Project description:RNA-seq analysis on CTR, miR-294, and siMbd2 transfected Dgcr8-/- ESCs showed that Mbd2 is responsible for the change of expression of a significant portion of genes that are regulated by miR-290 cluster mRNA profiles of CTR, miR-294 and siMbd2 transfected Dgcr8-/- ESCs were generated by deep sequencing, in duplicate, using pair-end Illumina.
Project description:RNA profiling of WT,Dgcr8-/- mESCs and miR-294 or siMbnl1/2 transfected Dgcr8-/- ESCs showed that miRNAs, especially miR-294 regulate a large number of alternative splicing events, among which muscleblind-like proteins are responsible for a significant portion. Besides, upon depletion of miRNAs, RBPs and SFs are globally downregulated in mESCs.
Project description:RNA-seq analysis on CTR, miR-294, and siMbd2 transfected Dgcr8-/- ESCs showed that Mbd2 is responsible for the change of expression of a significant portion of genes that are regulated by miR-290 cluster
Project description:We performed Fluidigm C1 single cell sequencing analysis of wild-type and microRNA deficient (Dgcr8 knockout) mouse embryonic stem cells mock treated or transfected with either miR-294 or let-7.
Project description:let-7c and miR-294 were transfected into Dgcr8 -/- miRNA deficient ES cells and RNA was harvested after 12 hours the goal of this study was to identify direct and indirect targets of the let-7c and miR-294 miRNAs, we chose to harvest RNA early at 12 hours to mimize secondary effects due to ES cell differentiation, prior to performing the array experiment we found that at this time point candidate miRNA targets were maximally downregulated by qPCR
Project description:Study designed to determine the immediate effects of supplementing OSK reprogramming with miR-294 or miR-181. MEFs were infected on day 0, and transfected with miR-294, miR-181 or control mimic on day 1. On day 3 RNA was extracted. OSK infected MEFs samples were compared to non-infected MEFs and to fully reprogrammed iPSCs.
Project description:Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays, and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-Sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs, and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes, and upregulating pluripotency-associated genes such as Lin28. For RNA-IP experiments, cells were transfected with 300 pmol of miR-294 or cel-239b control miRNA (Dharmacon) and harvested 12-16 hr after transfection for immunoprecipitation. Following RNA-immunoprecipitation of Ago2-myc, RNA from the INPUT (total RNA) and IP were subjected to library preparation and sequenced by SOLiD. There are 10 samples from Dicer1-null mouse ES cells, which are essentially 5 sample pairs of INPUT (A) and its corresponding RNA-IP (B): Ago2-myc transfected with miR-294 (sampe 1), 2 replicates of Ago2-myc-MUT (catalytically inactive) transfected with miR-294 (sample 2 & 5), 2 replicates of Ago2-myc transfected with cel-239b (sample 3 & 4).
Project description:Purpose: To compare the E9.5 Dgcr8 conditional knockout embryonic heart cells transfected with NC miRNA and miR-541 mimics Methods: In vitro cultured E9.5 Dgcr8 conditional KO heart cells transfected with miR-541-5p and NC miRNA were extracted with TRIZOL 48hrs after transfection, and 10ng total RNA was reverse transcribed and amplified by Smart-seq2 protocol as described (Picelli et al., 2014). Duplicated biological samples were analyzed using Illumina HiSeqX10, Clean reads were mapped to mouse genome (mm9) using BWA software. Results: Genes differentially expressed in E9.5 Dgcr8 cKO embryonic heart cells transfected with NC miRNA and miR-541 were identified. Conclusions: miRNA-541 significantly changes the gene expression profiles of E9.5 Dgcr8 cKO embryonic heart cells and promote the cardiac function
Project description:The goal of this study was to identify genes regulated by miRNA-29 in helper T cells. We compared gene expression in miRNA-deficient helper T cells (DGCR8-deficient) transfected with miR-29b versus control miRNA. We also compared gene expression in wildtype helper T cells transfected with miR-29 inhibitor to cells transfected with control inhibitor 3 biological replicates of each genotype (DGCR8-deficient or Wildtype C57BL/6 mice), with 2 conditions per genotype for 12 samples total. Cells from each DGCR8-deficient mouse were transfected with either miR-29b or control miRNA and cells from wildtype mice were transfected with either miR-29 inhibitors or control inhibitors
Project description:Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by Dicer. dgcr8delta/delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1delta/delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild type, dgcr8delta/delta, and dicer1delta/delta mESCs. Several of the DGCR8-independent, Dicer-dependent RNAs were non-canonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of short hairpin RNAs (shRNAs). Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small-RNA generating pathways and show that mammalian siRNAs exist in tissues outside of oocytes. Small RNAs were sequenced from wt, dgcr8(-), and dicer(-) mouse ES cells and the frequencies of small RNA types compared between the three. This record includes Illumina-platform-generated datasets from all three samples and 454-platform-generated datasets from wt and dgcr8(-) samples. [raw data files are unavailable]
