Single-cell RNA-seq of total cells from testes of adult WT and miR-202-/- mice
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ABSTRACT:
PROVIDER: PRJNA731348 | ENA |
REPOSITORIES: ENA
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Project description:We report to investigate the changes in cellular composition and gene expression upon miR-202 KO. One testis from each of three adult WT mice and three adult KO mice was collected, and the KO and WT testes were pooled respectively for subsequent analysis. A total of 15,310 WT and 12,935 KO cells passed standard quality control and were used for subsequent analysis.
2021-05-21 | GSE174731 | GEO
Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP).
2012-12-19 | GSE42981 | GEO
Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.
2012-12-19 | E-GEOD-42981 | biostudies-arrayexpress
Project description:In fish, female fecundity is tightly linked to the proper completion of oogenesis that takes place in the ovary. These cellular processes have been shown to involve both endocrine and intra-ovarian factors. However, the role of small non-coding miRNAs remains largely unknown. Here, we analyzed the role of miR-202, a miRNA specifically expressed in the vertebrate gonads. We first used fluorescent in situ hybridization to determine the precise cellular expression of miR-202 in the medaka ovary. We then generated a mutant fish line (using CRISPR/Cas9 technology) to determine its role in female fecundity and oogenesis. We performed quantitative image analyses to analyze cellular modifications and a genome-wide transcriptomic approach to analyze gene expression modifications. Our results show that miR-202-5p is predominantly expressed in granulosa cells. In addition, mutant females display a drastically reduced fecundity. Our cellular and molecular analyses of ovaries from mutant females indicate that miR-202 deficiency impairs the early steps of oogenesis/folliculogenesis, which ultimately reduce female fecundity. This provides the first functional evidence that miR-202 is necessary for the female reproductive success, in particular the female fecundity, and shed new light on the regulatory mechanisms that control the early steps of follicular development.
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Project description:RNAs that are enriched in AGO2 Immunoprecipitated (IP) products or PIWIL1 IP products were identified from mouse(BALB/C) adult testes by examine the ratio of total RNA signal intensity to AGO2 IP RNA or PIWIL1 IP RNA signal intensity. Two-condition experiment,Total RNA extracted from mouse adult testes vs. AGO2 IP RNA extracted from mouse adult testes and total RNA extracted from mouse adult testes vs. PIWIL1 IP RNA extracted from mouse adult testes.
2013-04-01 | E-GEOD-27582 | biostudies-arrayexpress
Project description:RNAs that are enriched in AGO2 Immunoprecipitated (IP) products or PIWIL1 IP products were identified from mouse(BALB/C) adult testes by examine the ratio of total RNA signal intensity to AGO2 IP RNA or PIWIL1 IP RNA signal intensity.
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Project description:The microARN-202 (miR-202) controls female fecundity by regulating oogenesis in medaka
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Project description:We have identified that miR-2b-1 of D. melanogaster causes testicular bulging (a tumour like phenotype) and hence have performed a transcriptomic analysis on these bulged testes. Adult flies overexpressing miR-2b-1 were used to perform transcriptomic analysis to identify the differentially expressed genes.
2022-08-31 | GSE211399 | GEO
Project description:We preformed RNA sequencing to identify differentiatially expressed mRNAs after miR-202 knockout in isolated spermatogonia and spermatocytes, and cultured spermatogonial stem cells from male mice.
2020-02-15 | GSE126936 | GEO
Project description:MiR-142 is dynamically expressed and plays a regulatory role in hematopoiesis. Based on the simple observation that miR-142 levels are significantly lower in CD34+CD38- cells from blast crisis (BC) chronic myeloid leukemia (CML). CML patients compared with chronic phase (CP) CML patients (p=0.002), we hypothesized that miR-142 deficit plays a role in BC transformation. To test this hypothesis, we generated a miR-142 KO BCR-ABL (i.e., miR-142−/−BCR-ABL) mouse by crossing a miR-142−/− mouse with a miR-142+/+BCR-ABL mouse. While the miR-142+/+BCR-ABL mice developed and died of CP CML, the miR-142−/−BCR-ABL mice developed a BC-like phenotype in the absence of any other acquired gene mutations and died significantly sooner than miR-142+/+BCR-ABL CP controls (p=0.001). Leukemic stem cell (LSC)-enriched Lineage-Sca-1+c-Kit+ cells (LSKs) from diseased miR-142−/−BCR-ABL mice transplanted into congenic recipients, recapitulated the BC features thereby suggesting stable transformation of CP-LSCs into BC-LSCs in the miR-142 KO CML mouse. Single cell (sc) RNA-seq profiling showed that miR-142 deficit changed the cellular landscape of the miR-142−/−BCR-ABL LSKs compared with miR-142+/+BCR-ABL LSKs with expansion of myeloid-primed and loss of lymphoid-primed factions. Bulk RNA-seq analyses along with unbiased metabolomic profiling and functional metabolic assays demonstrated enhanced fatty acid β-oxidation (FAO) and oxidative phosphorylation (OxPhos) in miR-142−/−BCR-ABL LSKs vs miR-142+/+BCR-ABL LSKs. MiR-142 deficit enhanced FAO in miR-142−/−BCR-ABL LSKs by increasing the expression of CPT1A and CPT1B, that controls the cytosol-to-mitochondrial acyl-carnitine transport, a critical step in FAO. MiR-142 deficit also enhanced OxPhos in miR-142−/−BCR-ABL LSKs by increasing mitochondrial fusion and activity. As the homeostasis and activity of LSCs depend on higher levels of these oxidative metabolism processes, we then postulate that miR-142 deficit is a potentially druggable target for BC-LSCs. To this end, we developed a novel CpG-miR-142 mimic oligonucleotide (ODN; i.e., CpG-M-miR-142) that corrected the miR-142 deficit and alone or in combination with a tyrosine kinase inhibitor (TKI) significantly reduced LSC burden and prolonged survival of miR-142−/−BCR-ABL mice. The results from murine models were validated in BC CD34+CD38- primary blasts and patient-derived xenografts (PDXs). In conclusion, an acquired miR-142 deficit sufficed in transforming CP-LSCs into BC-LSCs, via enhancement of bioenergetic oxidative metabolism in absence of any additional gene mutations, and likely represent a novel therapeutic target in BC CML.
2023-01-13 | ST002446 | MetabolomicsWorkbench