The fetal sheep heart transcriptome

Background: The sheep is an extensively studied animal model with unique potential for the study of developmental programming yet genomic resources are lacking. We aimed to identify the expressed genome from the fetal sheep heart in order to identify genes and gene variants relevant to developmental studies and to provide a genomic resource for development of sheep-specific genetic tools. We used high throughput sequencing technologies to achieve these goals. Results: Using the bovine genome as a reference for assembly and annotation of 75-bp paired-end reads, we identified 13,444 sheep fetal heart genes of which 13,064 are novel in the sheep. In addition, we identified 8,617 high confidence SNPs and 240 genes with exon splicing that differed between the cow and the sheep fetal heart transcriptome. The genes with the highest expression levels and genes with novel exons with the highest expression levels in the fetal heart transcriptome are known to play central roles in muscle development and function. Conclusions: In this study we show that high throughput sequencing methods can generate extensive transcriptome information in the absence of an assembled and annotated genome for that species. The gene sequence data obtained provides a resource for development of genetic tools and combined with the polymorphism data will enable annotation and assembly of the sheep genome. In addition, identification of novel splice variants in the fetal sheep heart transcriptome followed by KEGG pathway analysis is a first step towards revealing mechanisms of genetic variation and gene environment interaction during fetal heart development.

A global view of the complexity of the porcine transcriptome using a full-sib pair with extreme phenotypes in growth and fat deposit by deep RNA sequencing (mRNA)

Pig is an important animal model for human obesity and diseases. However, the complexity of the porcine transcriptome is not yet fully elucidated. Here we have used massively parallel high-throughput sequencing of cDNA (RNA-Seq) to generate a high-resolution map of the porcine transcriptome and miRNA in liver (LI), longissimus dorsi (LD) and abdominal fat (AF) from an F2 female full-sib pair with extreme phenotypes in growth and fat deposit. On the basis of the porcine annotated genes against the UCSC database, we identified 21,414 annotated genes in our RNA-Seq analysis and 48,045-122,931 novel transcript fragments, which could be clustered into 17,085-29,499 novel transcriptional active regions (nTARs). We found that ~18.8% of the detected known genes showed alternative splicing patterns, and alternative 3' splicing was the most common type of alternative splicing events in pigs. We also detected that more than 22.7% of the known genes identified here were extended at their 5' and/or 3' end. We identified 2,796, 1,551 and 835 differentially expressed genes (DEGs), respectively, in AF, LI and LD between the two individuals. Overall design: Examining the complexity of the pig transcriptome in three organs (liver, abdominal fat, longissimus dorsi muscle) from a female full-sib pair.

Comparative analyses of transcriptome during skeletal muscle development between pig breeds differing in muscle growth rate by deep-sequencing

Obese and lean pig show breed-specific traits in muscle growth and meat quality. However, the mechanisms underlying remains unclear. Here, we reported the first genome-wide muscle development research from embryonic to 6 months old between Lantang (obese) and Landrace (lean) using Solexa/Illumina's Genome Analyzer. We obtained 4.22±0.9×107 total reads per sample with approximately 5×106 distinct tags. Filtered adaptor tags, empty reads, low quality tags and tags of copy number =1, we obtained 3.84±0.9×107 clean total tags with 1.5±0.5×106 clean distinct tags. Overall design: 20 libraries of longissimus muscle samples were sequenced in Lantang and Landrace at days 35, 49, 63, 77, 91 prenatal and 2, 28, 90, 120, 180 postnatal days.

Whole transcriptome profiling of the rat pineal gland

10.1073/pnas.1207748109

The rat pineal gland is a highly dynamic tissue with many hundreds of genes changing more than two-fold in a 24-hr daily rhythm as measured by Affymetrix GeneChip analysis. We sought to more completely understand this dynamic transcriptome using RNA-Seq in order to capture information regarding alternative splicing, novel exons, unannotated mRNAs, non-coding RNAs, etc. We also wished to identify transcripts that were selectively expressed in the pineal glands relative to other tissues. Toward this end we performed RNA-Seq on three types of samples; 1) a pool of pineal glands sampled at mid-day (ZT7); 2) a pool of pineal glands sampled at mid-night (ZT19); and a pool of 15 different non-pineal tissues collected from 3 animals at mid-day (ZT7). Animals were housed in a 14:10 light:dark lighting cycle. PolyA-selected RNA was fragmented and sequenced on an Illumina GAii machine, yielding paired-end 51-mer reads.

