Aug 21, 2020
1: User-friendly protocol: Probe set design (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
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. 1: User-friendly protocol: Probe set design (SABER-FISH). protocols.io https://protocols.io/view/1-user-friendly-protocol-probe-set-design-saber-fi-bh9fj93n
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: 38919
Keywords: probe design, mRNA, detection, mouse ,
Abstract
This protocol offers step by step instructions for probe design for given organism (standard mRNA detection).
Note
Attachments
Guidelines
For full documentation on the application of Oligominer, check the initial publication by Brian Beliveau and the Yin Lab33 and Github: https://github.com/brianbeliveau/OligoMiner.
Genome-wide probe sets have already been generated for a variety of stringency levels. The probe set files with medium probes end in ‘b’, for instance the ‘mm10 chr16b.bed’ file. B stands for ‘balanced.’ Probe sets by chromosome for a variety of organisms are available on The Wu lab Oligopaints website: https://oligopaints.hms.harvard.edu/.
We also recommend installing:
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Note
Genome-wide probe sets have already been generated for a variety of stringency levels. The probe set files with medium probes end in ‘b’, for instance the ‘mm10 chr16b.bed’ file. B stands for ‘balanced.’ Probe sets by chromosome for a variety of organisms are available on The Wu lab Oligopaints website: https://oligopaints.hms.harvard.edu/.
Furthermore, a new online tool for automated FISH probe design is available at http://paintshop.io/ (https://www.biorxiv.org/content/10.1101/2020.07.05.188797v1 ).
Retrieve BED file for gene or gene region of interset from UCSC genome browser:50
Select appropriate genome (e.g. mm10 for mouse).
Bring gene (or region) of interest into field of view.
tools → table browser.
Fill in as follows:
- Group: genes and gene predictions
- Track: e.g. NCBI RefSeq
- Table: e.g. RefSeq
- Region: Position
- Output format: BED
Get output → exons (for exon FISH).
Check file.
If there are multiple isoforms (e.g. different names that start with NM), then manually delete all but the desired one.
Check the strand orientation. If (-), then no need for extra steps. If (+), then you must get the reverse complement after probe design.
Example UNIX commands for identifying overlapping probes:
- Ensure Biopython and BEDTools are installed and loaded (see above), or transfer BED file and probeset files to a cluster/machine that has these set up
- A symbolic link to the chromosome probe set file can be placed in the working directory using: ln -s ../oligopaints/mm10_chrNb.bed
- For the gene of interest (GOI) bed file, run intersectbed (bedtools): intersectBed -a mm10 chrNb.bed -b GOI.bed -f 1 > GOI_probes.bed
- If the gene is the (+) strand, take the reverse complement: python ../bin/probeRC.py -f GOI_probes.bed -o GOI_probesRC
Primer sequences are appended to the 3’ end of the primers with a linker of TTT, e.g.:
(probe sequence)TTT(9-mer primer sequence)
Note
The optimal number of required probes for each mRNA target varies based on considerations such as which fluor will be used for detection and whether branching will be employed as well as transcript length and sequence (e.g. homology to the genome). In tissues, we successfully detected RNAs across a variety of fluors using anywhere from 24 to 50 probes. We recommend starting with this range of probe set sizes.