: Denmark

institute: University of Copenhagen

summary: Transcriptomic changes in U2OS cells upon depletion of the RNA-binding protein ZNF598 were analyzed in non-treated and IL-1 beta treated cells in comparison to control U2OS cells

overall_design: U2OS cells transfected with a mis-match Control siRNA or ZNF598-specific siRNA were either left non-stimulated (ns) or were treated for 1h with 2ng/ml (final) IL-1 beta, transcriptomic changes were assessed by total RNASeq, biological duplicates of all conditions were measured

first: Maxim

last: Tollenaere


title: Gene expression profiling in neuroblastoma cell lines treated with THZ531 or DMSO

first: Ruben

last: Dries

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


title: DNA methylation in thymic epithelial tumors.

city: Tokushima city

first: Koichiro

last: Kajiura

department: Department of Thoraci,Endocrine Surgery and Oncology

country: Japan

institute: Institute of Biomedical Sciences, Tokushima University Graduate School

type: Methylation profiling by genome tiling array

summary:
Analysis of DNA methylation in thymic epithelial tumor.
The aim of this study is to identify the gene affected carcinogenesis by DNA methylation in thymic epithelial tumor.

overall_design: 22 samples were all exracted fresh frozen sample. 3 neuroendocrine tumors, 7 squamous cell carcinomas, 8 B3 thymomas, 3 type A thymomas and 1 normal thymus.


title: Nppb Neurons Are Sensors Of Mast Cell-Induced Itch

city: Bethesda

first: Hans Juergen

last: Solinski

state: MD

address: 9000 Rockville Pike Bldg. 35, Rm. 3F143

institute: NIH/NIDCR

summary: Itch is an unpleasant skin sensation which can be triggered by exposure to many chemicals including those released by mast cells. The Nppb-expressing class of sensory neurons when activated elicit scratching responses in mice, however, it is unclear which itch-inducing agents stimulate these cells and the receptors involved. Here, we identify receptors expressed by Nppb-neurons and demonstrate the functional importance of these receptors as sensors of endogenous pruritogens released by mast cells. Our search for receptors in Nppb-neurons revealed that they express leukotriene, serotonin, and sphingosine-1-phosphate receptors. Targeted cell ablation, calcium imaging of primary sensory neurons, and conditional receptor knockout studies demonstrate that these receptors induce itch by the direct stimulation of Nppb-neurons and neurotransmission through the canonical GRP-dependent spinal cord itch pathway. Together our results define a molecular and cellular pathway for mast cell-induced itch.

overall_design: Examination of transcriptome (RNA-seq) of 2 different populations of DRG neurons in quadruplicates.

first: Hans

first: Pang-Yen

last: Tseng

first: Mark

last: Hoon


title: RNAseq from FACS sorted HopX positive cells from mouse brain

status: Public on Mar 28 2019

city: Philadelphia

first: Yijing

last: Su

state: PA

address: Levy 326

department: Department of Oral Medicine

institute: University of Pennsylvania

summary: Analysis of FACS sorted HopX positive cells from mouse brain on expressional profiling

overall_design: RNAseq was performed on  FACS sorted HopX positive cells from mouse brain at E15, P3 and adult.

first: Hongjun

last: Song


title: Global gene expression analysis in DMSO and THZ531 treated IMR32 and Kelly cells

summary: We performed gene expression profiling by Affymetrix primeview array in both DMSO and THZ531 treated cells.

overall_design: Expression profiles of  DMSO and THZ531 treated cells using GeneChip® PrimeView™ Human Gene Expression Array, in duplicate,for a total of 12 individual samples.

first: Daniel

middle: S

last: Day

first: Malgorzata

last: Krajewska

first: Rani

last: George


title: RNA-seq expression profiling of individual clones sorted from mouse mammary tumors

first: Gordon

last: Smyth

summary: Breast tumors are characterized by inherent heterogeneity but the evolving cellular organization of breast tumors through progression remains poorly understood. Individual clones were tracked by combining mouse models of breast cancer with Confetti reporter strains. Expression profiling of individual clones sorted from tumors arising in K5- and Elf5-driven Pten/p53-deficient mice revealed distinct molecular signatures.

overall_design: K5-rtTA-IRES-GFP and ElF5-rtTA-IRES-GFP transgenic mice were crossed with TetO-cre (JAX) and R26R-Confetti reporter strains to generate triple genetically modified mice. The mice were treated with medroxyprogesterone acetate (MPA) and dimethylbenz(a)anthracene (DMBA) to induce carcinogenesis. Three K5-driven and five Elf5-driven mammary tumors were selected. Individual live cells from each tumor were FACS sorted by the four Confetti fluorescent markers (to select individual clones) and by CD24 expression (high or low). Cell subsets for the eight tumors, four fluorescent markers and positive or negative CD24 status were profiled by RNA-seq (38 samples in all). Expression was quantified by counting RNA-seq reads at the gene level and (separately) at the exon level.


title: RNAseq in protected BAP1 mESc

first: Rohit

last: Reja

overall_design:
RNA was extracted from following genotypes - BAP1 wt (WT_D4), knock-in BAP1 C91A mutant (C91A_D4_4OHT),
BAP1 C91A control (C91A_D4), BAP1 knockout (cKO_D4_4OHT), BAP1 knockout control (cKO_D4),and RNF2-BAP1 double knockout (dKO_D4)) in mouse embryonic stem cells.
Cultured cells were treated with Tamoxifen 100 nM 4-hydroxytamoxifen (4-OHT; EMD Millipore) for 4 days to induce Cre-mediated recombination.
To preotect cells from death due to BAP1 knockout, all cells were treated with emricasan.

first: Meng

last: He


title: ETHANOL ACTIVATES IMMUNE RESPONSE IN LYMPHOBLASTOID CELLS

status: Public on Mar 25 2019

city: Indianapolis

first: Jeanette

middle: Nance

last: McClintick

state: IN

address: 635 Barnhill Dr.

department: Biochemistry & Molecular Biology

institute: Indiana University School of Medicine

summary: The short term effects of alcohol on gene expression in brain tissue cannot directly be studied in humans. Because neuroimmune signaling is altered by alcohol, immune cells are a logical, accessible choice to study and might provide biomarkers. RNAseq was used to study the effects of 48 h exposure to ethanol on lymphoblastoid cell lines (LCLs) from 20 alcoholics and 20 controls. Ethanol exposure resulted in differential expression of 4,577 of the 12,526 genes detectably expressed in the LCLs (FDR ≤ 0.05); 55% of these showed increased expression. Cells from alcoholics and controls responded similarly. Ingenuity pathway analysis identified several pathways in which expression was altered: NFkB, neuroinflammation, IL8, and dendritic cell maturation pathways were activated, consistent with increased signaling by NFkB, TNF,  IL1, IL4, IL18, TLR4, and LPS. Signaling by Interferons A and B decreased, as did EIF2 signaling, phospholipase C signaling and Glycolysis. Baseline gene expression patterns were similar in LCLs from alcoholics and controls. At relaxed stringency (p<0.05), 438 genes differed, 223 of which were also affected by ethanol. There was a suggestion of compensation because baseline differences (no ethanol) were in the opposite direction of differences due to ethanol exposure in 75% of these genes. Ingenuity Pathway analysis Identified pathways wAldosterone,  phospholipase C, and opioid signaling as significant. The pattern of expression was consistent with increased signaling by several cytokines including interferons, and TLR2 and 3 in alcoholics. Expression of genes in the cholesterol biosynthesis pathway including the rate-limiting enzyme HMGCR was lower in alcoholics.

overall_design: LCLs from 20 alcoholics and 20 controls treated or not with 75mM ethanol for 48h

middle: N

first: Howard

last: Edenberg


title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors [Modifications - screen]

first: Caleb

last: Lareau

summary: Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities.

overall_design: HEK293T cells were transfected with pCAG-BE3-P2A-EGFP or variants thereof or control pCAG-nCas9(D10A)-UGI-NLS-P2A-EGFP or control pCAG-P2A-EGFP constructs with various gRNAs as described below. Cells were sorted for top 5% GFP or all GFP + cells based on FITC signal. RNA-seq was performed to measure transcriptional changes associated with different constructs and guides.

first: Julian

last: Grunewald


title: Microarray analysis of Codonopsis pilosula and Astragalus embranaceus Aqueous Extracts on HepG2 Cells

city: Taipei

first: Liang-Chuan

last: Lai

state: Taiwan

address: 1 Jen-Ai Road, Sec 1

department: Physiology

country: Taiwan

institute: National Taiwan University

summary: HepG2 celline shows dowstream pathways change after drug treament.

overall_design: Total RNA obtained from cultured HepG2 cells 24 hours after Codonopsis pilosula (D1-3) or Astragalus membranaceus (H1-3) aqueous extracts treament in vitro.

first: Pin-Hao

last: Ko


title: FACS sorted HopX positive cells from mouse brain


title: Intra-individual methylomics detects the impact of early-life adversity

status: Public on Mar 23 2019

city: IRVINE

first: Shan

last: Jiang

address: 2300E Biological Sciences III

institute: UNIVERSITY OF CALIFORNIA, IRVINE

type: Methylation profiling by high throughput sequencing

summary: Genetic and environmental factors interact during sensitive periods early in life to influence mental health and disease via epigenetic processes such as DNA methylation. However, it is not known if DNA methylation changes outside the brain provide an 'epigenetic signature' of early-life experiences. Here, we employed a novel intra-individual approach by testing DNA methylation from buccal cells of individual rats before and immediately after exposure to one week of typical or adverse life experience. We find that whereas inter-individual changes in DNA methylation reflect the effect of age, DNA methylation changes within paired DNA samples from the same individual reflect the impact of diverse neonatal experiences. Genes coding for critical cellular–metabolic enzymes, ion channels and receptors were more methylated in pups exposed to the adverse environment, predictive of their repression. In contrast, the adverse experience was associated with less methylation on genes involved in pathways of death and inflammation as well as cell-fate related transcription factors, indicating their potential upregulation. Thus, intra-individual methylome signatures indicate large-scale transcription-driven alterations of cellular fate, growth and function.

overall_design:
The experimental paradigm involved rearing pups and dams in “impoverished” cages for a week (P2-P9). Briefly, routine rat cages were fitted with a plastic-coated aluminum mesh platform sitting ~2.5 cm above the cage floor (allowing collection of droppings). Bedding was reduced to only cover cage floor sparsely, and one-half of a single paper towel was provided for nesting material, creating a limited bedding and nesting (LBN) cage. Control dams and their litters resided in standard bedded cages. For each experiment, pups form several litters were mixed, and then assigned randomly to a control or LBN dam. Control and experimental cages were undisturbed during P2–P9, housed in a quiet room with constant temperature and a strong laminar airflow. For technical reasons the study was conducted in two ‘batches’ (cohorts). These differed solely in the dates at which they were conducted.

