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Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development [miRNA-Seq]

ABSTRACT: Mammalian nephron progenitors undergo transcriptional changes over the course of nephrogenesis that sensitize them to signals for differentiation over time. This increases the rate at which they exit their multipotent, self-renewing mesenchymal state and become differentiated epithelial cells, ultimately depleting the progenitor population and leading to the cessation of nephrogenesis. We hypothesized that changes in chromain accessibility and miRNA expression would accompany these transcriptional changes, and that regions of changing chromatin accessibiltiy could reveal chaning regulatory features such as enhancers, including some that affect miRNA expression. To test this, we pooled kidneys from wild-type mouse litters sacrificed at embryonic day 14.5 (E14.5) and post-natal day zero (P0) using positive selection for the surface protein Integrin alpha 8 (Itga8), then sequenced the assay for transposase-accessible chromatin (ATAC-seq) using 50,000 cells, and used the remaining cells for small RNA sequencing (smRNA-seq). smRNA-seq libraries were generated using the QIAseq miRNA library preparation kit (Qiagen 331502), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh.

ORGANISM(S): Mus musculus

PROVIDER: GSE168342 | GEO | 2023/03/04

REPOSITORIES: GEO

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Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development [ATAC-Seq]

Project description:Mammalian nephron progenitors undergo transcriptional changes over the course of nephrogenesis that sensitize them to signals for differentiation over time. This increases the rate at which they exit their multipotent, self-renewing mesenchymal state and become differentiated epithelial cells, ultimately depleting the progenitor population and leading to the cessation of nephrogenesis. We hypothesized that changes in chromain accessibility and miRNA expression would accompany these transcriptional changes, and that regions of changing chromatin accessibiltiy could reveal chaning regulatory features such as enhancers, including some that affect miRNA expression. To test this, we pooled kidneys from wild-type mouse litters sacrificed at embryonic day 14.5 (E14.5) and post-natal day zero (P0) using positive selection for the surface protein Integrin alpha 8 (Itga8), then sequenced the assay for transposase-accessible chromatin (ATAC-seq) using 50,000 cells, and used the remaining cells for small RNA sequencing (smRNA-seq). ATAC-seq libraries were generated using the Nextera DNA Flex Library Prep Kit (Illumina FC-121-1030) with modifications according to a previously published method (Buenrostro et al., 2013), and all sequencing was performed using an Illumina NextSeq500 by the Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh.

2023-03-04 | GSE168339 | GEO

Transfer of small RNA from extracellular vesicles secreted by primary human airway epithelial cells to Pseudomonas aeruginosa

Project description:P. aeruginosa were exposed to extracellular vesicles (EVs) secreted by primary human airway epithelial cells from 3 donors or PBS vehicle control for 6 h. Total RNA was isolated, small RNA libraries were prepared with the QIAseq smRNA kit (Qiagen) and 75-bp single-end reads were generated using Illumina MiniSeq. To assess transfer of human EV small RNAs to P. aeruginosa, sequences that did not map to the PA14 reference genome were aligned to the human genome.

2023-04-11 | GSE228919 | GEO

Transfer of small RNA from extracellular vesicles secreted by primary human airway epithelial cells to Pseudomonas aeruginosa

2021-07-21 | GSE174710 | GEO

Characterisation of the in-vivo miRNA landscape in Drosophila ribonuclease mutants reveals Pacman mediated regulation of the highly conserved let-7 cluster during apoptotic processes

Project description:RNA extracted from Drosophila wandering L3 wing imaginal discs was 2S rRNA depleted and prepared for miRNA sequencing using the QIAseq miRNA Library kit. The experiment was performed on Pacman and Dis3L2 null mutants and their respective isogenic control lines with four replicates of each condition.

