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SRP071661
Mus musculus
6 Downloadable Samples
Illumina HiSeq 2500
Description
Myelination is essential for nervous system function. Schwann cells interact with neurons and with the basal lamina to sort and myelinate axons, using known receptors and signaling pathways. In contrast, the transcriptional control of axonal sorting and the role of mechano-transduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. Here, we describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice we show that Taz is required in Schwann cells for radial sorting and myelination. Yap is redundant with Taz as ablation of both Yap and Taz abolishes radial sorting. Yap/Taz regulate Schwann cell proliferation and transcription of basal lamina receptors, both necessary for proper radial sorting of axons, and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells. Overall design: 3 cKO and 3 control wild-type mice
Publication Title
YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 1000
Description
Purpose: The aim of this study is to evaluate the global gene expression induced by OGG1-BER product 8-oxoG in mouse airways. Methods: RNA extracted from individual mouse lungs (experimental group: n=5) were pooled and a total 1 µg RNA was used for Next-Generation Sequencing (NGS) analyses on an Illumina HiSeq 1000 sequencing system. Sequence analysis were performed in duplicate. First- and second-strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq Sample Preparation Kit as recommended by the manufacturer (Illumina). Library quality was evaluated by using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Library DNA templates were quantitated by qPCR using known reference starndards. Cluster formation of the library of DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation. Paired-end, 50-base sequencing was performed with a TruSeq SBS kit v3 (Illumina) on the Illumina HiSeq 1000 by protocols defined by the manufacturer. Base call conversion to sequence reads was performed using CASAVA-1.8.2. Sequence data were analyzed with the Bowtie2, Tophat2 and GFOLD programs. Processed data are presented as reads per kilobase transcript per million (RPKM), normalized to the experimental control (RNA from saline-challenged lungs) and reported as fold change (test/control). Results: We mapped an average of 24.76 million sequence reads per sample and identified 23,337 transcripts in total RNA extracted from lungs of Balb/cJ mice as described in Methods. Approximately 10% of the transcripts showed differential expression between the saline-challenged control and 8-oxoguanine-challeged mouse lungs, with a fold change =3.0. We validated the expression changes of 7 selected pro-inflammatory cytokines and chemokines of interest for our studies by qRT-PCR. Hierarchical clustering followed by Protein ANalysis THrough Evolutionary Relationships database (PANTHER) analysis of differentially expressed genes. Results showed overrepresentation of various biological functions (GO terms) including immune system process (GO:0002376; p=5.24e-12) among others. Pathway analysis (PANTHER) indicated that the most overrepresented pathway was inflammation mediated by chemokine and cytokine (P00031, p=<0.01). In addition to gene expression analysis, we confirmed OGG1•8-oxoG-dependent RAS activation in lungs by active RAS pull-down assays, airways neutrophil accumulation by bronchoalveolar lavage fluid (BALF) differential cell counts and airway inflammation by histological examination (H&E staining) of lung sections. Conclusions: This is the first study at the whole-transcriptome level to show induction of innate immune response gene expression in mouse lungs after exposure to OGG1-BER product 8-oxoG. Overall design: Balb/cJ mice (5 per group) were intranasally challenged with 8-oxoguanine (1 µM, 60 µl) for 30, 60 and 120 min. Control group mice were intranasally challenged with saline (60 µl). RNA from individual mice whithin the same group was pooled and subjected to deep-sequencing analysis in duplicate using NGS on an Illumina HiSeq 1000 sequencing system. After alignment and processing, the resulting RPKM from treatment groups (8-oxoG-challenged) were normalized to the control group (saline-challenged).
Publication Title
The Potential Role of 8-Oxoguanine DNA Glycosylase-Driven DNA Base Excision Repair in Exercise-Induced Asthma.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 1000
Description
Purpose: The aim of this study is to test whether global gene expression induced by multiple challenges with OGG1-BER product 8-oxoG in mouse airways is linked to airway remodeling. Methods: RNAs extracted from individual mouse lungs (experimental group: n=5) were pooled and a total 1 µg RNA was used for Next-Generation Sequencing (NGS) analyses on an Illumina HiSeq 1000 sequencing system. Sequence analyses were performed in duplicate. First- and second-strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq Sample Preparation Kit as recommended by the manufacturer (Illumina). Library quailty was evaluated by using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Library DNA templates were quantitated by qPCR using known reference standards. Cluster formation of the library of DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation. Paired-end, 50-base sequencing was performed with a TruSeq SBS kit v3 (Illumina) on the Illumina HiSeq 1000 by protocols defined by the manufacturer. Base call conversion to sequence reads was performed using CASAVA-1.8.2. Sequence data were analyzed with the Bowtie2, Tophat2 and GFOLD programs. Processed data are presented as reads per kilobase transcript per million (RPKM), normalized to the experimental control (RNA from saline challenged lungs) and reported as fold change (test/control). Results: We mapped an average of 31.41 million sequence reads per sample and identified 23,337 transcripts in total RNA extracted from lungs of Balb/cJ mice as described in Methods. Approximately 14% of the transcripts showed differential expression between the saline-challenged control and 8-oxoguanine-challeged mouse lungs, with a fold change =3.0. We