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Analysis to evaluate the presence of 5’ cap in intragenic lncRNAs

ABSTRACT: RNA aliquots were digested with 5’-phosphate-dependent exonuclease (5'-exo) following pre-treatment with tobacco acid pyrophosphatase (TAP) to release the 5’ cap and render the RNA susceptible to 5’ exonuclease digestion (TAP+/5'-exo+). Two color array hybridizations were performed and TAP+/5'-exo+ samples were compared with TAP-/5'-exo+ control samples.

ORGANISM(S): Homo sapiens

PROVIDER: GSE62984 | GEO | 2015/06/30

SECONDARY ACCESSION(S): PRJNA266342

REPOSITORIES: GEO

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Analysis to evaluate the presence of 5’ cap in intragenic lncRNAs

Project description:RNA aliquots were digested with 5’-phosphate-dependent exonuclease (5'-exo) following pre-treatment with tobacco acid pyrophosphatase (TAP) to release the 5’ cap and render the RNA susceptible to 5’ exonuclease digestion (TAP+/5'-exo+). Two color array hybridizations were performed and TAP+/5'-exo+ samples were compared with TAP-/5'-exo+ control samples. Two-condition experiment, TAP+/5'-exo+ vs. TAP-/5'-exo+ treated total RNA samples. Biological replicates: 4 with dye swap

2015-06-30 | E-GEOD-62984 | biostudies-arrayexpress

Nucleotide-resolution, genome-wide, 5'-end maps of the transcriptome of Propionibacterium acnes (cultured with and without potassium downshift)

Project description:The differential RNA-seq data contained within this entry is complemented by global RNA-seq and microarray data, which is also deposited in GEO. Duplicate cultures of Propionibacterium acnes strain KPA171202 were grown exponentially in batch culture under anaerobic growth. Samples were taken following subculture with and without potassium downshift (i.e. removal from medium). This produced 4 samples; 2 replicates x 2 conditions. Aliquots of each of the 4 samples were then incubated with or without incubation TAP (tobacco acid pyrophosphatase) before library construction. Thus, 8 libraries were analysed. TAP treatment allows the cloning and sequencing of 5' ends that were originally triphosphorylated.

2013-05-10 | E-GEOD-46810 | biostudies-arrayexpress

Comparison of alternative decapping enzymes to map transcription start sites genome-wide

Project description:The production of Tobacco Acid Pyrophosphatase (TAP), an enzyme commonly used for the removal of the 5â??cap of eukaryotic mRNAs, has been recently discontinued. Here we performed a comparison of current alternatives for the mapping of 5â??cap mRNAs and the associated transcription start sites in Sacharomyces cerevisiae. Specifically we compared TAP with Cap-clip and a Decapping Pyrophosphohydrolase. Our results suggest that Cap-clip is a good alternative for TAP. We used two biological replicates of S. cerevisiae that was grown to exponential phase (OD600 ~1) in rich media (YPAD). Samples where processed until the dephosphorylation step (CIP treatment). After that each sample was split in 4 aliquots: TAP treatment, Cap-Clip treatment, Decapping Pyrophosphohydrolase treatment or no treatment (negative control). From that step all samples are processed in parallel.

2016-01-06 | E-GEOD-73819 | biostudies-arrayexpress

Comparison of alternative decapping enzymes to map transcription start sites genome-wide

Project description:The production of Tobacco Acid Pyrophosphatase (TAP), an enzyme commonly used for the removal of the 5’cap of eukaryotic mRNAs, has been recently discontinued. Here we performed a comparison of current alternatives for the mapping of 5’cap mRNAs and the associated transcription start sites in Sacharomyces cerevisiae. Specifically we compared TAP with Cap-clip and a Decapping Pyrophosphohydrolase. Our results suggest that Cap-clip is a good alternative for TAP.

2016-01-06 | GSE73819 | GEO

RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

Project description:RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation.

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Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing

Project description:Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

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Transcription start site analysis of Streptococcus agalactiae strain BM110

Project description:Streptococcus agalactiae, also known as Group B streptococcus, emerged in the 1960s as a leading cause of septicemia and meningitis in neonates. It is also an increasing cause of infections in adults with underlying diseases. To characterize transcription start sites (TSS) in the hypervirulent ST17 lineage (strain BM110) we used a differential RNA-seq strategy, based on selective Tobacco Acid Pyrophosphatase (TAP) treatment and adapter ligation, which differentiates primary transcripts and processed RNAs

2021-08-23 | GSE158048 | GEO

Glucocorticoid receptor profiling in IMR90, K562 and U2OS (ChIP-exo)

Project description:To identify the sequences responsible for recruitment of Glucocorticoid receptor (GR) to individual loci, we performed ChIP-seq and ChIP-exo that combines chromatin immunoprecipitation with an exonuclease digestion step. We performed these experiments in three cell lines : IMR90 (ATTC:CCL-186), U2OS osteosarcoma cell lines, K562 (ATCC:CCL243), upon glucocorticoid treatment.

2015-02-25 | E-MTAB-2956 | biostudies-arrayexpress

Comparison of the nucleotide-resolution, genome-wide, 5'-end maps of the transcriptomes of Escherichia coli K12 strain BW25113 and Streptomyces coelicolor A3(2) strain M145

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Gene expression is a circular process

Project description:This study involves the role of yeast mRNA decay factors in transcription. The experiment included here are the ChIP-exo results of three decay factors: Xrn1, Dcp2 & Lsm1. Four experiments were made: Xrn1, Dcp2, Lsm1 and control (no-TAP tag), in two replicates.

2013-05-23 | E-GEOD-44312 | biostudies-arrayexpress

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