The first buccal swab was collected from both cheeks of each pup prior to randomization on P2, using Hydraflock swab (Puritan diagnostics, LLC). Pups were then randomized to controls or LBN cages. On P10, buccal swabs were collected as described for P2. The Buccal swab was placed into the DNA shields™ (Zymo Research) immediately after swabbing. DNA was prepared from the DNA shields solution using the Quick-gDNA™ MiniPrep kit (Zymo Research) following the manufacturer’s protocol. The quantity of double stranded DNA was analyzed using Qubit, and RRBS Libraries were prepared from 40 ng of genomic DNA digested with Msp I and then extracted with DNA Clean & Concentrator™-5 kit (Zymo Research). Fragments were ligated to pre-annealed adapters containing 5’-methylcytosine instead of cytosine according to Illumina’s specified guidelines (www.illumina.com). Adaptor-ligated fragments were then bisulfite-treated using the EZ DNA MethylationLightning™ Kit (Zymo Research). Preparative-scale PCR was performed and the resulting products were purified with DNA Clean & Concentrator for sequencing. Amplified RRBS libraries were quantified and qualified by Qubit, Bioanalyzer (Agilent), and Kapa library quant (Kapa systems), and then sequenced on the Illumina NextSeq 500 platform.

Adaptor and low quality reads were trimmed and filtered using Trim Galore! 0.4.3. Reads were aligned to the rat genome (RGSC 6.0/rn6) by using Bismark 0.16.3. CpG sites were called by “bismark_methylation_extractor” function from Bismark. Single CpG sites with more than 10 reads coverage were kept for DMR calling. Differential methylation sites (DMSs) were first called using MethyKit (R 3.3.2) between P2 and P10 from the same individual with a false discovery rate (FDR) lower than 0.05. DMSs were shared in at least two individuals in either control or LBN groups were kept and DMSs falling within 100 base pairs were then merged into DMRs. The methylation percentage/level was calculated as the ratio of the methylated read counts over the sum of both methylated and unmethylated read counts for a single CpG site or across CpGs for a region. The delta methylation was calculated using the log2 transformation of the ratio of methylation level in the P10 sample and the methylation level in the P2 sample, defined as log2(P10/P2). Increased methylation in P10 is shown as a positive value while decreased methylation in P10 is shown as a negative value.

first: Ali

last: Mortazavi

first: Tallie

middle: Z

last: Baram

first: Noriko

last: Kamei

first: Jassica

middle: L

last: Bolton

first: Xinyi

last: Ma

first: Hal

last: Stern


title: PolyA-sequencing in IMR-32 cells treated with THZ531 or DMSO

first: Ruben

last: Dries

summary: We performed RNA-seq analsysis of polA transcripts in IMR-32 cells treated with THZ531 for 2 and 6h and DMSO as a control

overall_design: Expression of  polA transcripts in IMR-32 treated with THZ531 using  the RNA-seq library kit (QuantSeq 3’ mRNA Sequencing REV, Lexogen)  in duplicate, for a total of 6 individual samples

first: Malgorzata

last: Krajewska

first: Rani

last: George


title: PolyA-sequencing in Kelly and Kelly E9R neuroblastoma cells treated with THZ531 or DMSO

summary: We performed RNA-seq analsysis of polA transcripts in Kelly and Kelly E9 resistant (E9R) cells treated with THZ531 for 6h and DMSO as a control

overall_design:
Expression of  polA transcripts in Kelly and Kelly E9R cells treated with THZ531 using  the RNA-seq library kit (QuantSeq 3’ mRNA Sequencing REV, Lexogen)  in duplicate, for a total of 8 indyvidual samples
Please note that the bigWig processed data was generated from both replicates and is linked to the corresponding rep1 (*_1) sample records.


title: Whole breast tissue gene expression in comparison to expression in epithelial and stromal tissues

status: Public on Mar 19 2019

first: Julia

last: Santucci-Pereira

address: 333 Cottman Ave, P2051

institute: Fox Chase Cancer Center

summary:
The breast tissue contains both epithelial and stromal tissues in its composition.  In this substudy we compared the gene expression of the whole core needle biopsy of the breast with the expression observed in its epithelia and stroma.
Laser capture microdissection was performed to compare the gene expression profile in the whole breast biopsy with that in the epithelial and stromal tissues.  In this study we had samples collected from nive premenopausal women, free of any breast abnormality.

overall_design: For each of nine women included in this substudy, three microarrays were performed in the same batch, using RNA extracted from 1) the epithelial cells of the mammary gland, 2) the stroma and 3) whole tissue. For each subject a linear regression model was fitted across all genes. The gene expression values in the whole tissue were modeled as a linear function of the gene expression in epithelial and stromal tissues.

first: Anne

last: Zeleniuch-Jacquotte

first: Yelena

last: Afanasyeva

first: Hua

last: Zhong

first: Michael

last: Slifker

first: Suraj

last: Peri

first: Eric

last: Ross

first: Ricardo

last: López de Cicco

first: Yubo

last: Zhai

first: Theresa

last: Nguyen

first: Fathima

last: Sheriff

first: Irma

last: Russo

first: Yanrong

last: Su

first: Alan

last: Arslan

first: Pal

last: Bordas

first: Per

last: Lenner

first: Janet

last: Åhman

first: Anna-Stina

first: Robert

last: Johansson

first: Göran

last: Hallmans

first: Paolo

last: Toniolo

first: Jose


title: The female-biased factor VGLL3 drives cutaneous and systemic autoimmunity: RNA-seq analysis of the K5-Vgll3 transgenic mouse model of cutaneous and systemic lupus

city: Ann Arbor

first: Lam

last: Tsoi

state: MI

address: Medical Sciences 1 Bldg. 1301 E. Catherine

institute: University of Michigan

summary: RNA-seq analysis of nonlesional skin, whole blood, and cultured primary keratinocytes of wild-type and autoimmunity-prone K5-Vgll3 mice, which overexpress the female-biased transcription cofactor VGLL3 in the epidermis and develop a progressive lupus-like rash and systemic autoimmune disease with features of systemic lupus erythematosus. These skin and blood data demonstrate that skin-directed overexpression of VGLL3 results in gene expression changes strikingly similar to those seen in skin and blood of patients with cutaneous and systemic lupus, respectively.

overall_design: RNA-seq analysis of nonlesional skin and whole blood from 4 age- and sex-matched wild-type and transgenic K5-VGLL3 mice and of primary keratinocytes isolated from tails of 3 wild-type and 3 transgenic K5-VGLL3 23-day-old littermates

first: Allison

last: Billi

first: Johann

last: Gudjonsson


title: Gene expression analysis of previously treated hormone receptor-positive metastatic breast cancer

city: San Diego

first: Zhou

last: Zhu

address: 10646 Science Center Drive (CB4)

institute: Pfizer Worldwide Research & Development

summary: A large-panel gene expression analysis was conducted to identify biomarkers associated with the effectiveness of adding palbociclib to fulvestrant

overall_design:
Baseline tumor tissue from PALOMA-3 trial were profiled using EdgeSeq Oncology BM Panel, with 302 tumor samples evaluable for analysis.
Targeted RNA-Seq.

first: Yuan

last: Liu


title: DMC1 ChIP-seq from B6xCAST F1 PRDM9-Humanized/CAST testes

status: Public on Mar 18 2019

city: Oxford

first: Anjali

middle: Gupta

last: Hinch

address: Roosevelt Drive

department: WCHG

country: United Kingdom

institute: Oxford University

summary: We report testis DMC1 enrichment at DSB (Double-Strand Break) hotspots in a B6xCAST F1 mouse, which is heterozygous at Prdm9, with one wild-type CAST allele and one allele found in human populations (bred from genetically engineered mother, Davies et al Nature 2012). We also report H3K4me3 intensities at these hotspots, based on the raw data published under GSE119727 (Li et al). Separately, we have reported 2,649 crossover locations identified by single-cell DNA sequencing of 217 sperm from the same hybrid mouse.   We identify several factors, including PRDM9 binding on the repair-template homologue, telomere proximity and local GC-content, that affect the probability that a DSB is repaired as a crossover. We further show that these factors also influence the time it takes for the site of a DSB to find and engage its homologue, with rapidly-engaging sites being more likely to be repaired as crossovers.

