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GSE5266
Rattus norvegicus
8 Downloadable Samples
Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
Pharmacological and gene ablation studies have demonstrated a crucial role of the cardiac natriuretic peptides (NP) hormones ANF and BNP in the maintenance of cardiovascular homeostasis. In addition, hypertension and chronic congestive heart failure are clinical entities that may be regarded as states of relative NP deficiency. Hence the study of the function of the endocrine heart is highly relevant.
Publication Title
Transcriptional analysis of the mammalian heart with special reference to its endocrine function.
Sample Metadata Fields
Sex, Specimen part
View Samples- Homo sapiens
- 124 Downloadable Samples
Illumina HiSeq 2000, Illumina HiSeq 2500
Description
We conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. Our approach was to dilute bulk total RNA (from a single source) to levels bracketing single-cell levels of total RNA (10 pg and 100 pg) in replicates and amplifying the RNA to levels sufficient for RNA sequencing. Overall design: We performed replicate transcriptome amplifications of Universal Human Reference RNA (UHR) and Human Brain Reference RNA (HBR) that were diluted to single-cell and ten-cell abundances (10 and 100 picograms (pg.) total RNA or ~200,000 and 2 million mRNA molecules, respectively) and were amplified using three single-cell RNA amplification methods. Methods included the antisense RNA IVT protocol (aRNA), a custom C1 SMARTer protocol (SmartSeq Plus) performed on a Fluidigm C1 96-well chip, and a modified NuGen Ovation RNA sequencing protocol (NuGen). Bulk ribo-depleted UHR and HBR RNA were sequenced and served as a reference. The general experimental scheme was consistent for all dilution replicates; however, there were differences across experimental groups in the specifics of experimental protocols, necessitated by particular methodologies. Because of these experimental differences, head-to-head comparison of methods is not appropriate and our goal is to provide quantitative analyses of factors affecting individual methods. Current results should be used in experimental planning, data analysis, and method optimization rather than as a performance test of any particular method. Detailed experimental design: Each collaborating center obtained reference RNA with the same lot number for Universal Human Reference (UHR) RNA (Agilent 740000, Lot 0006141415) and Human Brain Reference (HBR) (Ambion AM6050, Lot-105P055201A) and performed replicate amplification using a single amplification method, detailed below. SmartSeq Plus: Reference RNA was diluted to an intermediate stock solution by serial dilution. A final 1000-fold dilution occurred on the C1 chip, such that individual wells in a given batch contained 9.99 pg. sampled from a common intermediate dilution. ERCC spike-in RNA mix 1 (Ambion 4456740) was also added for a final mass of approximately 7 femtograms (fg.) per sample, a 4,000,000x dilution from stock. Samples for each source RNA were prepared in single batches. After amplification, cDNA from the entire C1 96-well plate was quantified using picogreen. C1 chips with an average yield of less than 3 nanograms were discarded. The top 15 reactor wells by cDNA concentration were selected as representative 10 pg. samples for sequencing library preparation. Another 50 wells were selected by the same criteria. These were pooled in sets of 10, generating 5 100 pg. samples for each HBR and UHR. All samples for a given source were prepared in a single sequencing library preparation batch using Nextera XT C1 protocol. NuGen: HBR samples were prepared in a single batch using amplification protocol 1, generating 4 10 pg. and 4 100 pg. amplified replicates. UHR samples were prepared in two batches, using either amplification protocol 1 or 2, generating 15 10 pg. and 11 100 pg. samples. A single sequencing library preparation was performed for each batch of samples using either Lucigen NxSeq or NuGen Ovation Rapid protocol. aRNA: Amplification was performed as previously described (Morris J, Singh JM, Eberwine JH. Transcriptome analysis of single cells. J. Vis. Exp. [Internet]. 2011; Available from: http://www.jove.com/video/2634/transcriptome-analysis-of-single-cells). HBR samples were prepared in 4 batches from separate dilutions of reference RNA, generating 19 10 pg. and 3 100 pg. amplified replicates. ERCC spike-ins were added to 5 of the 10 pg. replicates before amplification at a dilution of 4,000,000x from stock. UHR samples were diluted and amplified in 2 batches from separate dilutions of reference RNA, generating 12 10 pg. and 7 100 pg. amplified replicates. A single sequencing library preparation was performed using Illumina TruSeq Stranded mRNA protocol modified to begin with amplified aRNA. A small numbers of reads were assigned to ERCC transcripts in replicates from the batch where ERCCs had been added that did not have spike-ins added (average of 0.5% of the number of reads assigned in spiked samples). 