Transcriptome sequencing of two macaques, crab-eating macaque and Indian rhesus macaque

Deep sequencing of mRNA from two macaques, crab-eating macaque and Indian rhesus macaque Overall design: Analysis of ploy(A)+ RNA of different specimens:brain,ileum,kidney,liver,testes and white adipose for crab-eating macaque while brain,heart,,kidney,liver,quadriceps and testes for Indian rhesus macaque

Whole transcriptome profiling of the Eya2 knockout mouse pineal gland and retina

10.1111/jpi.12673

Mice were obtained in which the transcription co-factor, Eya2, was knocked out. The transcriptomes of the pineal gland and retina were measured to determine the effect of this gene deletion on these tissues. Samples were taken at both mid-day and mid-night. In addition, samples were taken of mixed non-pineal tissues at mid-day and mid-night.

Profiling of differential allelic expression in horse, donkey, mule and hinny placental tissue

The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes of which 10 were paternally expressed. An additional 78 candidate novel imprinted genes identified by RNA-seq also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the novel genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase (HAT1), the first example of an imprinted gene involved in chromatin modification, and LY6G6C, the first imprinted gene to be identified in the major histocompatibility complex. The 78 novel candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting. Overall design: Examine allelic expression from four individual samples of invasive trophoblast tissue of the chorionic girdle from gestation day 33 conceptuses of horse, donkey, mule and hinny.

Digital gene expression and global mapping of polyadenylation sites with PolyA-Seq

We describe PolyA-Seq, a strand-specific method for high-throughput sequencing of the 3' ends of polyadenylated transcripts. PolyA-Seq is as accurate for digital gene expression as existing RNA sequencing approaches, and superior to microarrays. We used the approach to map polyadenylation (polyA) sites in 24 samples from normal tissues in human, rhesus, dog, mouse, and rat. Overall design: Detection of polyA sites in a mixture of 24 tissues in human, mouse, rat, dog and rhesus. Samples included two replicates each of MAQC Human Brain Reference and MAQC Universal Human Reference. Two additional human sets of reads are included that were used to distinguish true polyA sites from internal priming sites.

Transcriptome sequencing of naked mole rat, Heterocephalus glaber

Deep sequencing of mRNA from naked mole rat Overall design: Analysis of ploy(A)+ RNA of different specimens: brain, kidney, liver from new born , 4 years old , 20 years old and 4 years old hypoxia-exposed naked mole rat

Transposon-mediated gene regulatory network rewiring contributed to the evolution of pregnancy in mammals

We report the gene expression profile of human endometriual stromal cells and armadillo endometrial samples Overall design: We report the gene expression profile of human endometriual stromal cells and armadillo endometrial samples generated with Illumina GA2

Comparative Analysis of RNA-Seq Alignment Algorithms and the RNA-Seq Unified Mapper (RUM).

RNA-Seq of mouse retinal RNA, as described.

PrimSeq

RNA Sequencing of 12 primates and 4 mammals.

Genes with structural flexibility in nucleosome organization and evolutionary flexibility in transcriptional initiation

Background: Transcription start sites (TSSs) with pronounced and phased nucleosome arrays downstream and nucleosome-depleted regions upstream of TSSs are observed in various species. Results: We have characterized sequence variation and expression properties of this set of TSSs (which we call *Nucleocyclic TSSs*) using germline and somatic cells of three medaka (Oryzias latipes) inbred isolates from different locations. We found nucleocyclic TSSs in medaka to be associated with higher gene expression and characterized by a clear boundary in sequence composition with potentially-nucleosome-destabilizing A/T-enrichment upstream (p<1E-60 ) and nucleosome-accommodating C/G-enrichment downstream (p<1E-40 ) that was highly conserved from an ancestor. A substantial genetic distance between the strains facilitated the in-depth analysis of patterns of fixed mutations, revealing a localization-specific equilibrium between the rates of distinct mutation categories that would serve to maintain the conserved sequence anisotropy around TSSs. Downstream of nucleocyclic TSSs, C to T, T to C, and other mutation rates on the sense strand increased around first nucleosome dyads and decreased around first linkers, which contrasted with genomewide mutational patterns around nucleosomes (p<5%). C to T rates are higher than G to A rates around nucleosome associated with germline nucleocyclic TSS sites (p<5%), potentially due to the asymmetric effect of transcription-coupled repair. Conclusions: Our results demonstrate an atypical evolutionary process surrounding nucleocyclic TSSs.

Extension of cortical synaptic development distinguishes humans from chimpanzees and macaques

We search for developmental changes specific to humans by examining gene expression profiles in the human, chimpanzee and rhesus macaque prefrontal and cerebellar cortex, using microarray. In both brain regions, developmental patterns were more evolved in humans than in chimpanzees. To test the robust of the developmental patterns measured by microarray, we measured mRNA expression patterns in chimpanzee and macaque brains at different age groups, using high-throughput RNA-seq, Our results show that human-specific developmental patterns were reproduced in the RNA-seq data.

