Project description:MYCN overexpression in the doxycline MYCN inducible cell line SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN) using the dynamic transcriptome analysis (DTA) protocol. Samples at different time-points after MYCN over-expression and uninduced controls (0h) were sequenced in duplicates. The experimental setup consists of [A] total mRNA at 0h, 1h, 4h, 24h, which corresponds to the standard mRNA-seq protocol, [B] 4-thioUridine (4sU) labelled mRNA 30 min before extraction at time-points 0h, 1h, 4h, which is the freshly transcribed mRNA within the last 30 min before the time-point and [C] the counter-part, 4sU unlabelled mRNA 30 min before extraction which corresponds to the mRNA from before 30 min of extraction. The samples were sequenced on an Illumina GA IIx using the Illumina protocols.
Project description:Circular RNAs (circRNAs) in animals are an enigmatic class of RNAs with unknown function. To systematically explore circRNAs, we sequenced and computationally analyzed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, with oftentimes tissue/developmental stage specific expression. Sequence analysis suggested important regulatory functions for circRNAs. Indeed, we discovered that human circRNA CDR1as is densely bound by miRNA effector complexes and harbors 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebra fish impaired midbrain development similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, indicating previously unrecognized regulatory potential of coding sequences. 1 Sample
Project description:Background & Aims: The influences of the maternal diet during gestation has been suggested to be involved in the development of different aspects of the metabolic syndrome. In our mouse model we characterised the role of maternal western diet in the development of non-alcoholic fatty liver disease (NAFLD) in the offspring. Methods: Female mice were fed either a western (W) or low-fat control (L) semi-synthetic diet before and during gestation and lactation. At weaning, male offspring were assigned either the W or the L diet, generating four experimental groups: WW, WL, LW and LL offspring. Biochemical, histological and epigenetic indicators were investigated at 29 weeks of age. Results: Male offspring exposed to prenatal western style diet and to a post-weaning W diet (WW) showed hepatomegaly combined with increased hepatic cholesterol and triglycerides accumulation, compared to LW offspring. This was associated with up-regulation of de novo lipid synthesis and dysregulation of beta oxidation and lipid storage. Elevated hepatic transaminases and increased expression of Tnfa, Cd11, Mcp1 and Tgfb underpin the severity of liver injury. Histological analysis supported the presence of steatohepatitis in the WW offspring. In addition alterations in DNA methylation in key metabolic genes (Ppara, Insig, Fasn) were detected. Conclusion: Maternal dietary fat intake during critical developmental phases programs susceptibility to liver disease in mouse offspring. This was mediated by shifts in lipid metabolism and inflammatory response. Long lasting epigenetic changes may underlie this dysregulation 4 groups of 6 male mouse were analysed , 1 experimental and 1 biological outlier was excluded , so n=6,5,5,6 in the 4 groups (LL,LW,WL,WW)
Project description:Self-inhibition of pollen tubes plays a key role in SI, but the underlying mechanism in Camellia oleifera is poorly understood. Collection of secreted proteins from Camellia oleifera pollen tubes and ovaries for high-throughput sequencing.
Project description:Purpose: The current study tests the hypothesis that peroxisome proliferator-activated receptor β (PPARβ) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. Methods: Wild-type (WT) and PPARβ-null mice (KO) male mice (3-5 month, C57BL/6) were kept in steel microisolator cages at 22°C with a 14-hr/10-hr light/dark cycle. Food and water were provided ad libitum throughout the entire feeding period. Standard PH was performed. All the animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under protocols approved by the University of California Davis Animal Care and Use Committee.Mouse liver RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and integrity were determined by the Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis Chip. RNA-sequencing library preparation and sequencing was carried out by the Genome Sequencing Facility at University of Kansas Medical Center (Kansas City, KS). cDNA libraries were prepared with 2 µg of total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). The libraries were clustered and sequenced on an Illumina HiSeq 2000 instrument with 100 bp paired end reads. Results: Differential gene expression profiling revealed the inhibition of expression in genes and pathways that are involved in metabolism and proliferation in regenerating KO livers. Specifically, PPARβ deficiency affected the activation of Akt and the expression of E2fs. Pathways that control glycolysis, FA synthesis as well as cell proliferation were de-regulated in regenerating PPARβ KO livers. Conclusions: The data suggest a role for PPARβ in regulating liver regeneration that is mediated at least in part through Akt and E2f-regulated pathways. mRNA profiles of 3-month old wild type (WT) and PPARbeta mice were generated by deep sequencing, one simple, using Illumina HiSeq 2000 instrument with 100 bp paired end reads
Project description:We analyzed transcriptome-wide gene expression and AS changes in etiolated Arabidopsis seedlings exposed to red, blue, and white light. Our study revealed that different types of light signals trigger rapid AS responses of numerous genes, including splicing factors and other functional groups. Among these candidates was RRC1, which was previously shown to function in PHYB signaling. The light signaling phenotype of an rrc1 mutant could only be complemented with the splicing variant being up-regulated upon light exposure, indicating a self-reinforcing circuit. Finally, we provide evidence that light-regulated AS can occur in a phytochrome-independent manner and is closely intertwined with the plantâ??s energy status. Analysis of alternative splicing patterns of dark-grown Arabidopsis seedlings exposed for 1 or 6 hours to white, blue, or red light, or kept in darkness; all samples in duplicates
Project description:The effect of the GSK3 inhibitor Azakenpaullone (Azak) and the differentiation agent retinoic acid (RA) was studied in low and high MYCN levels in the MYCN inducible neuroblastoma cell line SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN). Azak was dissolved in DMSO in order to apply it to the cells. Therefore a vehicle control consisting of SY5Y-MYCN cells treated with 24h 1 ul/ml DMSO only was used in duplicates. Doxycycline (Sigma) dissolved in water was used at a final concentration of 1ug/ml to induce MYCN expression in SY5Y-MYCN. A co-treatment study with Dox and Azak was conducted. SY5Y-MYCN cells were treated with 24h Azak, 24h Azak & 48h Dox and 48h Dox, with biological duplicates. 1 uM RA (dissolved in DMSO) and 1 ug/mL Doxycycline were given individually and in combination. SY5Y-MYCN cells were treated with 24h RA, 24h RA & 48h Dox, and 48h Dox and RNA was extracted in biological duplicates. For the 24h RA & 48h Dox co-treatment cells were treated with Dox for 24h and then with RA and fresh Dox for a further 24h.
Project description:This data set is part of a study where the genome of Malassezia sympodialis (strain ATCC 42132) was sequenced using long-read technology and annotated using RNA-seq and proteogenomics. RNA was extracted at two different culture times (2 and 4 days). Seven RNA-seq libraries were prepared from independent samples. Two samples (P2 and P3) were enriched for protein-coding RNA using poly(A)-selection. The remaining five samples were processed with RiboMinus to deplete ribosomal RNA, and thus retain both mRNA and non-ribosomal noncoding RNA for sequencing. In total, we obtained 71 million RNA-seq read pairs mapping to genomic regions other than the highly expressed ribosomal loci.