18 additional HBR 10 pg. replicates were amplified using aRNA for protocol optimization experiments. These samples were treated separately and were excluded from primary analysis. Bulk UHR and HBR: For each reference RNA, three sequencing libraries were generated from bulk material at the same laboratory as the SmartSeq Plus replicates. Cytoplasmic and mitochondrial ribosomal RNA (rRNA) were depleted using Ribo-Zero Gold as part of Illumina TruSeq Stranded Total RNA protocol. Samples were sequenced on Illumina HiSeq 2000. Because of differences in experimental design, direct comparison across methods of precision and the effect of input RNA abundance is difficult. For example, input RNA amount as a factor have different meanings for the different amplification methods: for SmartSeq Plus, because 100pg samples were constructed by pooling 10 pg. samples after cDNA amplification, any resulting effects involve library construction, while for aRNA and NuGen resulting effects reflect both cDNA amplification steps and library steps.
Publication Title
Assessing characteristics of RNA amplification methods for single cell RNA sequencing.
Sample Metadata Fields
Subject
View Samples- Rattus norvegicus
- 24 Downloadable Samples
- Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Ras dexamethasone-induced protein 1 is a modulator of hormone secretion in the volume overloaded heart.
Sample Metadata Fields
No sample metadata fields
View Samples- Rattus norvegicus
- 12 Downloadable Samples
Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
Pharmacological and gene ablation studies have demonstrated a crucial role of the caridac natriuretic peptides (NP) hormones ANF and BNP in the maintenance of cardiovascular homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying increased atrial ANF and BNP expression and secretion. These investigations are important because under chronic congestive heart failure, the secretion of NPs although increased and beneficial, is relatively insufficient as demonstrated by the fact that patients benefit form the unloading of the heart induced by therapeutic administration of either ANF or BNP.
Publication Title
Ras dexamethasone-induced protein 1 is a modulator of hormone secretion in the volume overloaded heart.
Sample Metadata Fields
No sample metadata fields
View Samples- Rattus norvegicus
- 12 Downloadable Samples
- Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
Pharmacological and gene ablation studies have demonstrated a crucial role of the caridac natriuretic peptides (NP) hormones ANF and BNP in the maintenance of cardiovascular homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying increased atrial ANF and BNP expression and secretion. These investigations are important because under chronic congestive heart failure, the secretion of NPs although increased and beneficial, is relatively insufficient as demonstrated by the fact that patients benefit form the unloading of the heart induced by therapeutic administration of either ANF or BNP.
Publication Title
Ras dexamethasone-induced protein 1 is a modulator of hormone secretion in the volume overloaded heart.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
The transcription factor Snail has been proposed to mediate epithelial-to-mesenchymal transition (EMT) and confer mesenchymal invasive phenotype to epithelial cancer cells
Publication Title
SNAIL-induced epithelial-to-mesenchymal transition produces concerted biophysical changes from altered cytoskeletal gene expression.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The E2A transcription factors promote the development of thymus-seeding cells but it remains unknown whether these proteins play a role in T lymphocyte lineage specification or commitment. By examining E2A-dependent genes in developing T cells, we will address whether these proteins are involved in these processes.
Publication Title
E2A transcription factors limit expression of Gata3 to facilitate T lymphocyte lineage commitment.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 2 Downloadable Samples
- Affymetrix Mouse Expression 430A Array (moe430a)
Description
to study the proliferation of PERK knockout mice islets.
Publication Title
PERK EIF2AK3 control of pancreatic beta cell differentiation and proliferation is required for postnatal glucose homeostasis.