Comprehensive identification of long non-coding RNAs expressed during zebrafish embryogenesis [RNA_seq]

Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in human and mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time series of RNA-Seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts, while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1,133 non-coding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first comprehensive identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic and evolutionary studies. Overall design: RNA-Seq for 8 zebrafish developmental stages, 2 lanes for each stage (3 for shield).

Identification of polyA sites in Drosophila melanogaster (modENCODE project)

The 3' ends of most Drosophila melanogaster genes are poorly annotated or are determined by only a single EST or cDNA clone. To enhance the annotation of poly(A) site use in Drosophila, we performed deep sequencing on RNA isolated from 29 dissected tissues using an approach designed to enrich for poly(A) spanning reads. From these experiments, we identified 1.4 million poly(A) spanning reads leading to the identification of many new poly(A) sites and the identification of many tissue-specific poly(A) sites.

Neolamprologus brichardi Transcriptome or Gene expression

Neolamprologus brichardi RNA sequencing from multiple tissues.

Latimeria chalumnae Transcriptome or Gene expression

Latimeria chalumnae (Coelacanth) RNA sequencing from multiple tissues.

Oryctolagus cuniculus Transcriptome or Gene expression

Oryctolagus cuniculus (Rabbit) RNA sequencing from multiple tissues.

RNA-seq profiling of theca and granulosa tissue of dominant follicle at 3 stages of follicular development in cows and heifers.

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status (such as lactatino) are largely unknown. To gain insight into the main metabolic mechanisms aff

Canis lupus familiaris Transcriptome or Gene expression

Canis familiaris (Dog) RNA sequencing from multiple tissues.

The transcriptome of embryos of the green anole, Anolis carolinensis, at 28 and 38 somite pair stages

Anolis carolinensis embryos were collected 0-1 days post egg laying, and total RNA was extracted for RNA-Seq analysis (Illumina Hi-Seq2000). Transcriptome sequence from these stages in the green anole, equivalent to mouse 9.5-10.5 dpc embryos, will help to improve gene annotations in A. carolinensis and provide expression level information for key organogenesis and patterning processes. Overall design: Anolis carolinensis embryos were collected 0-1 days post egg laying for RNA-Seq analysis. The two embryos collected were at 28 somite-pair (28S) and 38 somite-pair (38S), equivalent to mouse 9.5 dpc and 10.5 dpc embryos, respectively. Total RNA was extracted using the total RNA component of the mirVana (Ambion) kit, RNA-Seq library prep was carried out using the NuGEN Ovation RNA-Seq kit, and sequencing was carried out on an Illumina HiSeq 2000, following the manufacturer''s protocol. The untrimmed data was then aligned to the Anolis carolinensis reference genome (Anocar2.0) using tophat.

Transcriptome study in rhesus monkey

RNA-Seq technology was widely used to study gene expression, splicing structure and RNA editing. To facilitate transcriptome study in rhesus macaque, and provide an evolutionary context to understand human biology, we performed 90bp × 2, paired-end, strand-specific, polyA-postive RNA-Seq in 10 tissues of the same macaque animals. The data were deposited under accession number GSE42857 and GSE34426. Overall design: 5 tissue samples examined: prefrontal cortex, liver, skeletal muscle, adipose, testis

Anolis carolinensis Transcriptome or Gene expression

Anolis carolinensis (Green Anole) RNA sequencing from multiple tissues.

Maternal Influence on Exonic CpG Island Methylation in the Developing Hippocampus [RNA-Seq]

Examined transcriptomes of 5HT1A wild type offspring with 5HT1A wild type/heterozygous mother or 5HT1A KO offspring with 5HT1A of heterozygous/knock out mother

Deep sequencing the circadian and light-dependent transcriptome of Drosophila brain

RNA-Seq transcriptional profiling of Drosophila brains from wildtype and period loss-of-function (per0) flies with time points taken over two days in constant darkness. Time points at CT24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, and 68. 10-12 brains per time point.

