Transcriptional profiling of staurosporine-induced cell death in Vogel's minimal medium with different concentrations of calcium
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ABSTRACT:
ORGANISM(S): Neurospora crassa
PROVIDER: GSE53013 | GEO | 2014/12/05
SECONDARY ACCESSION(S): PRJNA230677
REPOSITORIES: GEO
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Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type cells grown with different availabilities of calcium. Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium (with the indicated concentrations of calcium) for 6 hours (26M-BM-:C, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. For the experiments in 0 Ca2+ or 20 mM CaCl2 media, the concentration of KH2PO4 in the 50x VogelM-bM-^@M-^Ys stock solution was limited to 10 mM to avoid precipitation with supplemental calcium. Cells were harvested using 0.45 M-NM-<m filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 M-NM-<g RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerM-bM-^@M-^Ys protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analyzed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: Differences in the concentration of extracellularly available Ca2+ result in distinct transcriptional programs. Conclusions: Our transcriptomic analysis provides a reference dataset for future investigations on the role of Ca2+ in fungal biology. Transcriptional profile of staurosporine-treated Neurospora crassa wild type cells growing with different concentrations of calcium was compared using illumina HiSeq2000 and single reads of 50 bp.
2014-12-05 | E-GEOD-53013 | biostudies-arrayexpress
Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type and Δczt-1 (ΔNCU09974) cells Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26ºC, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 μm filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 μg RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturer’s protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments/Reads Per Kilobase of transcript per Million mapped reads (FPKM/RPKM). Results: Transcriptional profiling of staurosporine-treated cells wild type by RNA-sequencing showed that genes encoding the machinery for protein synthesis are enriched among the genes repressed by the drug. Functional category enrichment analyses also show that genes encoding components of the mitochondrial respiratory chain are downregulated by staurosporine, whereas genes involved in endoplasmic reticulum activities are upregulated. In contrast, a staurosporine-treated czt-1 deletion strain is unable to repress the genes for the respiratory chain and to induce the genes related with the endoplasmic reticulum, indicating a role for CZT-1 on the regulation of activity of these organelles. Conclusions: This transcriptional profiling study is framed in a comprehensive study on the role of the novel transcription factor CZT-1 (NCU09974) in Neurospora crassa.
2014-07-01 | GSE52153 | GEO
Project description:Purpose: Compare the transcriptional profile of staurosporine-treated cells in Neurospora crassa wild type and M-NM-^Tczt-1 (M-NM-^TNCU09974) cells Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26M-BM-:C, 140 rpm, constant light) followed by the addition of staurosporine (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 M-NM-<m filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 M-NM-<g RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerM-bM-^@M-^Ys protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments/Reads Per Kilobase of transcript per Million mapped reads (FPKM/RPKM). Results: Transcriptional profiling of staurosporine-treated cells wild type by RNA-sequencing showed that genes encoding the machinery for protein synthesis are enriched among the genes repressed by the drug. Functional category enrichment analyses also show that genes encoding components of the mitochondrial respiratory chain are downregulated by staurosporine, whereas genes involved in endoplasmic reticulum activities are upregulated. In contrast, a staurosporine-treated czt-1 deletion strain is unable to repress the genes for the respiratory chain and to induce the genes related with the endoplasmic reticulum, indicating a role for CZT-1 on the regulation of activity of these organelles. Conclusions: This transcriptional profiling study is framed in a comprehensive study on the role of the novel transcription factor CZT-1 (NCU09974) in Neurospora crassa. Transcriptional profile of wild type and M-NM-^Tczt-1 cells staurosporine-treated cells in Neurospora crassa was compared using illumina HiSeq2000 and single reads of 50 bp.
2014-07-01 | E-GEOD-52153 | biostudies-arrayexpress
Project description:Purpose: Evaluate the transcriptional profile of Neurospora crassa cells treated with a group of novel antifungal compounds Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26ºC, 140 rpm, constant light) followed by the addition of the indicated compound (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 μm filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 μg RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturer’s protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: XP13 promoted a particularly active transcriptional response. Functional enrichment analysis of the genes with altered expression revealed novel pathways that are affected by these compounds. Conclusions: These compounds are active in Neurospora crassa and we anticipate that this and future studies will ascertain their potential as antifungal agents.