overall_design: Examination of DMC1 enrichment in the testes of a (C57BL/6J x CAST/Eij)F1 PRDM9-Humanized/CAST mouse

first: Peter

last: Donnelly


title: Novel risk variants affecting enhancers of TH1 and TREG cells in type 1 diabetes

city: Hershey

first: Yasin

last: Uzun

address: 700 HMC Crescent Rd

department: Department of Pediatrics

institute: Pennsylvania State University College of Medicine

overall_design: Refer to individual Series


title: Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq

city: ROCHESTER

first: JEONG-HEON

last: LEE

state: Minnesota

address: 200 1ST STREET S.W.

department: Epigenomics program/CIM

institute: MAYO CLINIC

summary: Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research.  However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin.

overall_design: During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65°C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with ChIP-seq for histone mark and chromatin-associated protein. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37°C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45 - 60°C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65°C is close to mononucleosomal size.

first: Jian

last: Zhong

first: Zhenqing

last: Ye

first: Jeong

last: Lee


title: Nascent RNA Sequencing after NMYC activation in SH-EP MYCNER cells

city: Kiel

first: Elmar

last: Wolf

state: Schleswig-Holstein

address: Rudolf-Höber-Str. 1

department: Biochemical Institute

country: Germany

institute: Christian-Albrechts-University Kiel

summary: In order to distinguish transcription changes from RNA modification and post transcription changed, nascent RNA seq via metabolic labeling of freshly synthesized RNA was carried out using 4sU labeling/biotin purification.

overall_design: nascent RNA was extractred post N-MYC activation and compared with untreated cells nascent RNA to gather fold changes of pre-mRNA on gene basis.

first: Apoorva

last: Baluapuri

first: Susanne

last: Walz

first: Martin

last: Eilers


title: Contribution of time of day and the circadian clock to the heat stress responsive transcriptome in Arabidopsis

city: Riverside

first: Dawn

last: Nagel

address: 3401 Watkins Avenue (Batchelor Hall Rm 1123)

department: Botany and Plant Sciences

institute: University of Califonia, Riverside

summary: In Arabidopsis, a large subset of heat responsive genes exhibits diurnal or circadian oscillations. However, to what extent the dimension of time and/or the circadian clock contribute to heat stress responses remains largely unknown. To determine the direct contribution of time of day and/or the clock to differential heat stress responses, we probed wild-type and mutants of the circadian clock genes CCA1, LHY, PRR7, and PRR9 following exposure to heat (37°C) and moderate cold (10°C) in the early morning (ZT1) and afternoon (ZT6). Thousands of genes were differentially expressed in response to temperature, time of day, and/or the clock mutation. Approximately 30% more genes were differentially expressed in the afternoon compared to the morning, and heat stress significantly perturbed the transcriptome. Of the DEGs (~3000) specifically responsive to heat stress, ~70% showed time of day (ZT1 or ZT6) occurrence of the transcriptional response. For the DEGs (~1400) that are shared between ZT1 and ZT6, we observed changes to the magnitude of the transcriptional response. In addition, ~2% of all DEGs showed differential responses to temperature stress in the clock mutants. The findings in this study highlight a significant role for time of day in the heat stress responsive transcriptome, and the clock through CCA1 and LHY, appears to have a more profound role than PRR7 and PRR9 in modulating heat stress responses during the day. Our results emphasize the importance of considering the dimension of time in studies on abiotic stress responses in Arabidopsis.

overall_design:
We used RNA-sequencing to measure transcriptomic changes in Columbia-0 (WT), cca1-1/lhy-20 (cca1lhy) and prr7-3/prr9-1 (prr7prr9) under 1 h of moderate cold (10°C) and 1 h of heat stress (37°C). Samples were collected at ZT1 and ZT6.

Seedlings were grown on plates containing Murashige and Skoog (MS) medium supplemented with 1.5% sucrose (wt/vol) in 12 h light and 12 h dark (LD) cycles for 12 days at constant 22°C and 90 µm light intensity.

Single-end 75 base pair sequences were generated for each mRNA library using the NextSeq 500 (Illumina).

first: Emily

last: Blair

first: Titouan

last: Bonnot

first: Maureen

last: Hummel

first: Erika

last: Hay

first: Jill

last: Marzolino

first: Ivan

last: Quijada


title: ChIP-nexus of mineralocorticoid receptors and glucocorticoid receptors in neuroblastoma cells

status: Public on Mar 15 2019

city: Bristol

first: Caroline

middle: Alison

last: Rivers

address: Dorothy Hodgkin Building, Whitson Street

department: Translational Health Sciences

institute: Bristol University

summary:
Purpose: To compare the binding profiles of mineralocorticoid and glucocorticoid receptors after corticosterone treatment in neuroblastoma cells.
Methods: Tagged glucocorticoid receptors (eGFP-rat GR) and mineralocorticoid receptors (mCherry-rat MR) were transiently transfected into Neuro2A mouse neuroblastoma cells and treated with (i) vehicle, (ii) corticosterone 100 nM for 20 mins and (iii) corticosterone 100 nM for 20 mins followed by media repacement twice and 40 mins further incubation in charcoal-stripped serum media. ChIP-nexus samples were prepared in triplicate for deep sequencing using Illumina NextSeq, as well as control samples taken from cell lysates before immunoprecipitation. Sequence data was analyzed and processed for the identification of enriched binding sites, using MACS2, and for the characteristic staggered ChIP-nexus borders, using the MACE pipeline (Wang et al., 2014).
Results: Corticosterone-sensitive glucocorticoid receptor (GR) binding sites and mineralocorticoid receptor (MR) binding sites showed considerable overlap, by MACS2 analysis. GR and MR binding sites were highly similar showing recruitment to the same DNA sites, by MACE analysis (Wang et al., 2014). Further analyses showed GR could tether MR to the DNA by GR in a functional manner such that gene expression could be altered by the presence of tethered MR.
Conclusions: MR can be recruited to DNA-binding sites by GR, even in the absence of MR DNA-binding. MR can interact in multiple modes at genomic DNA binding sites.

overall_design: ChIP-nexus samples were prepared in triplicate from expressed tagged glucocorticoid receptors (eGFP-mouseGR) and mineralocorticoid receptors (mCherry-mouseMR) transiently transfected into Neuro2A mouse neuroblastoma cells for deep sequencing using Illumina NextSeq, for each of 3 treatment groups: (i) vehicle, (ii) corticocsterone 100 nM 20 mins, (iii) corticosterone 100 nM 20 mins followed by 2 washed and incubation without steroid for a further 40 mins. Additional gene expression and ChIP testing was performed using DNA-binding domain mutants to confirm findings.


title: RNAseq in BAP1 KO primary mouse melanocytes

city: South San Francisco

first: Rohit

last: Reja

address: 1 DNA Way

institute: Genentech

summary: Malignancies arising from mutation of tumor suppressor genes display an unexplained tissue proclivity. For example, tumor suppressor BAP1 encodes a ubiquitously expressed deubiquitinase for histone H2A but germline mutations predominantly cause uveal melanomas and mesotheliomas. We show that BAP1 inactivation causes apoptosis in mouse embryonic stem cells, fibroblasts, liver and pancreas, whereas melanocytes and mesothelial cells remain viable. E3 ligase RNF2, which silences genes by monoubiquitinating H2A, promoted apoptosis in BAP1-deficient cells by suppressing the pro-survival genes Bcl-2 and Mcl-1. Our data argue that BAP1 modulates gene expression by countering H2A ubiquitination, but its loss only promotes tumorigenesis in cells that do not engage an RNF2-dependent apoptotic program. We propose that intolerance of BAP1 loss, and perhaps the loss of other tumor suppressors, restricts the mutant tumor spectrum.

overall_design: RNA was extracted from following genotypes - BAP1 wt (WT) and BAP1 knockout (BAP1 KO).

first: Meng

last: He


title: Inducible bronchus-associated lymphoid tissues (iBALT) serve as sites of B cell selection and maturation following influenza infection in mice

status: Public on Mar 14 2019

city: Melbourne

first: Hyon Xhi

last: Tan

state: Victoria

address: 792 Elizabeth St

department: Department of Microbiology and Immunology

institute: The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity

summary: GC-like structures within iBALT serve as a distinct niche that independently support the maturation and selection of B cells primarily targeted against the influenza virus nucleoprotein. Our findings suggest that iBALT, which are positioned at the frontline of the lung mucosa, drive long-lived and unique GC reactions that contribute to the diversity of the humoral response targeting influenza.

overall_design: GC and memory B cells were sorted from various tissues in influenza PR8-infected mice. B cells were identified as Live/Dead negative, F4/80-, Streptavidin-, CD3-, CD45+, B220+ and intravenous CD45.2-. GC (CD38lo/GL7+) or memory (CD38hi/GL7-) B cells were sorted. Transcriptional profiling and differential gene expression was performed using RNA sequencing.

first: Hyon-Xhi

first: Robyn

last: Esterbauer

first: Hillary

last: Vanderven

first: Jennifer

last: Juno

first: Stephen

last: Kent

first: Adam

last: Wheatley


title: Investigation of Bach2 function by heme-iron homeostasis in red-pulp macrophage.

city: Sendai

first: Kazuhiko

last: Igarashi

address: Seiryo 2-1

institute: Tohoku University

summary: Macrophages are central in regulating iron homeostasis. Transcription repressor Bach2 regulates by heme. Here we investigated the relationship between heme-regulated Bach2 and macrophage in spleen.  We found that gene expression were not many change between WT and Bach2 knock out mice in red-pulp macrophage.Our results suggest that the function of the red-pulp macrophage is not dependent on according to expression of Bach2.