Platypus fibroblast and ovary transcriptomes

As a result of sex chromosome differentiation from ancestral autosomes, male mammalian cells only contain one X chromosome. It has long been hypothesized that X-linked gene expression levels have become doubled in males to restore dosage balance between the X and autosomes, and that the resulting X overexpression in females then drove the evolution of X inactivation (XCI). However, this model has never been directly tested and patterns and mechanisms of dosage compensation across different mammals and birds generally remain little understood. We have traced the evolution of dosage compensation using extensive transcriptome data from males and females representing all major mammalian lineages and birds. Our analyses suggest that the X has become globally upregulated in marsupials but probably not in placental mammals, where instead at least a subset of autosomal genes interacting with X-linked were downregulated. Thus, different driving forces may underlie the evolution of XCI and the highly efficient equilibration of X expression levels between the sexes observed for both of these lineages. In the egg-laying monotremes and birds, which have homologous sex chromosome systems, partial upregulation of the X (Z in birds) evolved but is largely restricted to the heterogametic sex, which provides an explanation for the partially sex-biased X (Z) expression and lack of global inactivation mechanisms in these lineages. Our findings suggest that dosage reductions imposed by sex chromosome differentiation events in amniotes were resolved in strikingly different ways. Overall design: 3 samples (ovary, fibroblast male, fibroblast female) from different platypus individuals are analysed.

Profiling of differential allelic expression in horse, donkey, mule, and hinny placental tissues.

In eutherian mammals, dosage compensation of X-linked genes is achieved by X chromosome inactivation. X inactivation is random in embryonic and adult tissues, but imprinted X inactivation (paternal X silencing) has been identified in the extraembryonic membranes of the mouse, rat, and cow. Few other species have been studied for this trait, and the data from studies of the human placenta have been discordant or inconclusive. Here, we quantify X inactivation using RNA sequencing of placental tissue from reciprocal hybrids of horse and donkey (mule and hinny). In placental tissue from the equid hybrids and the horse parent the allelic expression pattern was consistent with random X inactivation, and imprinted X inactivation can clearly be excluded. We characterized horse and donkey XIST gene, and demonstrated that XIST allelic expression in female hybrid placental and fetal tissues is negatively correlated with the other X-linked genes chromosome-wide, which is consistent with the XIST-mediated mechanism of X inactivation discovered previously in mice. As the most structurally and morphologically diverse organ in mammals, the placenta also appears to show diverse mechanisms for dosage compensation that may result in differences in conceptus development across species. Overall design: Examine allelic expression from individual samples of invasive trophoblast tissue of the chorionic girdle from gestation day 33-34 conceptuses of 5 horses, 3 donkeys, 6 mules, and 1 hinny.

Salmo salar Genome sequencing and assembly

10.1038/nature17164

Atlantic salmon is one of 68 closely related species of salmonids which comprise eleven genera that include salmon, trout, charr, freshwater whitefishes, ciscos and graylings. Atlantic salmon are of considerable economic, social and environmental importance. Salmonids contribute to local and global economies through aquaculture, wild stock fisheries and recreational sport fisheries. In addition, they are a traditional food source for aboriginal peoples and play a central role in their culture. Salmon and trout are sentinel species for monitoring the aquatic environment, and therefore they are used extensively for eco-toxicology studies. Importantly, the common ancestor of salmon and trout experienced a whole genome duplication (60-100MYA), and modern species may be considered pseudo tetraploid as they are well along the process of reverting to a stable diploid state. There is a large salmonid research community working on the biology, life histories, population dynamics, biogeography, phylogenetic relationships, physiology and nutrition of salmonids. No other species group of fishes receives such comprehensive commercial and scientific attention. p An International Collaboration to Sequence the Atlantic Salmon Genome (ICSASG), representing researchers, funding agencies and industry from Canada, Chile and Norway, was formed to undertake a comprehensive genome sequence that would benefit all salmonids. p Note: Double haploid individual chosen for sequencing

Sex-dependent RNA expression in opossum brain and liver.

To compensate for the increased dosage of X chromosomes in females one of the two X chromosomes is inactivated in eutherian mammals in an Xist-dependent manner. Xist is, however, not found in metatherians. Here we describe long non-coding RNA Rsx (RNA-on-the-silent X) that exhibits properties consistent with a role in X-inactivation in opossum. Rsx is abundantly expressed in females but not males and is transcribed from, and coats, the inactive X chromosome copy. Furthermore, when both X chromosomes are active, Rsx is silenced. We used RNA-Seq to determine exon-intron structure and expression level of Rsx in males and females. Overall design: RNA sequencing was used to detect and characterize transcripts with strongly biased female expression.

Canis lupus familiaris Transcriptome or Gene expression

Canine cerebellum transcriptome sequencing

Next generation sequencing reveals differentially expressed genes associated with development of PSE turkey meat.