2024-02-28 | E-MTAB-13781 | biostudies-arrayexpress

Rapid differentiation of hiPSCs into functional oligodendrocytes using an OLIG2 synthetic modified messenger RNARapid differentiation of hiPSCs into functional oligodendrocytes using an OLIG2 synthetic modified messenger RNA

Project description:We show that a synthetic modified messenger RNA (smRNA)-based reprogramming method that leads to the generation of transgene-free OLs has been developed. An smRNA encoding a modified form of OLIG2, a key TF in OL development, in which the serine 147 phosphorylation site is replaced with alanine, OLIG2S147A, is designed to reprogram hiPSCs into OLs. We demonstrate that repeated administration of the smRNA encoding OLIG2 S147A lead to higher and more stable protein expression. Using the single-mutant OLIG2 smRNA morphogen, we establish a 6-day smRNA transfection protocol, and glial induction lead to rapid NG2+ OL progenitor cell (OPC) generation (> 70% purity) from hiPSC-derived neural progenitor cells (NPCs). The smRNA-induced NG2+ OPCs can mature into functional OLs in vitro and promote remyelination in vivo. Proteomic analysis of OLIG2-binding proteins indicates that OLIG2 is bound by the heat shock protein 70 (HSP70) complex. The HSP70 complex is bound more strongly to OLIG2 with the modified phosphorylation site than to wild-type OLIG2.

2023-03-10 | PXD036975 | Pride

Association of Presurgical Circulating MicroRNAs with 1-Year Postsurgical Pain Reduction in Lumbar Spinal Stenosis Patients with Spine Facet Osteoarthritis

Project description:OBJECTIVE: Our objective was to sequence the plasma miRNome from 56 patients with lumbar spinal stenosis (LSS) to determine whether miRNA signatures at time of surgery predicted pain responses one year later. MicroRNA libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the Illumina NextSeq550. Counts were produced for microRNAs captured in miRBase

2022-08-30 | GSE158825 | GEO

A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma

Project description:We compared 3 small RNA library prep kits (CleanTag, NEXTflex, QIAseq) and two RNA extraction methods (miRNeasy and MagnaZol) on plasma. We report that library preparation has a significant effect upon the miRNA profile detected, with QIAseq libraries exhibiting the least sequencing bias of the three library kits. RNA extraction methods also contribute, to a lesser extent, to the miRNA profile detected, with MagnaZol RNA extraction increasing the percentage of reads mapping to miRNAs and the number of individual miRNAs detected.

2019-05-21 | GSE118125 | GEO

Generation and comparative analysis of an Itga8-CreERT2 mouse with preferential activity in vascular smooth muscle cells

Project description:All current smooth muscle cell (SMC) restricted Cre mice recombine floxed alleles in vascular and visceral SMCs. We generated a tamoxifen-inducible CreERT2 mouse, Itga8-CreERT2, and compared its activity to the widely used Myh11-CreERT2 mouse. Both CreERT2 mice showed similar activity in vascular SMCs; however, Itga8-CreERT2 displayed low activity in visceral SMC-containing tissues (e.g., intestine). Myh11-CreERT2 (but not Itga8-CreERT2) mice exhibited high levels of CreERT2 protein, tamoxifen-independent activity, and an altered transcriptome. Whereas Myh11-CreERT2-mediated knockout of Srf resulted in a lethal intestinal phenotype, loss of Srf with Itga8-CreERT2 (SrfItga8) revealed viable mice with attenuated vascular SMC contractile gene expression, but no evidence of intestinal pathology. Male and female SrfItga8 mice presented with vascular contractile incompetence; however, only male SrfItga8 mice showed systemic changes in blood pressure. These results establish the Itga8-CreERT2 mouse as an alternative to existing SMC Cre strains where gene loss may result in visceral myopathies that obfuscate accurate phenotyping in vascular SMCs.

2022-09-13 | GSE199244 | GEO

Uncovering small RNA-mediated responses to cold stress in a wheat thermosensitive genic male sterile line by deep sequencing (Degradome-Seq)

Project description:The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNA) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 81 unique miRNA sequences from 30 families, and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. To identify smRNA targets in the wheat TGMS line, we applied the degradome sequencing method, which globally and directly identifies the remnants of smRNA-directed target cleavage. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing datasets and TaqMan qPCR results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, expressions of cold-stress responsive smRNAs integrated in the auxin-signaling pathway and their target genes were largely anticorrelated. We investigated tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway, and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress.

2012-04-11 | GSE37134 | GEO

Chromatin binding of RUNX1 in T-cell acute lymphoblastic leukemia (T-ALL) cells with depletion cBAF complex [ChIP-seq]

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2023-11-04 | GSE243007 | GEO

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