Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends

Ohtsubo, Yoshiyuki; Sakai, Keiichiro; Nagata, Yasunobu; Tsuda, Masashi

doi:10.1038/s42003-019-0660-7

Cited by 11 publications

(15 citation statements)

References 22 publications

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“…To examine nucleosome arrays in Fr-5 chromatin particles, DNA fragments in the chromatin were extended using terminal deoxynucleotidyl transferase (TdT), and observed by HS-AFM. Briefly, Fr-5 chromatin was immunoprecipitated as described above, and treated with T4 DNA polymerase (#311–02481, Nippon Gene) and exonuclease III (#2170A, Takara Bio) to repair DNA termini as described previously ( 41 ). After the chromatin was collected by re-immunoprecipitation, biotin-16-dUTP (#11093070910, Roche) was added to the DNA termini using TdT (#M828A, Promega).…”

Section: Methodsmentioning

confidence: 99%

Local states of chromatin compaction at transcription start sites control transcription levels

Ishihara

Sasagawa

Kameda

et al. 2021

Nucleic Acids Research

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The ‘open’ and ‘compact’ regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels.

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Section: Methodsmentioning

confidence: 99%

Local states of chromatin compaction at transcription start sites control transcription levels

Ishihara

Sasagawa

Kameda

et al. 2021

Nucleic Acids Research

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“…Therefore, we‍ ‍compared five types of oligonucleotide adaptors (U2-DNA-5P3P, U2-DNA-5P3H, U2-DNA-5P3am, U2-DNA-5OH3H, and U2-RNA-5P3H) for cDNA synthesis in the FLDS library construction. Based on the chemical structures of the 3′ end of mechanically-sheared dsDNA ( Ohtsubo et al , 2019 ), oligonucleotides with phosphorylated or hydroxylated 5′-ends were prepared. To prevent ligation among the oligonucleotide adaptors, appropriate chemical modifications were prepared for the 3′-ends.…”

Section: Resultsmentioning

confidence: 99%

“…In the initial test, 1‍ ‍ng of mechanically-sheared dsRNA was used as a template, and 25 and 35 PCR cycles were used for cDNA amplification. Unexpectedly, insufficient cDNA amplification was obtained using U2-DNA-5OH3H despite the expected dominance of a phosphate group in the 3′ end of mechanically-sheared dsRNA, as in the case of dsDNA ( Ohtsubo et al , 2019 ). In the sequencing libraries constructed using other U2 adaptors, the abundance of high-quality reads (depicted as Clean in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2 ( Urayama et al , 2018 ). A recent study reported that the 3′-end of mechanically-sheared dsDNA carries a phosphate group, hydroxyl group, and unidentified structures ( Ohtsubo et al , 2019 ). This finding suggests that a reassessment of the terminal residue modifications in oligonucleotide adaptors will increase the efficiency of adaptor ligation and subsequent cDNA synthesis in FLDS if similar structures occur in mechanically-sheared dsRNA.…”

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confidence: 99%

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RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS)

et al. 2021

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Fragmented and primer ligated dsRNA sequencing (FLDS) is a sequencing method applicable to long double-stranded RNA (dsRNA) that enables the complete genome sequencing of both double- and single-stranded RNA viruses. However, the application of this method on a low amount of dsRNA has been hindered by adaptor dimer formation during cDNA amplification and sequence library preparation. We herein developed FLDS ver. 3 by optimizing the terminal modification of an oligonucleotide adaptor and the conditions of adaptor ligation. We also examined the concentration of Mg 2+ in the PCR reaction for cDNA amplification and the purification method of amplified cDNA. Fine sequence reads were successfully obtained from metagenomic shotgun sequencing libraries constructed from 10 and 100 pg dsRNA, and these libraries exhibited weaker detection sensitivity for low-abundance dsRNAs (viral genomes and genome segments) than that constructed from 1‍ ‍ng of dsRNA. We also report the utility of capillary electrophoresis for dsRNA quantification. The FLDS ver. 3 package expands the frontiers of our knowledge in RNA virus diversity and evolution.

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“…Single-strand DNA breaks can be inflicted by numerous factors, of which DNA shearing, oxidative damage and various endonucleases appear most relevant to our isolation procedure. Shearing and oxidative damage to DNA often generate ‘dirty’ DNA ends that require further processing before ligation can occur (Bertoncini and Meneghini, 1995; Caldecott, 2008; Ohtsubo et al , 2019), whereas nucleases can leave ‘clean’ DNA ends that are directly ligatable. To determine whether the ssDNA breaks observed in our samples contained ligatable ends, we treated the DNA preparations from adult skeletal muscle with T4 DNA ligase and separated ligase-treated and untreated samples on a denaturing gel (Fig.…”

Section: Resultsmentioning

confidence: 99%

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method

Repolês

Gorospe

Tran

et al. 2021

Preprint

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The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR. We show that a commonly-used DNA isolation procedure preferentially introduces strand breaks into the mtDNA extracted from the skeletal muscle of aged mice, while mtDNA from adult animals is less affected. We present a comparison of mtDNA isolation methods and identify one that avoids this biased loss of muscle mtDNA integrity. Our results highlight the importance of a careful choice of mtDNA isolation method and serve as a resource to researchers planning analysis of mtDNA isolated from solid tissues.

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Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends

Cited by 11 publications

References 22 publications

Local states of chromatin compaction at transcription start sites control transcription levels

Local states of chromatin compaction at transcription start sites control transcription levels

RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS)

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method

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