overall_design: Gene expression in red-pulp macrophages cells of WT and Bach2 KO mice was measured. 3 independent mice ware used for each genotype.

first: Miki

last: Watanabe-Matsui


title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance

status: Public on Mar 21 2019

city: Los Angeles

first: Vasu

last: Punj

address: Biggy Street

institute: USC

summary: To Study the role of mutant Braf V600E and ERK dependent phosphorylation of TFEB in regulation of autophagy and lysosome biogenesis in the context of melanoma growth and metastasis, we used whole geneome RNA-seq to study role TFEB mediated  BRAFV600E autophagy regulation.

overall_design: For subcutaneous xenografts, A375 cells stably expressing WT TFEB or TFEB mutants (5 x 10^6) were injected subcutaneously into the lower flank of 5-6 six-week-old female NOD/SCID mice. After a three-weeks of post-inoculation, mice were sacrificed, and tumors were dissected and processed for RNA isolation.

first: Shun

last: Li

first: Christine

last: Quach

first: Hongrui

last: Guo

first: Gyu-beom

last: Jang

first: Hadi

last: Maazi

first: Shihui

last: Zhao

first: Nathaniel

last: Sands

first: Qingsong

last: Liu

first: Gino

last: In

first: David

last: Peng

first: Weiming

last: Yuan

first: Keigo

last: Machida

first: Min

last: Yu

first: Ashley

last: Hagiya

first: Yongfei

last: Yang

first: Liling

last: Tang

first: Chengyu

last: Liang


title: Impact of Immune and Stromal Infiltration on Outcomes Following Bladder-Sparing Trimodality Therapy for Muscle-Invasive Bladder Cancer

city: Vancouver

first: Elai

last: Davicioni

state: BC

address: 1152 Mainland St #430

country: Canada

institute: GenomeDx

summary:
Background: Bladder-sparing trimodality therapy (TMT) is an alternative to radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC), and biomarkers to inform therapy selection are needed.
Objective: To evaluate immune and stromal signatures in MIBC treated with TMT.

overall_design: Gene expression profiling was used to study molecular sub typing and the tumor microenvironment in the setting of bladder preservation therapy for muscle-invasive bladder cancer.

first: Jason

last: Efstathiou

first: Kent

middle: W

last: Mouw

first: Ewan

last: Gibb

first: Yang

first: Chin-Lee

last: Wu

first: Michael

middle: R

last: Drumm

first: Jose

middle: B

last: da Costa

first: Marguerite

last: du Plessis

first: Natalie

middle: Q

last: Wang

first: Felix

middle: Y

last: Feng

first: Roland

last: Seiler

first: Peter

middle: C

last: Black

first: William

middle: U

last: Shipley

middle: T

last: Miyamoto


title: Genome-wide validation of the direct regulated targets for 33 nitrogen-early response transcription factors

city: Urbana

first: Matthew

middle: D.

last: Brooks

state: Illinois

address: 1201 W Gregory Drive

department: GCPRU

institute: USDA

summary: The direct targets of 33 Nitrogen-early responsive TFs were identified using the TARGET system. Arabidopsis plants were grown on low nitrogen (1mM KNO3) and after 10 days protoplasts were generated from root tissue, Cells were transfected with a vector containing the GR-TF fusion and incubated overnight. Each experiment also included a GR-only empty vector control. In the morning, samples were pre-treated with 20 mM KNO3 and 20 mM NH4NO3 for 2 hours, and cycloheximide for 20 minutes, before TF entry into the nucleus was induced with dexamethasone. 3 hours after dex treatment samples were FACS sorted into RNA extraction buffer and RNA-seq libraries were generated. Transcriptional response to each TF compared to empty vector was assayed.

overall_design: Genome-wide direct regulated targets of 33 TFs were determined by comparison to an empty vector control. Vectors were expressed in protoplasts isolated from root tissue, samples were done in triplicate, and sequenced using the Illumina NextSeq platform.

middle: D

first: Gloria

last: Coruzzi


title: NUP98-HBO1-induced gene signature in human CB CD34+ cells

status: Public on Mar 13 2019

city: Hachioji

first: Yoshihiro

last: Hayashi

state: Tokyo

address: 1432-1 Horinouchi

institute: Tokyo University of Pharmacy and Life Sciences

summary: Chronic myelomonocytic leukemia (CMML) is an incurable hematopoietic stem cell malignancy. We identified a novel NUP98-HBO1 fusion from a patient with CMML. HBO1, a histone acetyltransferase (HAT) which belongs to the MYST family, is the first NUP98 fusion partner encodes HAT. To determine the effect of the NUP98-HBO1 fusion on downstream target gene regulation, we performed gene expression array analysis of NUP98-HBO1-transduced human cord blood (CB) CD34+ cells.

overall_design: FACS sorted CB CD34+ cells transduced with NUP98-HBO1 fusion or empty vector were subjected to RNA extraction and hybridization on the CodeLink Human Whole Genome Bioarray.

first: Hironori

last: Harada


title: Novel risk variants affecting enhancers of TH1 and TREG cells in type 1 diabetes [RNA-seq]

first: Yasin

last: Uzun

summary: We generated expression profiles of TH1 and TREG cells from T1D and healthy subjects by RNA-Seq. By integrating RNA-Seq dta with other data sets, we predicted and validated serveral T1D risk SNPs.

overall_design: Effector memory TH1 and effector memory TREG cells were isolated from 6 T1D patients and mached 5 helathy controls. Gene expression profilings were generated using RNA-Seq. By integrating RNA-Seq data, ChIP-Seq and other data sets, we predicted and validated several risk SNPs for T1D.

first: Kai

last: Tan

first: Peng

last: Gao

first: Bing

last: He

first: Sarah

middle: E

last: Salamati

first: Julie

last: Coffey

first: Eva

last: Tsalikian


title: In vivo microarray expression data from WT, Irf4-/- and checkpoint blockade treated Irf4-/- TEa CD4+ T cells

status: Public on Mar 11 2019

city: Houston

first: Hedong

last: Zhang

state: TX

address: 6670 Bertner Ave

department: Immunobiology & Transplant Science Center

institute: Houston Methodist Hospital Research Institute

summary:
Previously we found that Irf4-deficient T cells were dysfunctional and unable to reject heart allografts and checkpoint blockade treatment could reinvigorate dysfunctional Irf4-deficient T cells and enable them reject heart allografts. But shortly after the reinvigoration those T cells went back to dysfunctional state. TCR-transgenic TEa CD4+ T cells (B6 background) recognize a Balb/c I-Eα allopeptide presented by B6 antigen presenting cells, and were used to assess the effects of immune checkpoint blockades on Irf4-deficient alloreactive T cells. WT, Irf4-/- and checkpoint blockade treated Irf4-/- TEa CD4+ T cells were isolated from Balb/c heart transplanted B6 mice, followed by microarray analysis.
To define un-restored gene expressions in Irf4-deficient alloreactive T cells upon immune checkpoint blockades, we compared genes expression level among three groups. We revealed that checkpoint blockade restored the expression levels of the majority of wild-type T cell-expressed genes in Irf4-/- T cells, indicating the reinvigoration of Irf4-/- T cells. The remaining un-restored genes following checkpoint blockade, though minimal in number, may be responsible for the reinvigorated Irf4-/- T cells to become re-dysfunction.

overall_design: TCR(Vα2+Vβ6+)CD45.2+CD4+ TEa cells were isolated from splenocytes of WT TEa or Irf4−/− TEa mice by a FACSAria flow cytometer (BD Biosciences). B6.SJL CD45.1+ congenic mice were adoptively transferred with either 5 x 10e6 CD45.2+ WT TEa or 5 x 10e6 CD45.2+ Irf4‒/‒ TEa cells on day -1, and transplanted with Balb/c hearts on day 0. CD45.1+ mice adoptively transferred with either CD45.2+ Irf4‒/‒ TEa cells were i.p. injected with 400 μg Rat IgG, or 200 μg anti-PD-L1 plus 200 μg anti-CTLA-4 (9D9) mAbs on days 0, 3, 5. On day 6, adoptively transferred CD45.2+ TEa cells were sorted from splenocytes of transplant recipients by the FACSAria flow cytometer. Total RNA was extracted from sorted cells with RNeasy mini kit (Qiagen), followed by microarray analysis. Two independent experiments for each group were performed.

first: Jie

last: Wu

first: Wenhao

last: Chen


title: Gene expression profiling of novel glucocorticoids for severe asthma in RAW264.7 cells

city: Philadelphia

first: Hongbo

last: Liu

address: 3400 Civic Center Blvd

institute: University of Pennsylvania

summary: To investigate the anti-inflammation activity of novel glucocorticoids, we used microarray to profile gene expression pattern of novel GC VSG158 and VSG159, in parallel with FF and DEX, in the mouse macrophage RAW264.7 cells.

overall_design: RAW264.7 cells with various glucocorticoids (Dexamethasone, VSG158, VSG159, and fluticasone furoate) were treated with designated steroids at 100 nM one hour before overnight LPS stimulation of inflammation. Each treatment was duplicated. The following day, RNA samples were isolated using a Qiagen mini RNA extraction kit for gene profiling.