The success of turkey breeding for rapid growth rate and larger breast size has coincided with an increasing incidence of a meat quality defect described as pale, soft and exudative (PSE). We hypothesized that this defect, which is associated with an abnormally rapid rate of postmortem metabolism, derives from altered expression of genes involved in metabolic regulation. Our objective was to use deep transcriptome RNA sequence analysis (RNAseq) to identify differentially expressed genes between normal and PSE turkey breasts. Following harvest of turkey breasts (n = 43), the pH at 15 min post-slaughter and percent marinade uptake at 24h post-slaughter were determined. Breast samples were classified as normal or PSE based on marinade uptake (high = normal; low = PSE). Total RNA from samples with the highest (n=4) and lowest (n=4) marinade uptake were isolated and sequenced using the Illumina GAIIX platform. Of 21,340 gene loci discovered by RNAseq, 8480 loci completely matched the turkey reference genome, and 480 genes were differentially expressed (false discovery rate, FDR<0.05) between normal and PSE samples. Two highlights were the genes nephroblastoma overexpressed (NOV), upregulated about 38-fold and pyruvate dehydrogenase kinase isoform 4 (PDK4), downregulated 14-fold in PSE samples. Pathway analysis suggested that several biological functions, including carbohydrate metabolism and energy production, were affected by meat quality. Because PDK4 regulates conversion of pyruvate to acetyl CoA, differences in regulation of oxidative metabolism may exist among turkeys. Accelerated early postmortem metabolism would result in faster pH decline in PSE meat. This hypothesis was supported by the fact that decreased expression of PDK4 was associated with lower pH in PSE samples (pH[PSE] = 5.59±0.09, pH[normal] = 5.77±0.17). The RNAseq results provided a greater molecular mechanistic understanding of development of PSE turkey, which will be a foundation for new intervention strategies to prevent development of this defect. Overall design: The mRNA profiles of normal and PSE turkey breast muscle were generated by deep sequencing using Illumina GAIIx platform. Multiplexing was performed (2 samples/lane). Afterwards, difference in gene expression between normal and PSE samples were tested.

Transcriptomic analysis of the stress response to weaning at housing in bovine leukocytes using RNA-seq technology

Background: Weaning of beef calves is a necessary husbandry practice and involves separating the calf from its mother, resulting in numerous stressful events including dietary change, social reorganisation and the cessation of the maternal-offspring bond

RNA-seq analysis of differential gene expression in liver from lactating dairy cows divergent in negative energy balance

Background: The liver is central to most economically important metabolic processes in cattle. However, the changes in expression of genes that drive these processes remain incompletely characterised. RNA-seq is the new gold standard for whole transcriptome analysis but so far there are no reports of its application to analysis of differential gene expression in cattle liver. We used RNA-seq to study differences in expression profiles of hepatic genes and their associated pathways in individual cattle in either mild negative energy balance (MNEB) or severe negative energy balance (SNEB). NEB is an imbalance between energy intake and energy requirements for lactation and body maintenance. This aberrant metabolic state affects high-yielding dairy cows after calving and is of considerable economic importance because of its negative impact on fertility and health in dairy herds. Analysis of changes in hepatic gene expression in SNEB animals will increase our understanding of NEB and contribute to the development of strategies to circumvent it. Results: RNA-seq analysis was carried out on total RNA from liver from early post partum Holstein Friesian cows in MNEB (n=5) and SNEB (n=6). 12,833 genes were deemed to be expressed (>4 reads per gene per animal), 413 of which were shown to be statistically significantly differentially expressed (SDE) at a false discovery rate (FDR) of 0.1% and 200 of which were SDE (FDR of 0.1%) with a =2-fold change between MNEB and SNEB animals. GOseq/KEGG pathway analysis showed that SDE genes with =2- fold change were associated (P <0.05) with 9 KEGG pathways. Seven of these pathways were related to fatty acid metabolism and unexpectedly included ‘Steroid hormone biosynthesis’, a process which mainly occurs in the reproductive organs rather than the liver. Conclusions: RNA-seq analysis showed that the major changes at the level of transcription in the liver of SNEB cows were related to fat metabolism. ''Steroid hormone biosynthesis'', a process that normally occurs in reproductive tissue, was significantly associated with changes in gene expression in the liver of SNEB cows. Changes in gene expression were found in this pathway that have not been previously been identified in SNEB cows. Overall design: 11 liver RNA samples were analysed in total, 6 samples were from SNEB animals and 5 samples from MNEB animals

Genotype-Tissue Expression (GTEx) Common Fund Project

The aim of the Genotype-Tissue Expression (GTEx) Project is to increase our understanding of how changes in our genes affect human health and disease with the ultimate goal of improving health care for future generations. GTEx will create a database that researchers can use to study how inherited changes in genes lead to common diseases. GTEx researchers are studying genes in different tissues obtained from many different people. The GTEx project also includes a study of the GTEx donor consent process - this study will help ensure that the consent process and other aspects of the project effectively address the concerns and expectations of participants in the study. GTEx is a pioneering project that uses state-of-the-art protocols for obtaining and storing a large range of organs and tissues, and for testing them in the lab. Until now, no project has analyzed genetic variation and expression in as many tissues from the same person in... (for more see dbGaP study page.)

