Primer-based Chloroplast Sequencing and Assembly
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ABSTRACT:
PROVIDER: PRJEB17861 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:We describe a novel quantitative cDNA expression profiling strategy, involving amplification of the majority of mouse transcriptome using a defined set of 44 heptamer primers. The amplification protocol allows for efficient amplification from as low as 50pg of mRNA and did not alter the expression of the transcripts even with 200 fold dilution of the minimum requirement of the starting material (10ng of mRNA) for standard RNA-seq protocols. We implemented our methodology on embryological lineage segregation, achieved by graded activation of Activin A/TGFβ signaling in mouse embryonic stem cells (mESCs). The fold changes in transcript expression were in excellent agreement with quantitative RT-PCR and we observed a dynamic range spanning more than five orders of magnitude in RNA concentration with a reliable estimation of low abundant transcripts. Our transcriptome data identified key lineage markers, while the high sensitivity showed that novel lineage specific transcripts anticipate the differentiation of specific cell types. We compared our strategy with Std. RNA-seq (Mortazavi et al. 2008) and SMART-seq (Ramsköld et al. 2012). We also showed potential of our methodology to suppress the representation of highly expressing ribosomal transcripts.
2013-04-29 | GSE45474 | GEO
Project description:We describe a novel quantitative cDNA expression profiling strategy, involving amplification of the majority of mouse transcriptome using a defined set of 44 heptamer primers. The amplification protocol allows for efficient amplification from as low as 50pg of mRNA and did not alter the expression of the transcripts even with 200 fold dilution of the minimum requirement of the starting material (10ng of mRNA) for standard RNA-seq protocols. We implemented our methodology on embryological lineage segregation, achieved by graded activation of Activin A/TGFβ signaling in mouse embryonic stem cells (mESCs). The fold changes in transcript expression were in excellent agreement with quantitative RT-PCR and we observed a dynamic range spanning more than five orders of magnitude in RNA concentration with a reliable estimation of low abundant transcripts. Our transcriptome data identified key lineage markers, while the high sensitivity showed that novel lineage specific transcripts anticipate the differentiation of specific cell types. We compared our strategy with Std. RNA-seq (Mortazavi et al. 2008) and SMART-seq (Ramsköld et al. 2012). We also showed potential of our methodology to suppress the representation of highly expressing ribosomal transcripts. Sequencing was performed on day 4 differentiating mouse ESCs treated for two days with 3 different dosages of Activin A (3ng/mL, 15ng/mL and 100ng/mL). The cells were also treated with SB-431542. Serial dilutions of mRNA derived Activin A(3ng/mL) samples were used to detemine the minimum amount of mRNA required to construct relaible sequencing library. SMARTseq libraries were prepared for both Activin A(3ng/mL) and Activin A(100ng/mL) samples. Three Different primer sets were designed to suppress the representaiton of Ribosomal transcripts.
2013-04-29 | E-GEOD-45474 | biostudies-arrayexpress
Project description:Microbiome PCR primer model is a Named Entity Recognition (NER) model that identifies and annotates microbiome target gene primers in texts. This is the final model version used to annotate metagenomics publications in Europe PMC and enrich metagenomics studies in MGnify with primer metadata from literature. For more information, please refer to the following blogs: http://blog.europepmc.org/2020/11/europe-pmc-publications-metagenomics-annotations.html https://www.ebi.ac.uk/about/news/service-news/enriched-metadata-fields-mgnify-based-text-mining-associated-publications
2022-02-21 | MODEL2202170011 | BioModels
Project description:Evaluation of rRNA primers for metabarcoding of eukaryotes
| PRJEB47297 | ENA
Project description:A method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level.
