Single-molecule fluorescence in situ hybridisation (smFISH)

Thank you for requesting further detailed protocol used in our paper. 

We have attached step-by-step protocols for (1) the single-molecule fluorescence in situ hybridisation (smFISH) procedure in tissue culture cells and (2) the synthesis of smFISH probes. 

Kind regards. 

smFISH on tissue culture cells
 

Seeding adherent cells
1. Clean 12-13 mm round coverslips (#1.5) with 80% EtOH-soaked kimwipe
a. Store the coverslips in 100% EtOH
2. Place coverslips in 24-well plate and completely dry them before seeding cells
a. Alternatively, use 8-well chamber wells
3. Seed 0.5-2 million cells per well, and incubate for 24 h
 

smFISH procedure
Day 1
1. Aspirate growth medium, and wash cells 1x with PBS
2. Fix in 4% PFA (in PBS) for 30 minutes at RT
a. Fixed cells can be stored at 4˚C in PBS for a week or in 70% EtOH at 4˚C for several months
3. Rinse 1x → Wash 2x with PBS, 5 min ea.
4. Permeabilise the cells in PBSTx 0.1% for 10 minutes at RT
a. Or permeabilise in 70% EtOH overnight at 4˚C
5. Rinse 1x → Wash 2x with PBS, 5 min ea.
6. Rinse 1x → Wash 1x with 2x SSC for 5 min
7. Rinse 1x with pre-warmed wash solution
8. Pre-hybridise 2x in pre-warmed wash solution for 20 min ea. at 37˚C
9. Hybridise in hybridisation solution with FISH probes overnight at 37˚C
a. 1:50 of 25 µM COVID probes
b. 1:100 of 25 µM host RNA probes
 

Day 2
10. Rinse 1x → Wash 1x with pre-warmed wash solution, 20 min at 37˚C
11. Counterstain for 40 min at 37˚C (in wash solution)
a. DAPI (1:500 of 0.5 mg/ml stock)
b. Dye-coupled phalloidin (1:1,000) or CellMask green (1:1M)
12. Wash 1x with wash solution 20 min at 37˚C
13. Rinse 1x → Wash 2x with 2X SSC, 5 min ea
14. Mount using 7 µl hard-set Vectashield, dab off excess mounting media
a. IBIDI chamber samples can be mounted in 50-100 µl of wet-set mounting media
15. Store mounted samples at 4˚C until imaging up to 2 weeks

Materials
● Make all reagents using nuclease-free or DEPC-treated water!
● smFISH probes are synthesised according to (Gaspar et al., 2017) or bought from Biosearch technologies

References
Gaspar, I., Wippich, F., Ephrussi, A., 2017. Enzymatic production of single-molecule FISH and RNA capture probes. RNA 23, 1582–1591. https://doi.org/10.1261/rna.061184.117
Titlow, J.S., Yang, L., Parton, R.M., Palanca, A., Davis, I., 2018. Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction. Methods Mol Biol 1649, 163–175. https://doi.org/10.1007/978-1-4939-7213-5_10
Yang, L., Titlow, J., Ennis, D., Smith, C., Mitchell, J., Young, F.L., Waddell, S., Ish-Horowicz, D., Davis, I., 2017. Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains. Methods 126, 166–176. https://doi.org/10.1016/j.ymeth.2017.06.025
 

Enzymatic smFISH probe synthesis
 

Procedures
Prepare pooled oligos
1. Probe design - Use Stellaris probe designer with 3 nucleotide spacing
a. Aim for 20 - 48 oligos per set
b. 18 - 22 nt length, 3 nt spacing, avoid probes spanning exon-exon junctions
2. Order oligos from IDT with 200 µM normalised concentration in a 96-well plate format

a. IDTE buffer: 10 mM Tris pH 8.0, 0.1 mM EDTA
3. Spin down the plate and pool 10 µl of oligos from each well - Store as a pooled stock of 200 µM
 

Prepare TdT reaction mix
1. Prepare reaction mix (15 µl) in a PCR tube

2. Incubate overnight at 37˚C in a thermocycler with the hot lid on
 

Purify labelled probes
1. Follow Zymo Oligo clean & concentrator kit (D4060) protocol for purification
a. Add 100 µl oligo binding buffer directly to the reaction mix (this also stops the TdT reaction)
b. Elute in 25 µl TE buffer
2. Nanodrop measure OD260 and dye absorption maxima
a. Measure eluted probe undiluted and a 1:10 dilution of pooled unlabelled oligos
b. Calculate the degree of labelling and the probe concentration using Gasper et al. 2017 supplementary file
3. Dilute to 25 µM with TE buffer and store at -20 ˚C. Protect from light.

Materials
Make all reagents using nuclease-free or DEPC-treated water

Appendix 1: Preparing dye-ddUTP
Conjugation reaction (NHS chemistry)
1. Reconstitute Amino-11-ddUTP to 10 mM in 0.1 M NaHCO3 pH 8.3
a. 200 nmol Amino-11-ddUTP + 20 µl NaHCO3 = 10 mM conc.
b. Spin down the tube, and dissolve the small yellow aggregate at the bottom of the tube
2. Reconstitute the dye powder to 20 mM in anhydrous DMSO (use a new bottle)
3. Mix 20 µl of the reconstituted dye and dye to 20 µl of amino-ddUTP (2:1 molar ratio)
4. Incubate for 3 hrs in dark at RT with gently shaking
5. Add 1 M Tris-HCl (pH 7.4) to quench unreacted NHS ester groups (to final conc. of 10 mM)
a. Add 0.4 µl Tris-HCl
6. Aliquot and adjust concentration to 1 mM with nuclease-free water (5 mM → 1 mM)
 

Appendix 2: Note on producing a large amount of probes
● The capacity of Zymo IC columns are up to 10 µg of ssDNA
○ 15 µl reaction is approx. 6 µg for 20 nt probes
○ The reaction can be scaled up to 20 µl
● Zymo IC columns and oligo binding buffers can be bought separately
● Ethanol precipitation or SPRI beads can be considered for large-scale synthesis

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