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A simple but robust approach for isolating chromatin associated RNAs in Arabidopsis thaliana

ABSTRACT: A simple but robust approach for isolating chromatin associated RNAs in Arabidopsis thaliana

PROVIDER: PRJNA881599 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR21616068_1.fastq.gz	Fastqsanger.gz
	SRR21616068_2.fastq.gz	Fastqsanger.gz
	SRR21616069_1.fastq.gz	Fastqsanger.gz
	SRR21616069_2.fastq.gz	Fastqsanger.gz
	SRR21616070_1.fastq.gz	Fastqsanger.gz

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A simple but robust approach for isolating chromatin associated RNAs in Arabidopsis thaliana

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A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis.

Project description:BackgroundChromatin-bound RNAs are the primary product of transcription that undergo on-chromatin processing such as capping, splicing, and polyadenylation. These processing steps then determine the fate of RNAs. Albeit its vital importance, a simple and robust method for isolating different fractions of chromatin-bound RNAs is missing in plants.ResultHere, we describe our updated method and the associated step-by-step protocol for chromatin-bound RNAs isolation in A. thaliana. The chromatin-bound RNAs isolation is based on the 1 M UREA wash that removes the majority of non-chromatin-associated proteins from the nucleus, as previously developed in mammalian cells. On-demand, the isolated chromatin-bound RNAs can be either used directly for gene-specific analysis or subject to further rRNA removal and also the optional polyadenylated RNA removal, followed by high-throughput sequencing. Detailed protocols for these procedures are also provided. Comparison of sequencing results of chromatin-bound RNAs with and without polyadenylated RNA removal revealed that a small fraction of CB-RNAs is polyadenylated but not yet fully spliced, representing RNA-processing intermediate on-chromatin.ConclusionThis optimized chromatin-bound RNAs purification method is simple and robust and can be used to study transcription and its-coupled RNA processing in plants.

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Project description:Genomes of members of the family Enterobacteriaceae contain large repertoires of putative fimbrial operons. Since many of these operons are poorly expressed in vitro, a convenient method for inducing elaboration of the encoded fimbriae would greatly facilitate their functional characterization. Here we describe a new technique for identifying fimbriated bacteria from a library of transposon mutants by screening with immunomagnetic particles for ligand expression (SIMPLE). The SIMPLE method was applied to identify the T-POP mutants of Salmonella enterica serotype Typhimurium carrying on their surfaces filaments composed of PefA, the major subunit product of a fimbrial operon (pef) that is not expressed during growth in Luria-Bertani broth. Four such mutants were identified from a library of 24,000 mutants, each of which carried a T-POP insertion within the hns gene, which encodes a global silencer of horizontally acquired genes. Our data suggest that the SIMPLE method is an effective approach for isolating fimbriated bacteria, which can be readily applied to fimbrial operons identified by whole-genome sequencing.

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