title: Hepatitis B virus-mediated alterations to the primary hepatocyte transcriptome [Full Set]

summary:
Purpose:  Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer.  A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases.  Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale.  Because of this, we performed an RNA-seq analysis of primary rat hepatocytes either expressing HBV or not over a series of time points to determine the global changes HBV has on primary hepatocyte physiology.
Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes (PRH) over the course of 72hr.  PRH were collected immediately after isolation (0hr), or 24hr, 48hr, or 72hr after plating.  In addition, PRH were infected 24hr after plating with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV) and collected at 48hr after plating (24hr after infection) or 72hr after plating (48hr after infection).  cDNA libraries were sequenced using the Illumina NextSeq 500 platform to generate either 1x75bp reads.  Reads were mapped using the STAR aligner, and output BAMs were further analyzed in R using the GenomicAlignments package, to quantify number of reads per transcript, and DESeq2, to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample.
Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 48hr and 72hr.  In addition, we noted considerable change to the hepatocyte transcriptome as a direct result of the isolation/plating procedure, regardless of the presence of HBV.  Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes.

overall_design: mRNA profiles of cultured primary rat hepatocytes were generated, in triplicate, using the Illumina NextSeq 500 platform from freshly isolated cells (0hr), 24hr, 48hr, or 72hr after plating, and with or without expression of HBV 48hr or 72hr after plating.


title: Single-cell RNA-seq of bone marrow-derived mesenchymal stem cells reveals unique profiles of lineage priming

status: Public on Aug 06 2015

city: Minneapolis

first: Brenda

middle: M

last: Ogle

state: Minnesota

address: 312 Church St SE

department: Biomedical Engineering

country: USA

institute: University of Minnesota-Twin Cities

type: Expression profiling by high throughput sequencing

summary: The plasticity and immunomodulatory capacity of mesenchymal stem cells (MSCs) have spurred clinical use in recent years.  However, clinical outcomes vary and many ascribe inconsistency to the tissue source of MSCs.  Yet unconsidered is the extent of heterogeneity of individual MSCs from a given tissue source with respect to differentiation potential and immune regulatory function.  Here we use single-cell RNA-seq to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow.  We found genes associated with MSC multipotency were expressed at a high level and with consistency between individual cells. However, genes associated with osteogenic, chondrogenic, adipogenic, neurogenic and vascular smooth muscle differentiation were expressed at widely varying levels between individual cells. Further, certain genes associated with immunomodulation were also inconsistent between individual cells.  Differences could not be ascribed to cycles of proliferation, culture bias or other cellular process, which might alter transcript expression in a regular or cyclic pattern.  These results support and extend the concept of lineage priming of MSCs and emphasize caution for in vivo or clinical use of MSCs, even when immunomodulation is the goal, since multiple mesodermal (and even perhaps ectodermal) outcomes are a possibility.  Purification might enable shifting of the probability of a certain outcome, but is unlikely to remove multilineage potential altogether.

overall_design: Examination was performed using single-cell RNA-seq of sixteen mouse MSCs (mMSC1-mMSC16), non MSC single cell controls (HL1cm1-HL1cm5), and the population controls (mMSC-PC and HL1cm-PC).

first: Brian

middle: T

last: Freeman

first: Philip

middle: J

last: Jung


title: Identification of in vivo binding sites of human C2H2-ZF proteins

status: Public on Feb 17 2015

city: Montreal

first: Hamed

middle: S

last: Najafabadi

state: QC

address: 740 Dr. Penfield Avenue, Room 7202

department: Human Genetics

country: Canada

institute: McGill University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. We determined in vivo binding sites of 39 human C2H2-ZF proteins, and correlated them with potential functions for these proteins.

overall_design: We expressed GFP-tagged C2H2-ZF proteins in stable transgenic HEK293 cells. Chromatin immunoprecipitation was performed as described before (Schmidt et al., Methods, 2009), and ChIP samples along with several control samples from different experimental batches were sequenced on  Illumina HiSeq 2500. Reads were mapped to hg19 (GRCh37) assembly, and peaks were identified by MACS using an experiment-specific background that controls for various biases, such as the Sono-Seq effect as well as potential co-purification of targets of other (interacting) proteins.

first: Frank

middle: W

last: Schmitges

first: Ernest

last: Radovani

first: Jack

last: Greenblatt

first: Timothy

middle: R

last: Hughes


title: RNA-seq analysis of human heart failure

status: Public on Apr 28 2014

city: Valencia

first: Estefanía

middle: Tarazón

last: Melguizo

state: Valencia

address: Avenida Fernando Abril Martorell, 106

country: Spain

institute: Health Research Institute La Fe

summary: The goal of this study is to compare the transcriptome of heart failure patients (with ischemic or dilated cardiomyopathy) undergoing heart transplantation compared with healthy controls.