Sample Metadata Fields
Sex
View Samples- Hordeum vulgare
- 12 Downloadable Samples
- Affymetrix Barley Genome Array (barley1)
Description
Transcriptional changes were monitored in the barley cultivar Golden Promise 24 hours post inoculation (hpi) with the bacteria Pseudomonas syringae pv. tomato DC3000 avrRpm1 (PstavrRpm1) using the Affymetrix Barley genome array GeneChip. Seedlings of Golden Promise were grown to growth stage 12-13 (Zadoks et al., 1974) before inoculating with either PstavrRpm1 or water (for the mock inoculation control) by infiltration. Plants were grown under a 18 C / 16 h light period; 12 C / 8 h dark period, with artificial lighting (100 mol m-2 s-1) and a relative humidity of 75 85 %. Leaf samples from three seedlings were collected 24 hpi for RNA extraction and transcriptomics analysis from the area infiltrated (local) and from the area next to the infiltrated region (adjacent) from three biological replicates. Leaf tissue was ground under liquid nitrogen and total RNA extracted using the RNeasy miniprep kit (Qiagen), following the manufacturers instructions. RNA was DNase treated using Turbo DNase (Ambion) according to the manufacturer instructions. RNA integrity was confirmed using the Agilent 2100 Bioanalyzer (Agilent). The two cycle-target labeling method was used following the Affymetrix protocol. Affymetrix GeneChip processing, including RNA quality control, microarray hybridisation and data acquisition was performed through contract research services by Cogenics (North Carolina, U.S.A.). A total of twelve hybridisations were performed. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Ellen Colebrook. The equivalent experiment is BB92 at PLEXdb.]
Publication Title
Broad-spectrum acquired resistance in barley induced by the Pseudomonas pathosystem shares transcriptional components with Arabidopsis systemic acquired resistance.
Sample Metadata Fields
Specimen part
View Samples- Triticum aestivum
- 12 Downloadable Samples
- Affymetrix Wheat Genome Array (wheat)
Description
Transcriptional changes were monitored in the wheat cultivar Renan 24 hours post i noculation with adapted and non-adapted Magnaporthe isolates using the Affymetrix wheat genome array GeneChip. Wheat plants cv. Renan were grown in a peat and sand (1:1) mix at 23 C in a Sanyo Fitotron growth cabinet (Sanyo Gallenkamp PLC, Loughborough, U.K.) with a 16/8 h, light/dark cycle. Three Magnaporthe isolates were used in this expt, two wheat-adapted isolates (BR32, BR37) and one wheat non-adapted isolate (BR29). Magnaporthe isolates were grown for eleven days on Complete Media Agar at 25 C under a 16/8h, light/dark cycle. Conidia were harvested by flooding the plates with 5 mL of sterile inoculation solution [0.25% (w/v) gelatine and 0.01% (v/v) Tween 20] and scraping the conidia from the surface using a sterile glass rod. Conidia were filtered through sterile miracloth and the density adjusted to 1 x 10 5 conidia mL-1 with inoculation solution. Fourteen day old wheat seedlings mist inoculated with 4 mL of a Magnaporthe conidia suspension and plants were sealed in plastic propagators to maintain relative humidity c.100% and kept at 25 C in the dark for the first 24 hours post inoculation (hpi). Inoculation solution without Magnaporthe conidia was used as a mock-inoculation control. Leaf samples were collected 24 hpi for transcriptomics analysis from three independent biological experiments. Leaf tissue was ground under liquid nitrogen and total RNA extracted using a QIAquick RNeasy Plant Extraction Kit (Qiagen, Hilden, Germany), followed by TURBO DNaseTM (Ambion, Texas, U.S.A.) treatment. RNeasy Mini Spin column purification (Qiagen) was used to further purify RNA samples for array hybridisation. RNA quality checks, cRNA conversion and Affymetrix genome array hybridisation was carried out by the Nottingham Arabidopsis Stock Centre (NASC) array hybridisation service (http://affymetrix.arabidopsis.info/). ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Graham McGrann. The equivalent experiment is TA24 at PLEXdb.]
Publication Title
Wheat blast: histopathology and transcriptome reprogramming in response to adapted and nonadapted Magnaporthe isolates.
Sample Metadata Fields
Specimen part, Treatment
View Samples