Gasterosteus aculeatus RNA Sequencing

Gasterosteus aculeatus (Threespine stickleback) RNA sequencing from multiple tissues.

Whole transcriptome analyses of six thoroughbred horses before and after exercising using RNA-Seq

We sequenced the whole mRNA of six thoroughbred horse (Equus caballus) blood and muscle tissues before and after exercising, generating a total of 1.3 billion short reads with 90-bp pair-end sequences from 24 samples. Comparing with current genome annotation, we identified 32,361 unigene clusters spanning 51.83 Mb that contained 11,933 (36.87%) annotated genes. More than 60% (20,428) unigene clusters did not match any current equine gene model. We identified 189,973 single nucleotide variations (SNVs) from the aligned sequences against the horse reference. Most SNVs (171,558 SNVs; 90.31%) were novel compared with over 1.1 million equine SNPs from two databases. Some genes have significantly different expression levels under different environment. We define those identical genes which have different expression levels are *differentially expressed* and carried out differentially expressed gene analysis before and after exercise conditions. We discovered, 62 up- and 80 down-regulated genes in the blood and 878 up- and 285 down-regulated genes in the muscle from the 24 samples. Six out of 28 previously exercise-related known genes, HIF1A, ADRB2, PPARD, VEGF, TNC, and BDNF, were highly expressed in the muscle after exercise. We discovered 56 functionally unknown transcription factors that are probably associated with an early regulatory exercise mechanism from 91 differentially expressed transcription factors. We found interesting RNA expression patterns where different alternative splicing forms of the same gene showed reversed expressions before and after exercising. Overall design: whole mRNA sequencing profiles of six thoroughbred horse (Equus caballus) blood and muscle tissues before and after exercising

GSE37909: RNA-seq from ENCODE/Caltech (Mouse)

Overall Design: Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell/mouse). Cells were lysed in RLT buffer (Qiagen RNEasy kit), and processed on RNEasy midi columns according to the manufacturer's protocol, with the inclusion of the *on-column* DNAse digestion step to remove residual genomic DNA. A quantity of 75 µgs of total RNA was selected twice with oligo-dT beads (Dynal) according to the manufacturer's protocol to isolate mRNA from each of the preparations. A quantity of 100 ngs of mRNA was then processed according to the protocol in Mortazavi et al. (2008), and prepared for sequencing on the Illumina GAIIx or HiSeq platforms according to the protocol for the ChIP-Seq DNA genomic DNA kit (Illumina). Paired-end libraries were size-selected around 200 bp (fragment length). Libraries were sequenced with the Illumina HiSeq according to the manufacturer's recommendations. Paired-end reads of 100 bp length were obtained.Reads were mapped to the reference mouse genome (version mm9 with or without the Y chromosome, depending on the sex of the cell line, and without the random chromosomes in all cases) using TopHat (version 1.3.1) (http://tophat.cbcb.umd.edu/). TopHat was used with default settings with the exception of specifying an empirically determined mean inner-mate distance and supplying known ENSEMBL version 63 splice junctions.

Whole transcriptome profiling of the rat pineal gland using mid-day and mid-night samples (Experiment 2)

10.1073/pnas.1207748109

The transcriptome of the rat pineal gland is highly dynamic, with many hundreds of genes changing more than two-fold on a 24-hr daily rhythm, as revealed earlier using Affymetrix GeneChip analysis. We sought to more completely characterize this temporally dynamic transcriptome using RNA-Seq to capture information regarding alternative splicing, novel exons, unannotated mRNAs, non-coding RNAs and coding transcripts not represented on the Affymetrix chips. Toward this end we performed RNA-Seq on pools of rat pineal glands obtained at mid-day (ZT7) and mid-night (ZT19). Animals had been housed in a 14:10 light:dark lighting cycle. PolyA-selected RNA was fragmented and sequenced on an Illumina HiSeq 2000 machine, yielding paired-end, 101-mer reads. This is Experiment 2 of similar RNA-Seq experiments investigating the rat pineal transcriptome at ZT7 compared to ZT19. Experiments 1 through 4 are included in Coon et al., 2012 and have been archived as separate SRA entries: Experiment 1, SRA037284; Experiment 3, SRA052826; Experiment 4, SRA053154.

