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Efficient cellular fractionation of RNA

ABSTRACT: Efficient cellular fractionation of RNA

PROVIDER: PRJEB4845 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR358551.fastq.gz	Fastqsanger.gz
	ERR358552.fastq.gz	Fastqsanger.gz
	ERR358553.fastq.gz	Fastqsanger.gz
	ERR358554.fastq.gz	Fastqsanger.gz
	ERR358555.fastq.gz	Fastqsanger.gz

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Project description:The aim of this experiment was to develop new methods to extract pure fractions of nuclear and cytoplasmic RNA. We compared the results of nuclear and cytoplasmic RNA-sequencing to results of the total and polyA+ RNA-sequencing. Cytoplasmic, nuclear, total and polyA+ RNA was extracted from two human brain samples and sequenced on the SOLiD system. We analyzed the data to look for differences in levels of nascent transcription and mature mRNAs between the different samples.

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Project description:The response to DNA damage intersects with many other physiological processes in the cells, including initiation of DNA repair, regulation of transcription and translation, chromatin remodeling, and the cell cycle regulation to contend with the challenge. Transcription shutdown is not simply a consequence of the physical arrest of RNA polymerase II by DNA lesions, but also could be elicited by signaling in trans. While to date many of the specific molecular events are not fully elucidated. What is the signal that operates in trans to slow transcriptional elongation globally? Which factors are required for transcription restart after DNA repair? How is TC-NER controlled by the multifaceted transcriptional response? In this study, we sought to map the transcription restart and isolate the set of lncRNAs that are likely to function at the chromatin interface by using biochemical fractionation of the cellular compartment coupled to RNA-seq.

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Subcellular RNA localization during Drosophila embryonic neurogenesis [fractionation-Seq]

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Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues.

Project description:BackgroundThe starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression.ResultsWe present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics.ConclusionOur results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues.

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