first: Yuanzheng

first: H

last: Xu


title: 3' mRNA QuantSeq of microglia/macrophages during zebrafish retinal regeneration following ouabain-induced inner retinal lesion

city: Moscow

first: Diana

last: Mitchell

state: Idaho

address: 875 Perimeter Dr, MS 3051

department: Biological Sciences

institute: University of Idaho

summary: RNA-seq to identify transcriptome of microglia and macrophages during zebrafish retinal regeneration

overall_design: A retinal lesion causing death of inner retinal neurons was induced by injection of 2μm final concentration of ouabain using mpeg1:GFP transgenic fish. 32 fish total were included in the experiment. At 7 days post ouabain lesion (7 dpi), 4 biological samples were generated by pooling 8 retinas to create one sample, and single cell suspension was prepared from dissociated retinas as detailed in Chi et al. 2018 Experimental Eye Research DOI 10.1016/j.exer.2018.08.002. FACS sorting was then used to purify GFP+ and GFP- cells from each sample.  For each sample (#1-4), mpeg1:GFP+ and GFP- cells were isolated/purified by FACS, RNA was extracted,and libraries were made using Lexogen 3' mRNA-Seq Prep Kit FWD for Illumina. Sequencing was performed on a SE75 run using an Illumina HiSeq 4000 instrument. Reads were quantified/mapped using Salmon v0.9.1 using the quasi-mapping-based mode and the "--noLengthCorrection" setting against Ensembl release-90 Danio rerio transcriptome (GRCz10).

middle: M

first: Chi

last: Sun

first: Samuel

middle: S

last: Hunter

first: Deborah

last: Stenkamp


title: A Bayesian mixture model for clustering droplet-based single cell transcriptomic data from population studies

status: Public on Mar 09 2019

city: Pittsburgh

first: Zhe

address: 130 DeSoto Street

institute: University of Pittsburgh

summary:
Abstract: The recently developed droplet-based single cell transcriptome sequencing (scRNA-seq) technology makes it feasible to perform a population-scale scRNA-seq study, in which the transcriptome is measured for tens of thousands of single cells from multiple individuals. Despite the advances of many clustering methods, there are few tailored methods for population-scale scRNA-seq studies. Here, we develop a BAyesian Mixture Model for Single Cell sequencing (BAMM-SC) method to cluster scRNA-seq data from multiple individuals simultaneously. BAMM-SC takes raw count data as input and accounts for data heterogeneity and batch effect among multiple individuals in a unified Bayesian hierarchical model framework. Results from applications of BAMM-SC to in-house experimental scRNA-seq datasets using blood and lung cells from humans or mice demonstrate that BAMM-SC outperformed existing clustering methods with considerable improved clustering accuracy, particularly in the presence of heterogeneity among individuals.

Data purpose: To evaluate the performance of BAMM-SC for clustering droplet-based scRNA-seq data in population-based study, we performed single cell RNA-seq on peripheral blood mononuclear cells (PBMC) isolated from whole blood obtained from 4 healthy donors, and on lung cells isolated from streptococcus pneumonia (SP) infected and naïve mice.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Peripheral blood mononuclear cells (PBMC) from healthy donors, skin cells from the dorsal mid-forearm of healthy donors, and lung cells from streptococcus pneumonia (SP) infected and naïve mice

first: Kong

first: Wei


title: Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program

status: Public on Mar 08 2019

city: Amsterdam

first: Vincent

middle: M.

last: Christoffels

state: N/A (NA)

address: Meibergdreef 15  K2-159

department: Medical Biology

country: Netherlands

institute: Amsterdam UMC

summary: Expression analysis of mouse sinoatrial nodes and working cardiomyocytes

overall_design: RNA-seq on human and mouse sinoatrial node and working myocardial tissue

last: van Eif

first: Sonia

last: Stefanovic


title: Flura-seq identifies organ-specific adaptations in metastasis-initiating cells

city: New York City

first: Lin

last: Tian

state: New York

address: Mortimer B. Zuckerman Research Center, 417 East 68th Street

department: Cancer Biology & Genetics Program

institute: Memorial Sloan Kettering Cancer Center

summary: Metastasis-initiating cells dynamically adapt to the distinct microenvironments of different organs, but these early adaptations are poorly understood due to the limited sensitivity of in situ transcriptomics.  We developed fluorouracil-labeled RNA sequencing (Flura-seq) for in situ analysis with unprecedented sensitivity.  Flura-seq utilizes cytosine deaminase (CD) to convert fluorocytosine to fluorouracil, covalently labeling nascent RNA for purification and sequencing. Flura-seq revealed that breast cancer micrometastases in lung and brain exhibit unique, reversible gene signatures depending on the microenvironment. Specifically, the mitochondrial electron transport Complex I and the NRF2-driven antioxidant programs were induced in oxygen-rich pulmonary micrometastases, compared to mammary tumors or brain micrometastases. Loss of Complex I activity, and antioxidant supplementation potentiated pulmonary metastatic growth. We confirm lung metastasis-specific NRF2 overexpression in clinical samples, thus validating Flura-seq’s utility in identifying clinically actionable microenvironmental adaptations in early metastasis. The sensitivity, robustness and economy of Flura-seq are broadly applicable beyond cancer research.

overall_design: Examination of 5-FU labeled RNAs in cancer cells present in different organs

first: Harihar

last: Basnet

first: Joan

last: Massagué


title: Gene expression profiling of planarians after inhibition of hmcn-1

city: Cambridge

first: Peter

last: Reddien

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: Inhibition of hemicentin-1, which encodes a highly conserved ECM glycoprotein specifically expressed in muscle, leads to aberrant localization of internal parenchymal cells, including planarian stem cells (neoblasts) and differentiated cells, between the outer epidermis and sub-epidermal muscle.

overall_design: Polyadenylated RNA was isolated from tail fragments planarians following feeding with in vitro transcribed RNAi of a control gene (C.elegans unc-22; 3 replicates) or hmcn-1 (3 replicates). Worms were fed four times every other day and cut 5 days after final feeding for a total of 13 days, a timepoint before ectopic cells are visible.

first: Lauren

last: Cote


title: Nascent RNA sequencing in IMR-32 cells treated with THZ531 or DMSO

city: Boston

first: Ruben

last: Dries

address: 360 Longwood Ave

department: Computational Biology

institute: DFCI

summary: We performed RNA-seq analsysis of nascent transcripts obtained by 4sU labeling in IMR-32 cells treated with THZ531 for 30min and 2h and DMSO as a control

overall_design: Expression of  nascent  transcripts in IMR-32 treated with THZ531 using  standard RNA-seq library kit in triplicate, for a total of 9 individual samples

first: Malgorzata

last: Krajewska

first: Rani

last: George


title: Mycobacterium tuberculosis: DNA gyrase depleted Vs control

status: Public on Mar 07 2019

city: Faridabad

first: Nisheeth

last: Agarwal

state: Haryana

address: 3rd Milestone, Faridabad-Gurugram Expressway

department: Infection and Immunity

country: India

institute: Translational Health Science and Technology Institute

summary: Transcriptional profiling of gyr(-) strain of Mycobacterium tuberculosis H37Ra comparing 20ng/ml ATc treated cells with ATc-untreated cells after 4 days of treatment with shaking at 200rpm at 37°C.

overall_design: Two-condition experiment, ATc-treated (Test) Vs ATc-untreated cells (Control). Biological replicates: 1 test, 1 control. Dye swap.

first: Eira

last: Choudhary


title: Novel risk variants affecting enhancers of TH1 and TREG cells in type 1 diabetes [ChIP-seq]

status: Public on Mar 26 2019

city: Hershey

first: Yasin

last: Uzun

address: 700 HMC Crescent Rd

institute: Pennsylvania State University College of Medicine

summary: Most type 1 diabets (T1D) associated SNPs are located in non-coding regions, making it hard to understand their functional impact. We performed epigenomic profiling of two enhancer marks, H3K4me1 and H3K27ac, using primary TH1 and TREG cells from healthy  and T1D subjects. By integrating enhancers predicted using these ChIP-Seq data, T1D associated SNPs and additional supporting data, we found and validated several novel risk SNPs for T1D.

overall_design: Effector momory TH1 and effector memory TREG cells were purified using FACS from 5 health and 6 T1D subjects. With these cells, we generated the profiling of H3K4me1 and H3K27ac using a low-cell number ChIP-Seq protocol. Enhancers were predicted using these ChIP-Seq data and analyzed by integrating RNA-Seq, eQTL, T1D associated SNPs and additional data, we predicted and validated several novel T1D risk SNPs.

first: Kai

last: Tan

first: Peng

last: Gao

first: Bing

last: He

first: Sarah

last: Salamati

first: Julie

last: Coffey

first: Eva

last: Tsalikian


title: Evaluation of the potential of 4 micro algae extracts to modulate the immune response and metabolic state in intestinal epithelium cells

city: Lelystad

first: Marcel

last: Hulst

state: Flevoland

address: Edelhertweg 15

department: Livestock research

institute: Animal Sciences Group

summary: Micro algae's are used as alternative protein source in human and animal diets. Besides micro algae contain substantial amounts of proteins they also contain a high concentration of, often unique, biological and chemical substances with potential to induce beneficial and health promoting effects in humans and animals. This study was set up to evaluate the potential of these substances to improve (intestinal) health. The effect of extracts prepared from 3 monocultures of micro algae's (Chlorella vulgaris [C], Haematococcus pluvialis [H], and Spirulina platensis [S]) and a mixed culture of micro algae's (AM; a mixture of Scenedesmus sp. and Chlorella sp. ) was studied in the presence and absence of the enterotoxigenic bacterium Escherichia coli k99 strain (ETEC, [E]) as an in vitro challenge. The E.coli-k99 strain with adhesion factor F41 (41/32) was isolated from a mastitis-infected udder. Gene expression was measured in cultured intestinal porcine epithelium cells (IPECJ2 cell line) after 2 and 6 hours incubation with C, H, and S extracts, and after 6 hours with the AM extract, using “whole genome” porcine microarrays. Gene expression profiles were analysed using functional bioinformatics programs to provide insight in the biological processes induced by micro algae extracts.

