Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Library construction and sequencing

CL Changqing Liu
XS Xiaoli Shi
FW Fan Wu
MR Mingdong Ren
GG Guang Gao
QW Qinglong Wu
request Request a Protocol
ask Ask a question
Favorite

After the sample was qualitatively analyzed, the genomic DNA was sheared by using g-TUBEs (Covaris, USA) according to the expected sizes of the fragments for the library. The fragmented DNA of the target size was enriched and purified by using MegBeads. Next, the fragmented DNA was repaired for damage and then end-repaired. The stem-loop adaptor was linked on both ends of each DNA fragment, and the link-failed fragments were removed by exonuclease. Then, the target fragments were screened by BluePippin (Sage Science, USA) and purified to construct the library. Finally, an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to determine the sizes of the library fragments.

After the library was constructed, DNA templates and enzyme complexes of a certain concentration and volume were transferred to the ZMWs of the Sequel system (Pacific Biosciences, USA) for real-time single-molecule sequencing.

Copyright and License information: The Author(s) ©2020
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A