overall_design:
We analyzed 36 human samples. 13 from ischemic and 13 from dilated human hearts compared with 10 healthy control donors.
The 'processed_data_readme.txt' contains the list of the processed data column headers associated with each sample.

last: Tarazón

first: Esther

last: Roselló-Lletí

first: Ana

last: Ortega

first: Maria

last: Molina-Navarro

first: Juan

middle: C

last: Triviño

first: Francisca

last: Lago

first: José

last: González-Juanatey

first: Placido

last: Orosa

middle: A

last: Montero

first: Antonio

last: Salvador

first: Manuel

last: Portolés

first: Miguel

last: Rivera


title: An Atlas of Bovine Gene Expression Reveals Novel Distinctive Tissue Characteristics and Evidence for Improving Genome Annotation

status: Public on Sep 29 2010

city: Clay Center

first: Gregory

middle: Paul

last: Harhay

state: NE

department: Genetics and Breeding Research Unit

institute: USDA-ARS-USMARC

summary:
Background: A comprehensive transcriptome survey, or "gene atlas", provides information essential for a complete understanding of the genomic biology of an organism. We present an atlas of RNA abundance for 92 adult, juvenile and fetal cattle tissues and 3 cattle cell lines.

Results: The Bovine Gene Atlas was generated from 7.2 million unique digital gene expression tag sequences (300.2 million total raw tag sequences), from which 1.59 million unique tag sequences were identified that mapped to the bovine genome accounting for 85% of the total raw tag abundance. Filtering these tags yielded 87,764 unique tag sequences that unambiguously mapped to 16,517 annotated protein-coding loci in the genome accounting for 45% of the total raw tag abundance. Clustering of tissues based on tag abundance profiles generally confirmed ontology classification based on anatomy. There were 5,429 constitutively expressed loci and 3,445 constitutively expressed unique tag sequences mapping outside annotated gene boundaries that represent a resource for enhancing current gene models. Physical measures such as inferred transcript length or antisense tag abundance identified tissues with atypical transcriptional tag profiles.  We report for the first time the tissue specific variation in the proportion of mitochondrial transcriptional tag abundance. The Bovine Gene Atlas can be examined at http://www.agbase.msstate.edu/bovineatlas .

Conclusions: The Bovine Gene Atlas is the deepest and broadest transcriptome survey of any livestock genome to date.  Commonalities and variation in sense and antisense transcript tag profiles identified in different tissues facilitate the examination of the relationship between gene expression, tissue, and gene function.

overall_design: An atlas of mRNA abundance for 92 adult, juvenile and fetal cattle tissues and 3 cattle cell lines.

middle: P

last: Smith

first: Leeson

last: Alexander

first: Christian

middle: D

last: Haudenschild

first: John

last: Keele

first: Lakshmi

middle: K

last: Matukumalli

first: Steven

last: Schroeder

first: Curtis

last: Van Tassell

first: Cathy

last: Gresham

first: Susan

last: Bridges

first: Shane

last: Burgess

first: Tad

last: Sonstegard


title: Hyperthermophilic archaeon Pyrococcus furiosus response to hydrogen peroxide

status: Public on Feb 25 2010

city: Athens

first: Michael

middle: W. W.

last: Adams

state: GA

address: 120 East Green St.

department: Molecular Biology and Biochemistry

institute: University of Georgia

summary:
Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus
Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons.  More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 – 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I.  The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs.

overall_design: Pyrococcus furiosus (DSM 3638) was grown at 95°C in a 20-liter fermentor using maltose as the carbon and energy source. An exponential-phase culture of P. furiosus that had undergone three successive transfers in the experimental medium was used to inoculate the 20-liter fermentor. The culture was shocked with 0.5 mM of hydrogen peroxide (H2O2) when cell density was in mid-exponential phase (~ 5.0 ´ 107 cells/ml, as determined by direct microscopic cell count). To obtain RNA for microarray and for quantitative PCR (QPCR) analyses, samples (2 liter) were rapidly removed from the fermentor and cooled to 4°C. Total RNA was extracted using acid-phenol and stored at -80°C until needed.  A total of 3 biological replicates in triplicate (3 copies on the same slide) was used in the data set.

first: Kari

last: Strand

first: Chengjun

last: Sun

first: Ting

last: Li

first: Francis

middle: E

last: Jenney

first: Gerrit

last: Schut


title: Large-scale characterization of DNA binding landscape of eukaryotic C2H2 zinc fingers

first: Hamed

last: Najafabadi

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

Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors (TFs).  However, it is unknown whether most C2H2-ZFs even bind DNA, or what sequences they bind.  Using a combination of bacterial one-hybrid (B1H) assays, protein-binding microarrays (PBMs), and ChIP-seq, we have found that most natural C2H2-ZFs bind DNA both in vitro and in vivo. This SuperSeries contains the data for identification of C2H2-ZF binding preferences using these three approaches.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Refer to individual Series

first: Sanie

last: Mnaimneh

first: Frank

last: Schmitges

first: Ally

last: Yang

first: Ernest

last: Radovani

first: Mihai

last: Albu

first: Jack

last: Greenblatt

first: Timothy

last: Hughes


title: Comparison of the human oocyte to its sister polar body

status: Public on Oct 12 2011

city: Providence

first: Adrian

last: Reich

state: RI

address: 185 Meeting St

department: MCB

institute: Brown University

summary: Clinicians need additional metrics for predicting quality of human oocytes for IVF procedures. Human polar bodies reflect the oocyte transcript profile. Quantitation of polar body mRNAs could allow for both oocyte ranking and embryo preferences in IVF applications. The transcriptome of a polar body has never been reported, in any organism.