overall_design:
IPECJ2 cells were grown in 2 cm2 wells for 7 days at 37 ºC and 5% CO2 using 1:1 DMEM/Ham’s F10 1:1 medium supplemented with 5% FCS without antibiotics. For all tests, confluent monolayers were washed twice with medium without FCS (hereafter denoted as medium) and incubated for 1 hour with this medium. Hereafter, the medium was discarded and algae extracts suspended in medium were added to wells with IPECJ2 monolayers and incubated for a period of 2 and 6 hours in the absence and presence of ETEC (±10 cfu per cell). All incubations were tested in duplicate and for each type of additive duplicate control wells containing no additive (only culture medium) were incubated for 2 and 6 hours. After incubation total RNA was extracted. RNA's extracted from replicates were pooled (biological replicates) and this pool was hybridized in duplicate (technical replicates).
IPECJ2 enterocyte cell line derived from the jejunum of piglets, host-feed interaction

first: Arjan

last: Hoekman

first: Rommie

last: van der Weide

first: Marinus

last: van Krimpen


title: Single cell RNA-seq of Epidermal cells from newborn (P1) wildtype and BAP1 knockout mice

city: South San Francisco

first: Rohit

last: Reja

state: CA

address: 1 DNA Way

institute: Genentech

summary: BAP1 deletion in primary mouse epidermal cell culture leads to increased melanocytes population. However, it is unclear whether BAP1 exhibits its function in melanocytes or in the progenitor cells for melanocytes, since the epidermal culture contains a mixed cell types. We aim to carry out a single cell RNAseq profiling with this system, and dissect BAP1's roles in different cell types.

overall_design: Epidermal cells from newborn (P1) mice after culture for 7 days ex vivo - one wildtype (BAP1 WT) and one BAP1 knockout (BAP1 KO)  sample were dissociated into single cells and sequenced.

first: Meng


title: Induced Mesenchymal stromal cells derived from iPS cells from aged individuals acquire a rejuvenation signature and a MSC-specific secretome

city: Düsseldorf

first: Wasco

last: Wruck

address: Moorenstr. 5

institute: Universitätsklinikum Düsseldorf

summary: Human induced pluripotent stem cells (iPSCs) - derived mesenchymal stromal cells (iMSCs) are a potentially useful cell type for circumventing ageing-related shortfalls associated with primary mesenchymal or skeletal stromal cells (MSCs/SSCs). To date, the extent of the reflection of ageing-hallmarks in iMSCs differentiated from iPSCs derived from elderly donors remains unclear. In this study, we show that fetal (55 days post conception) femur-derived MSCs and MSCs isolated from aged (60 – 74 years) donors differ in their transcriptome and secretome profile. Yet, iMSCs irrespective of donor age and cell type acquired a rejuvenation gene signature, specifically, INHBE, DNMT3B, POU5F1P1, CDKN1C, GCNT2; also present in pluripotent stem cells but not observed in the parental MSCs. Furthermore, dendrograms generated from the transcriptomes of iMSCs derived from the human embryonic stem cell line H1, fetal and aged MSCs always clustered together with the parental fetal femur-derived MSCs. In addition, these were distinct from MSCs isolated from aged donors when comparing gene ontologies (GOs) related to ageing processes. Critically, in terms of regenerative medicine applications, iMSCs re-acquired a similar secretome (e.g. SERPINE1, SDF-1a, HGF, IL6, IL10, THBS1) to that of the parental fetal MSCs, thus re-enforcing their capabilities of imparting context dependent paracrine signaling.

overall_design: Total RNA obtained from induced mesenchymal stromal cells derived from iPS cells from aged and young individuals

first: Matthias

last: Megges


title: Investigation of the effect of 1,25(OH)2D3 on murine CD11+ dendritic cells

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

institute: Centre for Immunity, Infection and Evolution

summary: Gene expression was compared between CD11c+ bone marrow derived dendritic cells (BMDC) was compared between cells conditioned in 20nM 1,25(OH)2D3 (Vitamin D) or vehicle. A second analysis compared gene expression in 1,25(OH)2D3 or vehicle CD11c+  BMDCs which were matured in presence of 0.1ug/ml LPS.

overall_design: Murine bone marrow was harvested and cells grown for 9 days in presence of GM-CSF and either 20nM 1,25(OH)2D3 or vehicle. CD11c+ were isolated by FACS after which RNA was extracted. In some experiments the CD11c+ cells were stimulated with 0.1ug/ml LPS for an additional 18hours

first: Richard

middle: J

last: Mellanby


title: Genome-wide gene expression profiling of three Ethiopian insecticide resistant Anopheles arabiensis populations and two insecticide susceptible Anopheles arabiensis strains

status: Public on Mar 20 2019

city: Ghent

first: Wannes

last: Dermauw

address: Coupure Links 653

department: Crop Protection

country: Belgium

institute: University Ghent

summary: Illumina HiSeq 2500 technology was used to sequence  the transcriptome of three Ethiopian field populations of An. arabiensis (Asendabo (ASN), Chewaka (CHW) and Tolay (TOL)) and two An. arabiensis strains (Sekoru (SEK, from Ethiopia) and Moz (MOZ, from Mozambique)). Both ASN, CHW and TOL were previously shown to be resistant against delamethrin and DDT, while the two An. arabiensis strains (SEK and MOZ) were both susceptible to deltamethrin and DDT (Alemayehu et al. 2017, DOI: 10.1186/s13071-017-2342-y; Witzig et al. 2013, DOI: 10.1038/hdy.2012.112). Differentially expressed genes (absolute fold change (FC) ≥ 2 and Benjamini-Hochberg adjusted p-value < 0.05) were determined between each insecticide resistant field population (ASN, CHW or TOL) and one of the insecticide susceptible strains (SEK or MOZ).

overall_design: mRNA profiles consisting of four biological replicates each for each population (ASN, CHW, TOL) or strain (SEK, MOZ),  twenty samples in total, were generated with RNA collected from ten adult female An. arabiensis mosquitoes.

first: Eba

middle: A

last: Simma

first: Simon

last: Snoeck

first: Astrid

last: Bryon

last: Clark

first: Luc

last: Duchateau

first: Thomas

last: Van Leeuwen


title: MicroRNA-mediated suppression of the TGF-β pathway confers transmissible and reversible CDK4/6 inhibitor resistance

status: Public on Mar 04 2019

city: boston

first: Liam

last: Cornell

state: MASSACHUSETTS

address: 450 brookline avenue

institute: Dana-Farber Cancer Institute

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

platform_organism:
Homo sapiens
Human alphaherpesvirus 1
Human alphaherpesvirus 2
Cytomegalovirus
Lymphocryptovirus
Rhadinovirus
JC polyomavirus
Human immunodeficiency virus 1
Merkel cell polyomavirus
Betapolyomavirus hominis
Betapolyomavirus macacae

overall_design: Refer to individual Series


title: MicroRNA-mediated suppression of the TGF-β pathway confers transmissible and reversible CDK4/6 inhibitor resistance (RNA-Seq)

summary: CDK4/6 inhibition is now part of the standard armamentarium for patients with estrogen receptor (ER)-positive breast cancer, so that defining mechanisms of resistance is a pressing issue. Here, we identify increased CDK6 expression as a key determinant of acquired resistance after exposure to palbociclib in ER-positive breast cancer cells. Increased CDK6 in resistant cells was dependent on TGF-β pathway suppression via miR-432-5p expression. Exosomal miR-432-5p expression mediated transfer of the resistance phenotype between neighboring cell populations. We confirmed these data in pre-treatment and post-progression biopsies from a parotid cancer patient who had responded to ribociclib, demonstrating clinical relevance of this mechanism. Additionally, the CDK4/6 inhibitor resistance phenotype can be reversed in vitro and in vivo by a prolonged drug holiday.

overall_design: To analyse the binding targets of miR-432-5p we performed a mRNA pulldown using a synthetic biotin laballed miR-432-5p. RNAseq was performed to identify the captured mRNA.

first: Geoffrey

middle: I

last: Shapiro.


title: Genomic signature of parity in the breast of premenopausal women

first: Julia

last: Santucci-Pereira

state: PA

address: 333 Cottman Ave, P2051

institute: Fox Chase Cancer Center

summary:
Full-term pregnancy (FTP) at an early age confers long-term protection against breast cancer, in this study, we evaluated gene expression changes induced by parity in the breast of premenopausal women.
Gene expression profiling of normal breast tissue from 30 nulliparous (NP) and 79 parous (P) premenopausal volunteers were performed using Affymetrix microarrays. In addition to a discovery/validation analysis, we conducted analysis of gene expression differences in P vs. NP women as a function of time since last FTP.

overall_design: We compared the gene expression profile of normal breast core biopsies from 79 parous (women that had a full term pregnancy) and 30 nulliparous (women that never completed a pregnancy) premenopausal women from Sweden. All samples were hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays.