Whole transcriptome profiling of the rat pineal gland using mid-day and mid-night samples (Experiment 3)

10.1073/pnas.1207748109

The rat pineal transcriptome is highly dynamic, with many hundreds of transcripts changing more than two-fold on a 24-hr basis, as revealed earlier using Affymetrix GeneChip analysis. We sought to more completely characterize this temporally dynamic transcriptome using RNA-Seq to capture information regarding alternative splicing, novel exons, unannotated mRNAs, non-coding RNAs, and coding transcripts not represented on the Affymetrix chips. Toward this end we performed RNA-Seq on pools of pineal glands at mid-day (ZT7) and at mid-night (ZT19) from animals that were housed in a 14:10 light:dark lighting cycle. We also wished to identify transcripts that were selectively expressed in the pineal glands relative to other tissues; to do this, RNA was sequenced from a pool of 15 different tissues collected from 3 animals at mid-day (ZT7). PolyA-selected RNA was fragmented and sequenced on an Illumina HiSeq 2000 machine, yielding paired-end, 101-mer reads. This is Experiment 3 of several similar RNA-Seq experiments investigating the rat pineal transcriptome at ZT7 compared to ZT19. Experiments 1 through 4 are included in Coon et al., 2012 and have been archived as separate SRA entries: Experiment 1, SRA037284; Experiment 2, SRA052823; Experiment 4, SRA053154.

Whole transcriptome profiling of the rat pineal gland using mid-day and mid-night samples (Experiment 4)

10.1073/pnas.1207748109

The rat pineal transcriptome is highly dynamic, with many hundreds of transcripts changing more than two-fold on a 24-hr basis, as revealed earlier using Affymetrix GeneChip analysis. We sought to more completely characterize this temporally dynamic transcriptome using RNA-Seq to capture information regarding alternative splicing, novel exons, unannotated mRNAs, non-coding RNAs, and coding transcripts not represented on the Affymetrix chips. Toward this end we performed RNA-Seq on pools of pineal glands at mid-day (ZT7) and at mid-night (ZT19) from animals that were housed in a 14:10 light:dark lighting cycle. We also wished to identify transcripts that were selectively expressed in the pineal glands relative to other tissues; to do this, RNA was sequenced from a pool of 15 different tissues collected from 3 animals at mid-day (ZT7). PolyA-selected RNA was fragmented and sequenced on an Illumina HiSeq 2000 machine, yielding paired-end, 101-mer reads. This is Experiment 4 of several similar RNA-Seq experiments investigating the rat pineal transcriptome at ZT7 compared to ZT19. Experiments 1 through 4 are included in Coon et al., 2012 and have been archived as separate SRA entries: Experiment 1, SRP006893; Experiment 2, SRP013114; Experiment 3, SRP013116. SRP013378(SRA053154)is actually 'Experiment 4', not 'Experiment 3'

Monodelphis domestica RNA Sequencing

Monodelphis domestica (Gray short-tailed opossum) RNA sequencing from multiple tissues.

Transcriptomes of germinal zones of human and mouse fetal neocortex suggest a role of extracellular matrix in progenitor self-renewal.

Total RNA was isolated from the VZ, inner SVZ (ISVZ), outer SVZ (OSVZ) and CP of six 13-16 weeks post-conception (w.p.c.) human fetuses and from the VZ, SVZ and CP of five E14.5 mouse embryos using laser capture microdissection of Nissl-stained cryosections of dorsolateral telencephalon. Poly A+ RNA was used as template for the preparation of cDNA which were then subjected to single-end 76-bp RNA-Seq.

Bos taurus Transcriptome or Gene expression

Sequencing of a pool of 9 bulls of varying conception rate (CR) scores from -2.9 to 3.5.

Comparative transcriptome analysis of various adipose depots in Hanwoo

*Background: Adipocytes mainly function as energy storage and endocrine cells. The amount and distribution of fat are important factor that influence the meat quality in the beef industry. Fat depot can be found around internal organ (ometal), beneath the skin (subcutaneous), and between muscles (intramuscular). Different adipose depot showed the biological and genetic difference depending on their location. This inter-depot variation might be influenced by the inherent genetic programing for development of adipose depots. In this study, we used RNA-seq data to investigate the difference in transcriptome of various adipose depots in Hanwoo. *Results: Using RNA-seq, we identified 5797, 2156, and 5455 DEGs in the comparison between OI, OS, and IS respectively (FDR<0.01) and found 853, 48, and 979 DEGs specific to subcutaneous, intramuscular and omental fat respectively. DEGs in intramuscular fat were highly enriched the metabolism related pathways compared to other fat depots. DEGs specific to the omental fat is significantly enriched in PPAR signaling pathway and cell-junction related pathway. In subcutaneous fat, cytokine-cytokine receptor interaction with chemokines (CXC and CC subfamily) was the most significantly enriched the pathways. Interestingly, melanogenesis pathway was associated with the subcutaneous depot. Even though the adipose tissues shared the same pathways for adipocyte differentiation, the regulation of genes were different based on the depot. *Conclusions: We comparatively analyzed the transcripome profile from different adipose tissues using NGS and identified DEGs between adipose depot and specific to depot in Hanwoo animals. The functional annotation analysis of DEGs found that transcriptome profile difference in various adipose tissue of intramuscular, subcutaneous, and ometal fat. Overall design: whole mRNA sequencing profiles of nine Korean native cattle (nine profiles of omental fat tissue, nine profiles of intramuscular fat tissue, nine profiles of subcutaneous fat tissue and eight profiles of muscle tissue)

