Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS transcriptome profiling (RNA-seq) from whole eye, after removal of the lens and cornea from 1-2 month old miR-211-/- mice and compare it with wt mice Methods: Whole eye (after removal of the lens and cornea) mRNA profiles of 1-2 month old wild-type (WT) and neural miR-211-/-mice were generated by deep sequencing, in multiple biological replicates, five for WT and six for miR-211-/- animals, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays RNA-Seq libraries were prepared from whole eye, after removal of the lens and cornea from miR-211-/- mice. Results: Each library was sequenced using 100 bp paired-end sequencing on the Illumina HiSeq 1000 system. Gene abundances from RNA-Seq data were quantified using RSEM45. Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome. This approach yielded read count values for a total of 38253 mouse genes annotated in GenCode. We only considered genes that had at least 1 count per million in at least five out of 11 samples as expressed, yielding a total of 15590 genes. Next we performed differential gene expression analysis to determine the transcriptional effects of miR-211 deletion. This analysis yielded a total of 63 genes that were differentially expressed with a False Discovery Rate (FDR) <0.1 (Fig. 4). Of these, the expression levels of 61 genes were significantly decreased upon miR-211 deletion, while only 2 genes were upregulated. Conclusions: Our study represents the first detailed analysis of whole eye transcriptomes, with biologic replicates, generated by RNA-seq technology on miR-211-/-. Overall design: Whole eye (after removal of the lens and cornea) mRNA profiles of 1-2 month old wild-type (WT) and neural miR-211-/-mice were generated by deep sequencing, in multiple biological replicates, five for WT and six for miR-211-/- animals, using Illumina GAIIx.
MiR-211 is essential for adult cone photoreceptor maintenance and visual function.
Specimen part, Subject
View SamplesHerein we compare mouse (C57B6/J background, 16 week old) adherent bone marrow stromal cell gene expression after 4 weeks of adipogenic differentiation in 3D versus 2D culture.
Development of a 3D bone marrow adipose tissue model.
Sex, Disease
View SamplesWe performed microarray analysis to evaluate differences in the transcriptome of type 2 diabetic human islets compared to non-diabetic islet samples.
Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells.
Sex, Age, Specimen part, Disease, Disease stage
View SamplesDynamical response to oxygen downshift under fermentation conditions was tested by taking sample before (S1) and after (S2, S3 and S4) the oxygen downshift. The dynamical changes relevant for ongoing research on physiology were applied.
Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110.
No sample metadata fields
View SamplesGenome-wide gene expression was measured in peripheral blood mononuclear cells (PBMCs) from patients with cystic fibrosis (CF) after treatment in vitro with the flagellin protein fliC, and/or synthetic peptide IDR-1018 to assess patterns of gene expression. The patterns of gene expression suggest that CF cells have a hyperinflammatory phenotype including dysfunctional autophagy processes. The synthetic peptide IDR-1018 attentuates this hyperinflammatory phenotype. Overall design: Total RNA was obtained from PBMCs obtained from CF patients after treatment with the fliC flagellin protein (that is known to play a role in CF lung inflammation), and/or the peptide IDR-1018 that has anti-inflammatory properties. Comparison of genes and pathways affected by these treatments indicated the role of autophagy process in CF disease.
Rescue of dysfunctional autophagy attenuates hyperinflammatory responses from cystic fibrosis cells.
