DNA-seq of ELMSeq reporter cassette
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB23364 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2192260.fastq.gz | Fastqsanger.gz | |||
| ERR2192251.fastq.gz | Fastqsanger.gz | |||
| ERR2192261.fastq.gz | Fastqsanger.gz | |||
| ERR2192252.fastq.gz | Fastqsanger.gz | |||
| ERR2192262.fastq.gz | Fastqsanger.gz |
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Project description:A ELMSeq reporter cassette was created to monitor Dam levels by methylation, and introduced in the genome. Some regions upstream dam were randomized to see their effect on gene expression. So the cassette consists in: 4xGATC sites - (promoter) - random region - dam. The amplicon was spanning the whole cassette, including some bases inside the dam. The cassette was introduced in Mycoplasma pneumoniae.
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Project description:A ELMSeq reporter cassette was created to monitor Dam levels by methylation, and introduced in the genome. The regions of 6 nt upstream and 6 nt downstream the stop codon were randomized to study their effect on gene expression. The ELMSeq reporter cassette was composed of: promoter - dam - random N6 - stop codon TAA - random N6 - spacer - 4xGATC. The amplicon was spanning the C-terminal region. The cassette was introduced in Mycoplasma pneumoniae.
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Project description:DNA-seq of an ELMSeq reporter cassette to study impact of C-terminal sequence variations on protein abundance
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Project description:Quantitative analysis of the sequence determinants of transcription and translation regulation is of special relevance for systems and synthetic biology applications. Here, we developed a novel generic approach for the fast and efficient analysis of these determinants in vivo. ELM-seq (expression level monitoring by DNA methylation) uses Dam coupled to high-throughput sequencing) as a reporter that can be detected by DNA-seq. We used the genome-reduced bacterium Mycoplasma pneumoniae to show that it is a quantitative reporter. We showed that the methylase activity correlates with protein expression, does not affect cell viability, and has a large dynamic range (~10,000-fold). We applied ELM-seq to randomized libraries of promoters or 5’ untranslated regions. We found that transcription is greatly influenced by the bases around the +1 of the transcript and the Pribnow box, and we also identified several epistatic interactions (including the +1 and the “extended Pribnow”). Regarding translation initiation, we confirmed that the Shine-Dalgarno motif is not relevant, but instead, that RNA secondary structure is the main governing factor. With this in hand, we developed a predictor to help tailor gene expression in M. pneumoniae. The simple ELM-seq methodology will allow identifying and optimizing key sequence determinants for promoter strength and translation. The ELM-seq methodology allows both researchers and companies to identify and optimize in an easy and comprehensive manner, key sequence determinants for promoter strength and translation.
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Project description:In order to characterize precisely the T-DNA insertion(s) in three reporter lines, we performed whole genome sequencing and searched for insertion sites and T-DNA copy numbers.
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Project description:Integron gene cassette metagenome
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Project description:We report the generation and analysis of high-throughput DNA methylation profiles at nucleotide resolution in a subset of targeted gene trap mouse mutants. Using high-throughput sequencing of bisulfite treated DNA, we generated DNA methylation percentage for CpG islands, and LacZ (reporter) gene in mice with the apparent silencing of the targeted gene promoter reflected by reduced reporter mRNA level. These results were contrasted with findings for a set of mutants with no silencing or CpG methylation following targeted mutagenesis using the same gene trap vector. Our findings supports the hypothesis that presence of the exogenous DNA in the targeting vector may influence the expression of genes in close proximity or may lead to promoter silencing of the target where the promoter is marked by CpG methylation. Examination of CpG methylation profiles in Knock-out and wild type mice We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. CpG Islands (samples labeled as CpG) and trans gene (samples labeled as LacZ) were amplified after Bisulfite treatment. Please note that the same gDNA was used to amplify CpG Island (Gene_CpG_KO ) and LacZ (Gene_LacZ_KO) reporter for the same gene. PCR product of amplification was gel separated, isolated and pooled. After libraries were prepared and sequenced, the alignment was performed. CpG island and LacZ alignments were done separately resulting in three different Processed Data files per gene investigated: Gene_CpG_KO, Gene_LacZ_KO and Gene_CpG_WT. LacZ reference is included in the submission, but is also available for download from KOMP Phenotype website (www.kompphenotype.org). Also please note that the libraries were prepared using Illumina TruSeq RNA Sample Prep Kit starting from adapter binding step as samples were double stranded Bisulfite treated DNA amplicons. So the library preparation was done as with RNASeq, but samples investigated were bisulfite treated.
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Project description:Purpose: The goal of this study is to compare the transcriptome of pancreatic cancer stem cells (Musashi2-reporter positive) to pancreatic cancer non-stem cells (Musashi2-reporter negative). Methods: Musashi2-reporter mice contain a GFP-cassette knocked into the first exon of the endogenous Musashi2 locus; heterozygous mice are used to track cells expressing Musashi2. These mice were crossed to the KPf/fC (Ptf1a-Cre; KrasG12D; p53f/f) mouse model of pancreatic cancer. Tumors from endstage Musashi2-reporter KPf/fC mice were dissociated and FACS sorted for EpCAM+/GFP+ (Musashi2+ stem cells) and EpCAM+/GFP- (Musashi2- non-stem cells). For sequencing, mRNA libraries were generated from total RNA and sequenced with 50 basepair (bp) single end reads (SR50) to a depth of approximately 30 million reads per sample on an Illumina HiSeq2500 using V4 sequencing chemistry. Conclusions: Our study provides comparative transcriptomic data from primary isolated pancreatic cancer stem and non-stem cells in biologic replicates.
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Project description:We designed 4 oligonucleotide libraries containing either a retained intron, a cassette exon, tandem 5' or tandem 3' splice sites, cloned them into dedicated reporter constructs, transfected and integrated these constructs in the genome of K562 cells, and performed targeted RNA sequencing to determine RNA splicing ratios and a FACSseq approach to determine protein isoform ratios.
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