2009-08-31 | GSE17221 | GEO
Project description:For analysis of mRNA expression levels, total RNA was harvested from each cell-line in replicate with Trizol™ (Thermo scientific). Total RNA was purified using Direct-zol™ columns according to the manufacturers specifications (Zymo Research). For cDNA synthesis 1 μg of total RNA was process as the T12VN-PAT assay (Jänicke et al., RNA 2012), except that this was adapted for multiplexing on the Illumina MiSeq instrument. We refer to this assay as mPAT for multiplexed PAT. The approach is based on a nested-PCR that sequentially incorporates the Illumina platform’s flow-cell specific terminal extensions onto 3’ RACE PCR amplicons. First, cDNA was generated using the anchor primer mPAT Reverse, next this primer and a pool of 50 gene-specific primers were used in 5 cycles of amplification. Each gene-specific primer had a universal 5’ extension (see supplementary file primers) for sequential addition of the 5’ (P5) Illumina elements. These amplicons were then purified using NucleoSpin columns (Macherey-Nagel), and entered into second round amplification using the universal Illumina Rd1 sequencing Primer and TruSeq indexed reverse primers from Illumina. Second round amplification was for 14 cycles. Note, that each experimental condition was amplified separately in the first round with identical primers. In the second round, a different indexing primer was used for each experimental condition. All PCR reactions were pooled and run using the MiSeq Reagent Kit v2 with 300 cycles (i.e. 300 bases of sequencing) according to the manufacturers specifications. Data were analysed using established bioinformatics pipelines (Harrison et al., RNA 2015)
2016-09-08 | GSE84913 | GEO
Project description:The studies were performed using Arabidopsis thaliana: wt Col (N6000) and SALK_116579 (geralt, GERMINATION ALBINO TRANSIENT) mutant. At2g47590 gene encodes GERALT protein. This protein was not characterized so far. It is localized in chloroplasts, influences chloroplast biogenesis and chlorophyll biosynthesis and its role decreases with the plant age.
2025-09-18 | GSE271033 | GEO
Project description:Long interspersed nuclear element-1 (LINE1) retrotransposons are classified into different subfamilies based on evolutionary history. For human biology, the L1PA lineage of LINE1s is particularly significant. This lineage contains both retrotransposition-competent and inactive subfamilies. PCR-based methods are widely used to monitor LINE1s in genomic DNA. However, PCR analysis distinguishing between L1PA lineage subfamilies of different evolutionary age is thus far challenging due to the difficulty to design primers that are sufficiently specific. Here, we developed and applied a workflow to design PCR primers that discriminate between different subfamilies of the L1PA lineage. Amplicon sequencing after PCR amplification of genomic DNA confirmed that the primers differentiate between groups of L1PA subfamilies of different evolutionary age. We validated the primers using RT-qPCR and verified consistency with publicly available RNA-seq data. Moreover, inhibition of DNA methyltransferase activity produced a dose-dependent increase in signals from primers selective for the younger subfamilies, consistent with transcriptional de-repression. These primers enable PCR-based surveys that can stratify expression, copy number or epigenetic modification for L1PA subfamilies of differing evolutionary age.
2025-07-09 | GSE301759 | GEO
Project description:Chloroplasts were purified by centrifugation in Percoll gradient from low temperature (4 C), control (22 C) and high temperature (30 C) grown Malus x domestica microshoots. The proteins were digested using filter aided sample processing (FASP) and analyzed using nano-LC MS/MS. Constant scan rate of 2 Hz and range from 100 to 2,200 m/z was used for the MS spectra acquisition. Data-dependent auto-MS/MS method (Compass 1.5 acquisition and processing software, Bruker Daltonics) was used for selecting the 10 most abundant precursor ions for fragmentation. The MS/MS summation time was adjusted based on precursor intensity within the range 1-10 Hz. Active exclusion of already analyzed precursors was used with a release time of 30 s. Precursor ion isolation window and the CID collision energy were adjusted between 3-9 Th and 34-55 eV as a function of the m/z value, respectively. The data files (9 low temperature samples, 12 control samples and 9 high temperature sampes) are described in the "metadata.txt" file.
2019-10-03 | PXD014212 | Pride