first: Anne

last: Zeleniuch-Jacquotte

first: Yelena

last: Afanasyeva

first: Hua

last: Zhong

first: Michael

last: Slifker

first: Suraj

last: Peri

first: Eric

last: Ross

first: Ricardo

last: López de Cicco

first: Yubo

last: Zhai

first: Theresa

last: Nguyen

first: Fathima

last: Sheriff

first: Irma

middle: H

last: Russo

first: Yanrong

last: Su

first: Alan

last: Arslan

first: Pal

last: Bordas

first: Per

last: Lenner

first: Janet

last: Åhman

first: Anna-Stina

last: Landström-Eriksson

first: Robert

last: Johansson

first: Göran

last: Hallmans

first: Paolo

last: Toniolo

first: Jose


title: MicroRNA-mediated suppression of the TGF-ß pathway confers transmissible and reversible CDK4/6 inhibitor resistance (miRNA array)

first: Liam

last: Cornell

overall_design: T47D cells were cultured to palbociclib resistance. We extracted RNA from parental, resistant, and parental T47D cells cultured in resistant cell media and analysed the expression of 3100 miRNAs using the Exiqon 7th generation miRCURY LNATM microRNA

first: Geoffrey

last: Shapiro


title: MicroRNA-mediated suppression of the TGF-β pathway confers transmissible and reversible CDK4/6 inhibitor resistance (miRNA-Seq)

overall_design: We performed miRNAseq in 44 tumor biopsies taken from patients who received CDK4/6 inhibitor treatment. Samples were grouped based on the patients response into sensitive (S), inherent (IR) or aquired (AR) CDK4/6 inhibitor resistance.

last: Shapiro.


title: B6xCAST F1 PRDM9-Humanized/CAST testes

city: Oxford

first: Anjali

middle: Gupta

last: Hinch

address: Roosevelt Drive

institute: Oxford University

type: Genome variation profiling by high throughput sequencing


title: Nitrogen and cycloheximide effects on protoplast and TF-target identification in the TARGET assay

city: Urbana

first: Matthew

last: Brooks

address: 1201 W Gregory Drive

institute: USDA

summary: The effects of nitrogen and cycloheximide treatments on nitrogen response and TF-target identification was tested in the cell-based TARGET assay. For protoplast experiments, Arabidopsis plants were grown on low nitrogen (1mM KNO3) and after 10 days protoplasts were generated from root tissue. Cells were transfected with a vector containing the GR-TGA4 fusion or GR-only empty vector and incubated overnight. In the morning, samples were pre-treated with nitrogen as specified for 2 hours, and +/-cycloheximide for 20 minutes, before TF entry into the nucleus was induced with dexamethasone. 3 hours after dex treatment samples were FACS sorted into RNA extraction buffer. For whole roots, Arabidopsis were grown in liquid media with low nitrogen (1 mM KNO3) in Phytatrays for 11 days. In the morning and at the same time as the protoplasts, the plants were transferred to fresh media containing either 20 mM KCL or 20mM KNO3 + 20mM NH4NO3 for 5 hours. Roots were harvested and immediately frozen in liquid N2.

overall_design: Three replicates were used for effect of cycloheximide and nitrogen source/dose on TGA4-target identification, control samples were transfected with a GR-only vector. Four replicates were collected for root vs. protoplast N-response.

first: Gloria

last: Coruzzi


title: RNA-Seq analysis of HIF-2a-responsive genes in clear-cell renal cell carcinoma

city: Hangzhou

first: Yilong

last: Zou

state: Zhejiang

address: Shilongshan Road #18, Cloud Town, Xihu District

department: School of Life Sciences

country: China

institute: Westlake University

summary: We performed RNA-Seq analysis of wildtype and three EPAS1-/- 786-O single cell clones generated by CRISPR/Cas9 to identify the HIF-2a-responsive genes in this cell line. Samples from wildtype 786-O cells treated with DMSO or HIF-2a antagonist compound C2 were also included in this analysis.

overall_design: In this experiment, we analyzed the transcriptomic profiles of 2 replicates of wildtype (WT) EPAS1+/+ 786-O cells, 1 replicate for each of the three independent EPAS1-/- 786-O single cell clones, 1 replicate of WT-786-O cells treated with DMSO and 1 replicate of WT-786-O cells treated with 10uM HIF-2a antagonist C2.

first: Stuart

middle: L

last: Schreiber


title: Defining the transcriptional and post-transcriptional landscapes of Mycobacterium smegmatis in aerobic growth and hypoxia

status: Public on Mar 17 2019

city: Worcester

first: Scarlet

last: Shell

address: 60 Prescott St

department: Biology and Biotechnology

institute: Worcester Polytechnic Institute

summary: The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5’-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.

overall_design: RNAseq expression libraries were constructed from M. smegmatis RNA extracted from cultures in log phase growth and in hypoxia.  RNA 5' ends were mapped and quantified in both conditions using an approach that involves ligating adapters to 5' ends with and without prior treatment by an enzyme to convert 5' triphosphates to monophosphates.

first: M

last: Martini

first: Ying

last: Zhou

first: Huaming

last: Sun


title: Metatranscriptomics of the macaque juvenile gut microbiome in health and chronic diarrhea

city: Davis

first: Samuel

middle: Taler

last: Westreich

address: 1 Shields Ave

department: Genome Center

institute: University of California, Davis

summary: Background: Idiopathic Chronic Diarrhea (ICD) is a common cause of morbidity and mortality among juvenile rhesus macaques. Characterized by chronic inflammation of the colon and repeated bouts of diarrhea, ICD is largely unresponsive to medical interventions including corticosteroid, antiparasitic and antibiotic treatments. Although ICD is accompanied by large disruptions in the composition of the commensal gut microbiome, no single pathogen has been concretely identified as responsible for the onset and continuation of the disease.  Results: Fecal samples were collected from twelve ICD-diagnosed macaques and twelve age and sex-matched controls. RNA was extracted for metatranscriptomic analysis of species and activity within the gut microbiome. Using SAMSA2, these samples were contrasted to identify shifts both in overall organism activity and functional activity. Bacterial, fungal, archaeal, protozoan, and macaque (host) transcripts were simultaneously assessed. ICD-afflicted animals were characterized by increased activity of known bacterial pathogens and by decreased activity of archaeal methanogens. Interestingly, known fungal opportunists were not increased in ICD, nor were the usual enteric protozoans, although Trichomonas activity is up-regualted. Known mucin degrading organisms and mucin-degrading enzymes were up-regulated in the fecal microbiomes of ICD-afflicted animals. Assessment of colon sections using immunohistochemistry confirmed differential mucin composition between healthy control and ICD animals. Finally, assessment of host-derived transcripts confirms colonic inflammation and suggests that the lumen is infiltrated by granulocytes. Conclusions: The simultaneous profiling of bacterial, fungal, archaeal, protozoan, and macaque transcripts from stool samples suggests that  ICD of rhesus macaques is associated with increased pathogen activity and altered mucin degradation.

overall_design: 24 total samples, 12 ICD-diagnosed rhesus macaque juveniles, 12 age-matched and sex-matched healthy controls

first: Amir

last: Ardeshir

first: Mary

last: Kable

first: Zeynep

last: Alkan

first: Ian

last: Korf

first: Danielle

middle: G

last: Lemay


title: ChIP-seq from GM12878 (ENCSR436QMA)

status: Public on Mar 03 2019

city: Stanford

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

summary:
Reverse Crosslink Control ChIP-seq on human GM12878 (fragmentation date 01-30-14)

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf


title: Single-Cell Transcriptome Analysis of CD34+ Stem Cell-Derived Myeloid Cells Infected With Human Cytomegalovirus

status: Public on Mar 12 2019

city: Oakland

first: Laura

last: Hertel

address: 5700 Martin Luther King Jr Way

institute: Children's Hospital Oakland Research Institute

summary: Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34+ hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of susceptible and resistant cell types, and the cellular features characterizing permissive cells, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes of ~ 7000 individual cells at day one post-infection using the 10X genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit – Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells susceptible to CMV replication, and provide a possible rationale for their preferential infection.

overall_design: The 10x Genomics Chromium platform was used for single cell RNA sequencing of a population of myeloid cells differentiated from CD34+ hematopoietic progenitors and infected with CMV strain TB40/E.

first: Melissa

last: Galinato

first: Kristen

last: Shimoda

first: Alexis

last: Aguiar

first: Fiona

last: Hennig

first: Dario

last: Boffelli

first: Michael

last: McVoy


title: RdDM-independent de novo and heterochromatin DNA methylation by plant CMT and DNMT3 orthologs [Bisulfite-Seq]

city: Tel-Aviv

first: Katherine

last: Domb

address: Haim Levanon st.

department: Gray Faculty of Medical and Health Sciences

country: Israel

institute: Tel Aviv University

summary: To properly regulate the genome, cytosine methylation is established by animal DNA methyltransferase 3s (DNMT3s). While altered DNMT3 homologs, DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs), have been shown to establish methylation via the RNA directed DNA methylation (RdDM) pathway, the role of true-plant DNMT3 orthologs remains elusive. Here, we profile de novo (RPS transgene) and genomic methylation in the basal plant, Physcomitrella patens, mutated in each of its PpDNMTs. We show that PpDNMT3b mediates CG and CHH de novo methylation, independently of PpDRMs. Complementary de novo CHG methylation is specifically mediated by the CHROMOMETHYLASE, PpCMT. Intragenomic, PpDNMT3b functions preferentially within heterochromatin and is affected by PpCMT. In comparison, PpDRMs target active-euchromatic transposons. Overall, our data resolve how DNA methylation in plants can be established in heterochromatin independently of RdDM; suggest that DRMs have emerged to target euchromatin; and link DNMT3 loss in angiosperms to the initiation of heterochromatic CHH methylation by CMT2.