overall_design: Eight total samples. There are 2 biological replicates of the following four conditions: pooled oocytes and their sister polar bodies and a single oocyte and its sister polar body.

first: Peter

last: Klatsky

first: Sandra

last: Carson

first: Gary

last: Wessel


status: Public on May 25 2007

first: Michael

last: Adams

summary:
Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus:  Characterization of a Coenzyme A-Dependent NAD(P)H Sulfur Oxidoreductase

The hyperthermophilic archaeon Pyrococcus furiosus, uses carbohydrates as a carbon source and produces acetate, CO2 and H2 as end products.  When S° is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected.  After one hour cells contain high NADPH- and coenzyme A-dependent S° reduction activity (0.7 units/mg, 85°C) located in the cytoplasm.  The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR).  NSR is a homodimeric flavoprotein (Mr 100 kDa) and is encoded by PF1186.  This was previously assigned to an enzyme that reduces coenzyme A disulfide, which is a side-reaction of NSR.  Whole genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to 7-fold within 10 min of S° addition.  This primary response to S° also involves the up-regulation (> 16-fold) of a 13 gene cluster encoding a membrane-bound oxidoreductase (MBX).  MBX is proposed replace the homologous 14 gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S° addition.  Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S°.  A secondary response to S° is observed 30 min after S° addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins, termed SipA and SipB.  This novel S°-reducing system involving NSR and MBX is so far unique to the heterotrophic Thermococcales, and is in contrast to the cytochrome- and quinone-based S°-reducing system in autotrophic archaea and bacteria.
Keywords: time course, kinetic, sulfur metabolism, archaea, Pyrococcus furiosus, hyperthermophile

overall_design: Each kinetic experiment (growth date) containing 5 timepoints (0, 10, 20, 30 and 60 minutes) was performed twice. Each hybridization was performed twice and in case of 30 min 3 times.

first: Stephanie

middle: L

last: Bridger


title: Ludwig Institute/UCSD ENCODE project June 2007 Release

status: Public on Jun 01 2007

city: La Jolla

first: Bing

last: Ren

state: CA

address: 9500 Gilman Drive

department: Department of Cellular and Molecular Medicine

institute: Ludwig Institute for Cancer Research

type: Genome binding/occupancy profiling by genome tiling array

summary:
The readout of genome information is controlled by transcriptional regulatory elements, but a comprehensive view of the combinatorial control by these DNA sequences, which bind regulatory protein and/or the modified histones in regulating gene transcription, is clearly preliminary. We have developed an experimental strategy for comprehensive determination of such functional elements in human DNA. This strategy involves the application of genome-wide location analysis, also known as ChIP-chip, to a panel of well-characterized regulatory proteins and histones with specific modifications, known to generally associate with transcriptional regulatory elements in vivo. Identification of their genomic binding sites will allow us to determine the sequence features in the human genome that carry out transcriptional regulatory function. We have designed and produced DNA microarrays to represent all the non-repetitive sequences in the 30 million basepair ENCODE regions of the human genome to map various types of transcriptional regulatory elements in three model cell types. We have identified promoters by mapping the genomic sequences associated with RNA polymerase II and the general transcription factor TFIID in cells, enhancer elements by mapping the genomic sequences associated with transcriptional co-activators and enhancer-specific chromatin modifications, and insulators by mapping the genomic sequences associated with the insulator binding protein CTCF. The raw microarray results are released periodically prior to publication.
Keywords: ChIP-chip ENCODE

overall_design:
ChIP-chip analyses were performed for various transcription factors and chromatin modifications in various cell types. Details to be described in publications.

	Ren B, Glass CK, Rosenfeld MG, Webster N, Ching KA, Harp L, Ye Z, Stuart RK, Calcar SV, Kim TH, Hawkins RD, Hon G, Barrera LO, Luna R, Wang K

first: Christopher

last: Glass

middle: G

last: Rosenfeld

first: Nicholas

last: Webster

first: Keith

last: Ching

first: Lindsey

last: Harp

first: Zhen

last: Ye

first: Rhona

last: Stuart

first: Sara

middle: V

last: Calcar

first: Tae

middle: H

last: Kim

first: R

last: Hawkins

last: Hon

first: Leah

middle: O

last: Barrera

last: Luna

first: K

last: Wang