AICAR can modulate the expression of microRNA in J1 mESC
ABSTRACT: We have demonstrated AICAR can maintain J1 mouse ES cells pluripotency in our previous research, yet its effects on ES cells miRNAs expression remain a mystery. In this study, we performed small RNA (sRNA) High-throughput sequencing using Illumina HiSeq 2000 to investigate the influence of AICAR on J1 mouse ES cells miRNAs expression and further found the mechanism of how miRNAs affect ES cells pluripotency maintenance. Samples that treated by DMSO is used as a control. Compare microRNA expression of J1 mESC cultured with or without small molecule AICAR
Project description:PD0325901 is involved in improving reprogramming efficiency during inducing pluripotent stem cells (iPSC) or maintain a blastocyst-like state in embryonic stem cells (ESC). However, the knowledge about this small molecule regulating miRNAs in ESC was limited. To understand the role of miRNAs during PD03-induced ESC maintenance and gain an insight how PD0325901 regulates miRNAs expression; we performed small RNA sequencing using Illumina HiSeq 2000 under the compounds treatment. The data show the miRNAs regulated by PD0325901. J1 mESCs maintained in medium containing 1000 U/mL LIF and supplemented with PD0325901 for 24 hours, J1 treated with DMSO was set as control. Then total RNA was extracted for analysis.
Project description:It has been demonstrated that CHIR99021 promotes self-renewal of mouse embryonic stem cells, however, the target genes of CHIR99021 is not fully understood. AICAR is the activator of AMP-activated protein kinase. It is reported that AICAR plays important role in mouse embryonic stem cells, however the moleculor mechanism of this phenomenon is unknown. To better understand the downstream target genes of CHIR99021 and AICAR, we performed Microarray analyses to identify their downstream targets. The data show the genes regulated by CHIR99021 or AICAR. J1 mESCs maintained in medium containing 1000 U/mL LIF and supplemented without or with CHIR99021 or AICAR for 24 hours, then total RNA was extracted for analysis.
Project description:We report the miRNA profiling in MEF cells, ES cells and three Pluripotent Stem Cells obtained by three different reprogramming approaches from MEF cells based on Solexa sequencing. iPS cells are reprogrammed by four factors (OSKM) from MEF cells. NT-ESCs were established by reprogramming MEF cells into ESCs using nuclear transfer. NT-iPSCs were established to reflect the combination of nuclear transfer and iPS technologies. iPSCs, NT-ESCs, and NT-iPSCs were exactly derived from the same MEF cells. The results indicate NT-ESCs give expression to the unique miRNAs other than both ESCs and iPSCs while pluripotent cells acquire or retain the pluripotent specific miRNAs compared with MEF. Furthermore, the comparison of different reprogramming cells suggests that several miRNAs have key roles in distinctly developmental potential reprogrammine cells. Small RNA profiles of MEF, ES, iPS, NT-ES and NT-iPS cells were generated by Solexa sequencing. MEF and ES cells were performed in triplicate. iPS, NT-ES and NT-iPS cells were sequenced in duplicate.
Project description:It has been demonstrated that vitamin C enhances reprogramming efficiency during inducing pluripotent stem cells, however, the underlying mechanisms are not fully understood. To find the downstream target genes of vitamin C and investigate the mechanism of vitamin C on reprogramming promotion, we performed Microarray analyses to identify its downstream targets. Retinoic acid (RA), a stimulus molecule for cellular differentiation, is set as negative control. The data show the genes regulated by vitamin C or RA. J1 mESCs maintained in medium containing 1000 U/mL LIF and supplemented without or with vitamin C or RA for 24 hours, then total RNA was extracted for analysis.
Project description:In this study we developed MPE-seq, a method for the genome-wide characterization of chromatin that involves the digestion of nuclei with methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] followed by massively parallel sequencing. Like micrococcal nuclease (MNase), MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes. We also performed MNase-seq as a comparison. We further performed ChIP-seq using chromatin samples obtained by MPE-Fe(II) or MNase digestion of nuclei. Nuclei from J1 mouse embryonic stem cells were treated with MPE-Fe(II) or MNase. The isolated DNA was sequenced by Illumina HiSeq sequencers. Some of the digested chromatin was studied by performing ChIP-seq using antibodies against histone H2B or H3.
Project description:sRNA profiling from adult Taenia multiceps by Illumina high-throughput sequencing was used to increase our understanding of the molecular regulation mechanisms of the cestode,find novel biomarkers for the parasitic disease and search new strategies to control these parasites. examination of 6 individuals of adult stage of Taenia multiceps (pooled)
Project description:To understand how microRNA are involved in the complex biology of this zoonotic parasite, we describe our initial attempts to characterize small RNA in adult Dirofilaria immitis by using Illumina/Solexa deep-sequencing technology. Examination of 4 different adult Dirofilaria immitis (pooled)
Project description:To know exactly how SB431542 contribute to mouse embryonic stem cells (mESCs) undifferentiated state maintenance, microarray experiment for DMSO mock treated and SB431542 treated J1 mESCs was performed J1 mESCs maintained in medium containing 1000 U/mL LIF and supplemented without or with SB431542 for 24 hours, then total RNA was extracted for analysis.
Project description:This study was undertaken in order to characterize the functions of Rex-1 and identify potential Rex-1 target genes.Both alleles of the Rex-1 gene were disrupted in J1 mouse embryonic stem cells. Gene expression levels in one of the resulting Rex-1 knockout cell lines was compared to that of J1 wild type cells. Experiment Overall Design: Gene expression in Rex-1 knockout mouse embryonic stem cells compared to that of J1 wild type cells. Cells were cultured in the presence and absence of LIF. This series utilized eight samples.There are two biological replicates per cell type cultured under this condition. The J1 samples serve as the control.
Project description:Methylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the DNA methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET family of enzymes and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide distribution map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation. Our method exploits the unique reactivity of 5fC to link a biotin tag for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, a histone mark associated with active transcription, and were frequently bound by RNA Polymerase II. Downregulation of TDG led to accumulation of 5fC in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation and in mouse embryonic fibroblasts derived from TDG knockout embryos. Collectively, our data suggest that 5fC plays a role in epigenetic reprogramming. The formation and removal of this cytosine modification are confined to specific genomic regions, which are in part controlled by TDG. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation, and hence, for appropriate patterns of gene expression during development. We devised a method to map 5-formylcytosine (5fC) by linking a biotin tag to 5fC for pulldown and high-throughput sequencing. We mapped 5fC in the following samples of mouse embryonic stem cells (J1): Wild-type ES cells (two replicates); ES cells transfected with siRNA targeting TDG (two replicates); ES cells transfected with non-targeting siRNA (two replicates). One genomic input library was also sequenced to detect and correct biases in fragment enrichment.