Sus scrofa strain:Duroc and wild boar Transcriptome or Gene expression

Pig RNA-seq.

Evolution of mammalian miRNA genes

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression, yet their origins and functional evolution in mammals remain little understood due to the lack of appropriate comparative data. Using RNA sequencing, we have generated extensive and comparable miRNA data for five organs in six species that represent all main mammalian lineages and birds (the evolutionary outgroup), with the aim to unravel the evolution of mammalian miRNAs. Our analyses reveal an overall expansion of miRNA repertoires in mammals, with three-fold accelerated birth rates of miRNA families in placentals and marsupials, facilitated by the de novo emergence of miRNAs in host gene introns. Generally, our analyses suggest a high rate of miRNA family turnover in mammals, with many newly emerged miRNA families being lost soon after their formation. Selectively preserved mammalian miRNA families gradually evolved higher expression levels as well as altered mature sequences and target gene repertoires, and were apparently mainly recruited to exert regulatory functions in nervous tissues. However, miRNAs that originated on the X chromosome evolved high expression levels and potentially diverse functions during spermatogenesis, including meiosis, through selectively driven duplication-divergence processes. Overall, our study thus provides detailed insights into the birth and evolution of mammalian miRNA genes and the associated selective forces. Overall design: 30 main samples from five adult tissues (brain, cerebellum, heart, kidney and testis) collected in six species (human, macaque, mouse, opossum, platypus and chicken) + 5 biological replicates + 3 samples from spermatogenic cells in adult mouse testis (Sertoli cells, spermatocytes and spermatids)

Canis lupus familiaris Transcriptome or Gene expression

Identify upregulated therapeutic pathways in canine lymphoma.

Transcriptome study in rhesus monkey

RNA-Seq technology was widely used to study gene expression, splicing structure and RNA editing. To facilitate transcriptome study in rhesus macaque, and provide an evolutionary context to understand human biology, we performed 90bp × 2, paired-end, strand-specific, polyA-postive RNA-Seq in 10 tissues of the same macaque animals. The data were deposited under accession number GSE42857 and GSE34426. Overall design: This submission represents RNA-Seq: cerebellum, lung, kidney, heart and spleen component of study.

Deep Sequencing of the Porcine Endometrial Transcriptome on Day 14 of Pregnancy

In pigs, conceptus attachment to the uterine surface epithelium starts around day 14 of pregnancy preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of initial conceptus attachment deep sequencing of endometrial RNA samples of pregnant animals (n=4) and corresponding cyclic controls (n=4) was performed using Illumina RNA-Seq. The obtained sequence reads were mapped to the porcine genome and relative expression values were calculated for the analysis of differential gene expression. Statistical analysis revealed 1,933 differentially expressed genes (FDR 1%), 1,229 with higher and 704 with lower mRNA concentration in the samples from pregnant animals. Expression of selected genes was validated by the use of quantitative real-time RT-PCR. The RNA-Seq data were compared to results of a microarray study of bovine endometrium on day 18 of pregnancy and additional related data sets. Bioinformatics analysis revealed for the genes with higher mRNA concentration in pregnant samples strong overrepresentation particularly for immune-related functional terms but also for apoptosis and cell adhesion. Overrepresented terms for the genes with lower mRNA concentration in pregnant samples were related to extracellular region, ion transport, cell adhesion and lipid and steroid metabolic process. In conclusion, RNA-Seq analysis revealed comprehensive transcriptome differences in porcine endometrium between day 14 of pregnancy and corresponding cyclic endometrium and highlighted new processes and pathways probably involved in regulation of non-invasive implantation in the pig. Overall design: In total, 8 samples were analyzed, 4 biological replicates for pregnant animals (samples from 4 different animals) and 4 biological replicates for cyclic controls (samples from 4 different animals)