overall_design: Whole-genome bisulfite sequencing of the following P. patens samples: wild type, drm1, drm2, drm1/drm2, dnmt3a, dnmt3b, dnmt3a/dnmt3b, met, cmt, and rdr2 containing the repetitive petunia sequence (RPS) transgene.

first: Rafael

last: Yaari

first: Aviva

last: Katz

first: Keith

last: Harris

first: Assaf

last: Zemach

first: Nir

last: Ohad


title: MicroRNA-mediated suppression of the TGF-β pathway confers transmissible and reversible CDK4/6 inhibitor resistance (array I)

first: Liam

last: Cornell

overall_design: T47D cells were cultured to palbociclib resistance. RNA was extracted parental, and palbociclib resistant cells. We used the affymetrix Human Genome U133+ 2.0 array to analyse gene expression.

first: Geoffrey

last: Shapiro


title: ChIP-seq from GM12878 (ENCSR542FLV)

summary:
ZBTB33 ChIP-seq on human GM12878

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf


title: Neurofibroma

city: Paris Cedex 05

first: Nicolas

last: Servant

address: 26 rue d'ulm

country: France

institute: Institut Curie


title: ChIP-seq from GM12878 (ENCSR177VFS)

first: ENCODE

last: DCC

summary:
MEF2B ChIP-seq on human GM12878

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf


title: Genome-wide maps of the H3K27me3 histone modification in wild-type or SUZ12-mutant ipNF05.5 cells.

summary: Using ChIP-seq, we profiled the H3K27me3 histone mark in wild-type or SUZ12-mutant ipNF05.5 cells.

overall_design: Examination of the H3K27me3 histone modification in wild-type or SUZ12-mutant ipNF05.5 cells.

first: Michel

last: Wassef

first: Armelle

last: Luscan

first: Dina

last: Zielinski

first: Setareh

last: Aflaki

first: Pascal

last: Jansen

first: Aude

last: Battistella

first: Carole

last: Kersouani

first: Margaret

middle: R

last: Wallace

last: Vermeulen

last: Vidaud

first: Eric

last: Pasmant

first: Raphaël

last: Margueron


title: NanoString analysis of immune genes in the tumor at day 10 after injection into WT and Rnf5-/- mice

city: Rockville

first: Tongwu

last: Zhang

state: MD

address: 9609 Medical Center Drive

department: Division of Cancer Epidemiology & Genetics

institute: National Cancer Institute

summary:
The NanoString analysis of 770 genes expressed by 24 immune cell types in the tumors at 10 days after injection were performed. The Nanostring analysis uncovered marked difference in key immune regulatory networks associated with

T, DC, NC and macrophage function

overall_design: Yumm1.5 tumor cells were injected into WT or Rnf5-/- mice. Tumor were collected at day 10 and RNA were isolated from the tumor. Then Nanostring nCounter assay for immune genes were performed

first: Yan

last: Li

first: Ze'ev

last: Ronai


title: Sec14l3 potentiates VEGFR2 signaling to regulate zebrafish vasculogenesis

city: Beijing

first: Bo

last: Gong

address: Qinghua Yuan street No.1

department: School of Life Science

institute: Tsinghua University

summary: We isolated GFP-positive and negative cell populations, by fluorescence-activated cell sorting (FACS), from Tg(fli1a:EGFP)y1 embryos  at 22-24 hpf, then extracted their RNAs for high-through put sequencing based on Illumina HiSeq2000 to find out enriched genes in vascular, hematopoietic and pharyngeal arch cells

overall_design: mRNA profiles of zebrafish endothelial and non-endothelial cells at 24 hpf

first: Shunji

last: Jia


title: RNA-seq of microglia isolated from aged mice treated with IgG or anti-CD22.

status: Public on Mar 01 2019

city: palo alto

first: John

last: Pluvinage

state: California

address: 3801 Miranda Ave Rm D3-131

institute: Stanford

summary: Sequencing data related to the manuscript entitled, "CD22 blockade restores homeostatic microglial phagocytosis in the aging brain."

overall_design: To assess the transcriptional effects of CD22 blockade, we implanted aged mice with osmotic pumps to continuously infuse a CD22 blocking antibody or an IgG control antibody directly into the cerebrospinal fluid for one month. Following one month of continuous infusion, we performed RNA-seq on purified microglia from the hemi-brains of these mice contralateral to the cannulation site to minimize injury-induced confounding factors. Primary mouse microglia were isolated by gentle dounce homogenization of the brain, magentic myelin removal, and FACS-purification of ~20,000 live CD11b+CD45lo cells. Microglia were sorted into RLT Plus buffer (Qiagen) containing beta-mercaptoethanol. RNA was extracted using a RNeasy Micro Plus kit (Qiagen) according the manufacturer’s protocol. RNA integrity was assessed on a Bioanalyzer (Agilent), and high quality samples were used for library preparation. cDNA synthesis and amplification was performed using the SmartSeq v4 Ultra-low input kit (Takara), and libraries were tagmented, adaptor ligated, and indexed using the Nextera XT kit (Illumina). After normalization and pooling, libraries were sequenced on a Hiseq 4000 (Illumina) using paired-end 100bp reads. Raw sequencing files were demultiplexed with bcl2fastq, reads were aligned using STAR, the count matrix was generated using SummarizedExperiment, and differential expression analysis was performed using DESeq2 with standard settings.


title: Testing antigen recognition of human monoclonal antibodies to PvDBPII  on PfPv500.1 array

status: Public on Mar 02 2019

city: Cleveland

first: Christopher

last: King

state: Ohio

address: 2109 Adelbert Road 4th Floor

department: Center for Global Health & Diseases

institute: Case Western Reserve University

summary: Established that 11 human anti-Plasmodium vivax Duffy Binding protein II monoclonal antibodies are not cross reactive with other plasmodium antigens as represented by PfPv500.1 array.

platform_organism:
Plasmodium falciparum
Plasmodium vivax

overall_design: 11 human monoclonal antibodies to Plasmodium vivax Duffy Binding protein region II,  an isotype control, a polyreactive isotype control and a human plasma pool from Papua New Guinea tested for recognition of PfPv500.1 array

first: Lenore

last: Carias


title: Modeling PD in midbrain-like organoids

city: Dresden

first: Lydia

last: Reinhardt

address: Fetscherstraße 105

institute: CRTD

summary: We have developed a method for derivation of mfNPCs from human induced pluripotent stem cells (hIPSC), that are only dependent on small molecules stimulating WNT and SHH signalling for their propagation.

overall_design: Human pluripotent stem cells were differentiated into the neural lineage using double SMAD inhibition, together with stimulation of WNT and SHH pathways. This led to expansion of neural epithelium, which was further cultured in N2B27 medium supplemented with 2.5uM SB and 150nM LDN plus CHIR, ascorbic acid (AA) and SAG (for mfNPC) or CHIR, AA and SAG (for smNPC), respectively.

first: Michael

last: Glatza


title: Spontaneously slow-cycling subpopulations of human cells originate from activation of stress response pathways

status: Public on Feb 28 2019

city: Boulder

first: Mingwei

last: Min

state: CO

address: 3415 Colorado Ave

institute: University of colorado-boulderB

summary: Slow-cycling subpopulations exist in bacteria, yeast, and mammalian systems. In the case of cancer, slow-cycling subpopulations have been proposed to give rise to drug resistance. However, the origin of slow-cycling human cells is poorly studied, in large part due to lack of markers to identify these rare cells. Slow-cycling cells pass through a non-cycling period marked by low CDK2 activity and high p21 levels. Here, we use this knowledge to isolate these naturally slow-cycling cells from a heterogeneous population and perform RNA-sequencing to delineate the transcriptome underlying the slow-cycling state. We show that cellular stress responses – the p53 transcriptional response and the integrated stress response – are the most salient causes of spontaneous entry into the slow-cycling state.

overall_design: mRNA profiling of spontaneously quiescent human cells and cells forced into quiescence by four different methods

first: Sabrina

last: Spencer


title: A genome-wide CRISPR screen identifies mediators of ferroptosis sensitivity in clear-cell carcinomas

first: Yilong

last: Zou

summary: Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCC tumors are distinguished by aberrant lipid and glycogen accumulation and are inherently refractory to a broad range of anti-cancer therapies. In the study associated with this dataset, we identified an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCC cells. However, the mediators of this sensitivity to ferroptosis are unknown. Here we perform a genome-wide CRISPR screen to identify genes that mediate the sensitivity to ML210, a selective inhibitor of glutathione peroxidase 4 (GPX4) and a potent inducer of ferroptotic cell death in 786-O, a clear-cell renal cell carcinoma cell line.

overall_design: 786-O cells with Cas9 expression were infected with a genome-wide lentiviral sgRNA library, selected with puromycin, and expanded for treatment with DMSO or ML210 for 4, 6, or 8 days, respectively. sgRNA-barcodes that were enriched in ML210 treated conditions were identified by high-throughput sequencing.

first: Mudra

last: Hegde

last: Doench

first: Stuart

last: Schreiber


title: Cellular response to small molecules that selectively stall protein synthesis by the ribosome

status: Public on Mar 06 2019

city: Berkeley

first: Jamie

middle: HD

last: Cate

address: 748 Stanley Hall

institute: University of California Berkeley

summary: We use ribosome profiling to compare the selectivity of two small molecules, PF-06446846 and PF-06378503 that inhibits translation of their target by selectively inducing ribosome-stalling in a nascent chain sequence dependent manner.

overall_design: Huh7 cells were treated with 1.5 µM PF-06446846, 1.5µM PF-06378503 or vehicle for 60 minutes, and subjected to ribosome profiling. This study included three biological replicates

first: Nadège

last: Liaud