Specimen part, Treatment, Subject
View SamplesSynthetic, innate defense regulators (IDR) peptides, designed based on natural host defenses peptides, have enhanced immunomodulatory activities and reduced toxicity leading to protection in infection and inflammation models that is dependent on macrophages/monocytes. Here we measured the effect of IDR-1018 on macrophage gene expression during differentiation. Differentiation in the presence of IDR-1018 induced a unique signature of immune responses suggesting that IDR-1018 drives macrophage differentiation towards an intermediate M1-M2 state, enhancing anti-inflammatory functions while maintaining certain pro-inflammatory activities important to the resolution of infection. Overall design: RNA-seq was performed using the Illumina Genome Analyzer IIx platform. Monocytes were isolated from 3 healthy donors, and left unstimulated or stimulated for 4 hours with 20 µg/ml IDR-1018. For library preparation, 500 ng of total RNA was processed according to the Illumina TruSeq RNA sample preparation guide (Illumina catalogue number FC-122-1002). Briefly, mRNA was purified using poly-dT beads, followed by synthesis of the first and second cDNA strands, end repair addition of an poly-A overhang, and ligation of adapters and unique barcodes, as per the manufacturer’s instructions. DNA enrichment was carried out via a 15-cycle PCR. Following quantification, 8 pM of dsDNA was used for cluster generation on a CBOT instrument (Illumina, San Diego, CA). RNA sequencing was done on a GAIIx instrument (Illumina), performed as a single read run with 51 amplification cycles. Data processing was carried out in house, using CASAVA to convert raw data and demultiplex to FASTQ sequence files. Reads were aligned to the reference genome using TOPHAT, and then mapped to genes using the Bioconductor package GenomeRanges.
Synthetic cationic peptide IDR-1018 modulates human macrophage differentiation.
Specimen part, Disease, Treatment, Subject
View SamplesBackground: Dendritic cells (DCs) are critical for regulating CD4 and CD8 T cell immunity, controlling Th1, Th2, and Th17 bias, generating inducible Tregs, and inducing tolerance. Multiple DC subsets have been identified in the mouse that are thought to have evolved to control these different immune outcomes. However, how these subsets differentially respond to inflammatory and/or tolerogenic signals in order to accomplish their divergent functionality remains unclear. Results: We analysed the responses of murine, splenic CD8 and CD11b DC subsets to in-vivo stimulation with lipopolysaccharide using RNA-Seq and systems biology approaches and observed responses are highly subset-specific. We reanalysed multiple datasets from the literature and show that these subset responses are obscured when analysing signaling at the population level. We show that the subset-specificity is due to the unique regulation of distinct TLR4 pathway modulators that ‘fine-tune’ a common TLR4 cascade rather and not due to major differences in signaling pathways or transcription factors. Conclusions: We propose the Pathway Modulation Model wherein common signaling pathways are regulated by unique sets of modulators allowing for distinct immune responses in closely related DC subsets. We extend these observations using analagous datasets from the literature and show that our model provides a global mechanism for generating cell subset-specific signaling in multiple subpopulations in mouse and man. Overall design: Splenic CD8 and CD11b DC subsets from LPS stimulated (10 pooled animals) and Control (5 pooled animals) mice were analysed by RNA-Seq.
A systems biology approach to the analysis of subset-specific responses to lipopolysaccharide in dendritic cells.
Specimen part, Cell line, Subject, Time
View SamplesInsulin like growth factor 1 (IGF-1) has a central role in mammalian hearing and hearing loss. The auditory and vestibular systems form the inner ear and have a common developmental origin. During chicken early development IGF-1 modulates neurogenesis of the cochleovestibular ganglion but no further studies have been conducted to explore the potential role of IGF-1 in the vestibular system.
Comparative gene expression study of the vestibular organ of the Igf1 deficient mouse using whole-transcript arrays.
Specimen part
View SamplesCultured organotypic cerebellar slices were exposed for different time points with either prions (RML) versus non-infectious brain homogenate (NBH) or ligands to the globular domain of the prion protein (POM1) vs IgG
Prion infections and anti-PrP antibodies trigger converging neurotoxic pathways.
Specimen part, Treatment
View SamplesP1 encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize silks and red phlobaphene pigments in pericarps and other floral tissues, which contributed to making P1 an important visual marker since the dawn of modern genetics. We conducted RNA-Seq using pericarps at two different stages, 14 and 25 days after pollination (DAP). High-throughput sequencing using the Illumina platform resulted in the generation of ~20 million high quality reads, from which ~90% aligned to the recently completed maize genome sequence corresponding to ~5 million reads for each one of the four samples. Overall design: Examination of two different RNA samples from two different stages of maize pericarp tissues.
A genome-wide regulatory framework identifies maize pericarp color1 controlled genes.
Specimen part, Subject
View Samples