Aug 21, 2020

Public workspace3: 30mer branch melting temperatures (SABER-FISH)

This protocol is a draft, published without a DOI.
  • Jocelyn Y. Kishi1,2,3,
  • Sylvain W. Lapan4,3,
  • Brian J Beliveau1,2,5,3,6,
  • Emma R. West4,3,
  • Allen Zhu1,2,
  • Hiroshi M. Sasaki1,2,
  • Sinem K Saka1,2,
  • Yu Wang1,2,
  • Constance L Cepko4,7,6,
  • Peng Yin1,2,6
  • 1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA;
  • 2Department of Systems Biology, Harvard Medical School, Boston, MA, USA;
  • 3These authors contributed equally;
  • 4Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA;
  • 5Present address: Department of Genome Sciences, University of Washington, Seattle, WA, USA;
  • 6Correspondence: py@hms.harvard.edu (P.Y.), cepko@genetics.med.harvard.edu (C.L.C.), beliveau@uw.edu (B. J. B.);
  • 7Howard Hughes Medical Institute, Chevy Chase, MD, USA
  • Human Cell Atlas Method Development Community
Jocelynkishi
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External link: http://saber.fish/
Protocol Citation: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem K Saka, Yu Wang, Constance L Cepko, Peng Yin 2020. 3: 30mer branch melting temperatures (SABER-FISH). protocols.io https://protocols.io/view/3-30mer-branch-melting-temperatures-saber-fish-bh9hj936
Manuscript citation:
Kishi, J.Y., Lapan, S.W., Beliveau, B.J. et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods 16, 533–544 (2019). https://doi.org/10.1038/s41592-019-0404-0
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: July 06, 2020
Last Modified: August 21, 2020
Protocol Integer ID: 38921
Keywords: branch melting temperature, temperature,
Abstract
This protocol describes the design of 30mer branch sequences.



Note
This protocol is part of the SABER-FISH collection.

Attachments
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Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
It's recommended to use a temperature at least 1 degree lower than the lowest melting temperature of all branch sequences you plan to use (see plot of melting temperatures Fig. S2).

You can find these melting temperature curves reported for each sequence, as well as those computed for 20mer imagers, 42mer barcode sequences, and an example set of FISH probes, reported in Download Supplementary Table 2.xlsxSupplementary Table 2.xlsx (available on the Nature Methods website).

Figure S2: Melting temperatures of branches under different formamide conditions. Melting temperatures of 30mer branch binding sequences are shown for the 50 designed PER primers in 1×PBS with different concentrations of formamide. Modeled with Biopython.69