Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Reverse transcription with template switching

YL Yusheng Liu
HZ Han Zhao
FS Fanghong Shao
YZ Yiwei Zhang
HN Hu Nie
JZ Jingye Zhang
CL Cheng Li
ZH Zhenzhen Hou
ZC Zi-Jiang Chen
JW Jiaqiang Wang
BZ Bing Zhou
KW Keliang Wu
FL Falong Lu
request Request a Protocol
ask Ask a question
Favorite

Each sample was added with 0.4 µl of RT primer (100 µM, Supplementary Table 18) and 1 µl of dNTP mix (10 mM each), incubated at 72 °C for 3 minutes, and put on ice immediately, to anneal the RT primer to the 3′-end adapter of RNAs. After adding 11.6 µl of the RT mix (final concentration: 1× SuperScript II first-strand buffer, 10 U/µl of SuperScript II reverse transcriptase (Invitrogen, 18064-014), 1 U/µl of RNase inhibitor (TaKaRa, 2313A), 5 mM DTT (Invitrogen, 18064-014), 1 M Betaine (Sigma-Aldrich, 61962), 6 mM MgCl2 (Invitrogen, AM9530G), and 0.98 µM TSO (Supplementary Table 18)), the sample was incubated at 42 °C for 90 minutes; 10 cycles of 50 °C for 2 minutes and 42 °C for 2 minutes; 70 °C for 15 minutes; and held at 4 °C.

Copyright and License information: The Author(s), under exclusive licence to Springer Nature America, Inc. ©2023
Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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