The improvewment of DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing.
10.12122/j.issn.1673-4254.2019.06.10
- Author:
Anghui PENG
1
;
Zhaoqiang LI
1
;
Yan ZHANG
1
;
Delong FENG
1
;
Bingtao HAO
1
Author Information
1. Cancer Research Institute, Southern Medical University, Guangzhou 510515, China.
- Publication Type:Journal Article
- Keywords:
DNA library construction;
Tn5 transposase library construction;
chromatin immunoprecipitation;
histone modification;
next-generation sequencing
- MeSH:
Chromatin Immunoprecipitation;
DNA;
Gene Library;
High-Throughput Nucleotide Sequencing;
Humans;
Reproducibility of Results;
Sequence Analysis, DNA
- From:
Journal of Southern Medical University
2019;39(6):692-698
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data.
METHODS:Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis.
RESULTS:Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%.
CONCLUSIONS:Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.