2015-11-25 | GSE53040 | GEO
Project description:Purpose: Evaluate the transcriptional profile of Neurospora crassa cells treated with a group of novel antifungal compounds Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26ºC, 140 rpm, constant light) followed by the addition of the indicated compound (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 μm filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 μg RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturerâs protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: XP13 promoted a particularly active transcriptional response. Functional enrichment analysis of the genes with altered expression revealed novel pathways that are affected by these compounds. Conclusions: These compounds are active in Neurospora crassa and we anticipate that this and future studies will ascertain their potential as antifungal agents. The transcriptional profile of Neurospora crassa wild type cells exposed with a group of novel compounds with antifungal activity was analysed using illumina HiSeq2000 and single reads of 50 bp.
2015-11-25 | E-GEOD-53040 | biostudies-arrayexpress
Project description:PAO1 was cultured planktonically to stationary phase with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.
2015-12-02 | GSE74444 | GEO
Project description:CD3+ T cells were enriched using the EasySep human T cell isolation kit (Stem cell technology). T cells from normal controls or patients with CARD9 mutations were co-cultured with monocytes of one normal control in the presence of heat killed candida. After 3 days, T cell were enriched again to remove the monocytes and total RNA was extracted from the T cells for the RNA-sequencing (RNASeq) evaluation. Targeted RNA sequencing library preparation was carried out using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies), which profiles more than 20,000 human genes; each amplicon (~150 bp) represents a unique targeted gene (one transcript per gene). For library preparation, each sample was run in duplicate, and a cDNA library was generated from a minimum of 10 ng of total RNA. The cDNA was barcoded and amplified with Ion AmpliSeq technology, and the amplified cDNA Libraries were evaluated for quality and quantified with Agilent Bioanalyzer high-sensitivity chip. Libraries were then diluted to 100 pM and pooled equally, with 4 individual samples per pool. Pooled libraries were amplified and enriched with the Ion Chef System (Life Technologies). Templated libraries were then sequenced on an Ion Torrent Proton sequencing system (Life Technologies) with Ion PI HiQ kit and chip version 3. We performed gene-level differential expression analysis of targeted RNASeq data using R (v.3.5.3) and the Bioconductor packages DESeq2 (v.1.22.2).
2021-08-12 | GSE181718 | GEO
Project description:Participants were recruited from the California Lupus Epidemiology Study (CLUES). CLUES was approved by the Institutional Review Board of the University of California, San Francisco. Peripheral blood mononuclear cells were isolated from patient donors. Cells were isolated from peripheral blood utilizing magnetic beads (CD14+monocytes, B cells, CD4+T cells and NK cells) using EasySep protocol from STEM cell technologies on 120 patients. RNA and DNA were extracted using Qiagen column and quality was assessed on Agilent bioanalyzer. 1ng - 5ng of RNA was used with SmartSeqv2 protocol to get cDNA, followed by Illumina Nextera XT DNA library prep with input of 0.8ng cDNA and 15 cycles of PCR amplification. Both cDNA and DNA libraries qualities were controlled on Agilent bioanalyzer. Libraries were sequenced on HiSeq4000 PE150.
2021-03-04 | GSE164457 | GEO
Project description:PAO1 and FRD1 were cultured planktonically and in biofilms with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined for PAO1 cultured planktonically, for FRD1 cultured planktonically, for PAO1 cutured in biofilms, and for FRD1 cultured in biofilms. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.
2015-12-02 | GSE74445 | GEO
Project description:Calcium is an essential macronutrient and plant requires it in large amounts for normal growth and development. This ion participated in innumerable processes and affects nearly all aspects of plant growth and development such as signal transduction, metabolism of lipids, proteins, and carbohydrates, cell growth, cell wall and membrane stabilization. We used whole genome microarrays to determine the transcriptomic profile of rice seedlings exposed to short-term and long term Ca2+ deficiency followed by Ca2+ resupply. Calcium treated seedlings were used for for RNA extraction and hybridization on Agilent microarray platform. Three biological replicates of each sample were used for microarray analysis. We wanted to know the altered expression patterns of calcium-responsive genes majorly involved in metabolic processes, signal transdction pathways, transcriptional regulation, and transport of multiple molecules including Ca2+. Seeds of indica rice were surface sterilized and grown hydroponically in rice growing medium. Plants were grown in the rice growth room under the condition of 16 h light/8 h dark (28M-BM-0C) photoperiod with 70 % humidity. After 5 days of normal growth, some of the seedlings were transferred to nutrient solution lacking CaCl2 (-Ca2+). After 5 and 14 days, calcium (0.798 mM CaCl2) was resupplied to the plants grown in -Ca2+ medium for 6 h.
2014-01-17 | E-GEOD-50917 | biostudies-arrayexpress