validated the expression changes of 18 selected EMT-related genes of interest for our studies by qRT-PCR. Hierarchical clustering followed by Protein ANalysis THrough Evolutionary Relationships database (PANTHER) analysis of differentially expressed genes was done using GENE-E online software from Broad Institute (http://www.broadinstitute.org/cancer/software/GENE-E/). Results from PANTHER analysis of upregulated transcripts (fold change =3.0) showed overrepresentation of various biological functions (GO terms) including developmental process (GO:0032502, P=4.58E-33), system development (GO:0048731, P=9.16E-33), cellular process (GO:0009987, P= 5.52E-31), cell adhesion (GO:0007155, P= 8.63E-28) among others. Pathway analysis (PANTHER) indicated that the most overrepresented pathways were: cadherin signaling (P00012, P=6.62E-07), wnt signaling (P00057, P= 5.81E-06), integrin signaling (P00034, P= 1.09E-05) among others. In addition to gene expression analysis, we confirmed airway remodeling by histological examination (Hematoxylin and Eosin, Masson's trichrome staining) of lung sections at seven days from the last challenge (day 11). Conclusions: This is the first study showing a link between gene expression at whole-transcriptome level induced by chronic OGG1-BER (mimicked by multiple challenges with 8-oxoG) and airway remodeling, supported by histological structural changes in lungs. Overall design: Balb/cJ mice (5 per group) were intranasally challenged with 8-oxoguanine (1 µM, 60 µl) for three times at days 0, 2 and 4. Control group mice were intranasally challenged with saline (60 µl). At 30, 60 and 120 min after the third challenge (day 4), mice were sacrificed and lungs were processed for RNA extraction. RNAs from individual mice within the same group were pooled and subjected to deep-sequencing analysis in duplicate using NSG on an Illumina HiSeq 1000 sequencing system. After alignment and processing, the resulting RPKM from treatment groups (8-oxoG-challenged) were normalized to control group (saline-challenged).
Publication Title
The Potential Role of 8-Oxoguanine DNA Glycosylase-Driven DNA Base Excision Repair in Exercise-Induced Asthma.
Sample Metadata Fields
No sample metadata fields
View Samples- Drosophila melanogaster
- 13 Downloadable Samples
- Illumina Genome Analyzer
Description
In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide. Overall design: Sequencing of small RNAs (either total small RNAs or Ago1-associated small RNAs) in wild-type, dcr-2 and r2d2 mutant flies. Small RNA sequencing, Small RNAs (18-29 nt long), Size selection (18 to 30 nt).
Publication Title
Target RNA-directed trimming and tailing of small silencing RNAs.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 27 Downloadable Samples
- Illumina HiSeq 2500
Description
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and provides quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner. Overall design: Wildtype mouse embryonic stem cells (mES cells) were subjected to s4U metabolic RNA labeling for 24 h (pulse, 100 µM s4U), followed by washout (chase) using non-thiol-containing uridine. Total RNA was prepared at various time points along the chase (0h, 0.5h, 1h, 3h, 6h, 12h, and 24h). Total RNA was then subjected to alkylation and mRNA 3' end sequencing library preparation (QuantSeq, Lexogen).
Publication Title
Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Mus musculus
- 18 Downloadable Samples
- Illumina HiSeq 2500
Description
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and provides quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner. Overall design: 5 µg/ml Actinomycin D was added to wildtype mouse embryonic stem (mES) cells and total RNA was prepared at various time points after addition of Actinomycin D (0h, 0.25h, 0.5h, 1h, 3h and 10h). Total RNA was subjected to mRNA 3' end library preparation (QuantSeq, Lexogen) and high througput sequencing.
Publication Title
Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Mus musculus
- 18 Downloadable Samples
- Illumina HiSeq 2500
Description
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and provides quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner. Overall design: Wildtype (wt) mouse embryonic stem (mES) cells, clonal mES cells that had been transfected with non-targeting control guide RNAs (ctr), or Exportin-5 depleted (Xpo5KO) mES cells were subjected to 3h and 12h s4U-pulse labeling followed by total RNA extraction, alkylation, mRNA 3' end library preparation (QuantSeq, Lexogen) and high throughput sequencing.
Publication Title
Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Mus musculus
- 18 Downloadable Samples
- Illumina HiSeq 2500
Description
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and provides quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner. Overall design: Wildtype (wt) mouse embryonic stem (mES) cells, clonal mES cells that had been transfected with non-targeting control guide RNAs (ctr), or Mettl3 depleted (Mettl3KO) mES cells were subjected to 3h and 12h s4U-pulse labeling followed by total RNA extraction, alkylation, mRNA 3´ end library preparation (QuantSeq, Lexogen) and high throughput sequencing.
Publication Title
Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina HiSeq 2500
Description
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and provides quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner. Overall design: Mouse embryonic stem (mES) cells were subjected to 45 min s4U-pulse labeling followed by total RNA extraction, alkylation, mRNA 3' end library preparation (Quant-seq, Lexogen) and high throughput sequencing.
Publication Title
Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina HiSeq 2500
Description
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and provides quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner. Overall design: Total RNA from wildtype mouse embryonic stem (mES cells) was extracted and subjected to alkylation or mock treatment prior to mRNA 3' end library preparation (QuantSeq, Lexogen) and high throughput sequencing.
Publication Title
Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples