7. HEK-293T cell line for lentiviral production.
8. 1Â Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM
KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 (without Ca
2+ and
Mg
2+
).
9. Trypsin (0.25%)/EDTA (1 mM).
10. Transfection reagent: JetPRIME
® (Polyplus transfection).
11. Syringes for filtering (5 mL).
12. Sterile falcon tube (15 mL).
13. 0.45 μm Cellulose acetate or polyethersulfone filters.
14. Lenti-X™ Concentrator (Clontech).
15. Polybrene.
16. 10% Bleach.
17. Live-cell imaging medium (showing lower background fluorescence) supplemented with 10% FBS.
18. 32% (w/v) Paraformaldehyde (PFA): Store at room temperature, protected from light.
19. Vectashield mounting medium with DAPI.
20. Glass microscopy slides.
21. Noncoated 22 Â 22 mm coverslips.
22. 25 mm diameter non-coated coverslips (0.17 mm thick).
23. Epifluorescent microscope of choice for acquisition of still
pictures (see Note 5).
24. Fluorescent microscope of choice for live-cell image acquisition
(see Note 6).
25. smFISH analysis software (FISH-quant [82], see above) Fiji
image-processing software (freely available at https://fiji.sc/).
3 Methods
3.1 Visualizing
MBSV6-Labeled
mRNAs in Yeast
3.1.1 Yeast
Transformation
1. Grow yeast expressing the mRNA of interest tagged with
24xMBSV6 in 5 mL of YPD at 26
C until an OD 600 of
0.6–0.8 is reached. Do not use cells grown over OD 600 >1.
The transformation efficiency will be significantly reduced. The
method to tag the mRNA with the MS2 system and to verify
that the expression of the mRNA is not affected is described in
more details in [43, 58] (see Note 7).
2. Centrifuge the cells for 3 min at 7000 Â g. Discard the supernatant and resuspend the cells in 5 mL of lithium-TE.
3. Centrifuge for 3 min at 1000 Â g and resuspend in 150 μL of
lithium-TE.
4. Put 450 μL of lithium-TE-PEG in an Eppendorf tube.
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Xavier Pichon et al.
8. 1Â Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM
KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 (without Ca
2+ and
Mg
2+
).
9. Trypsin (0.25%)/EDTA (1 mM).
10. Transfection reagent: JetPRIME
® (Polyplus transfection).
11. Syringes for filtering (5 mL).
12. Sterile falcon tube (15 mL).
13. 0.45 μm Cellulose acetate or polyethersulfone filters.
14. Lenti-X™ Concentrator (Clontech).
15. Polybrene.
16. 10% Bleach.
17. Live-cell imaging medium (showing lower background fluorescence) supplemented with 10% FBS.
18. 32% (w/v) Paraformaldehyde (PFA): Store at room temperature, protected from light.
19. Vectashield mounting medium with DAPI.
20. Glass microscopy slides.
21. Noncoated 22 Â 22 mm coverslips.
22. 25 mm diameter non-coated coverslips (0.17 mm thick).
23. Epifluorescent microscope of choice for acquisition of still
pictures (see Note 5).
24. Fluorescent microscope of choice for live-cell image acquisition
(see Note 6).
25. smFISH analysis software (FISH-quant [82], see above) Fiji
image-processing software (freely available at https://fiji.sc/).
3 Methods
3.1 Visualizing
MBSV6-Labeled
mRNAs in Yeast
3.1.1 Yeast
Transformation
1. Grow yeast expressing the mRNA of interest tagged with
24xMBSV6 in 5 mL of YPD at 26
C until an OD 600 of
0.6–0.8 is reached. Do not use cells grown over OD 600 >1.
The transformation efficiency will be significantly reduced. The
method to tag the mRNA with the MS2 system and to verify
that the expression of the mRNA is not affected is described in
more details in [43, 58] (see Note 7).
2. Centrifuge the cells for 3 min at 7000 Â g. Discard the supernatant and resuspend the cells in 5 mL of lithium-TE.
3. Centrifuge for 3 min at 1000 Â g and resuspend in 150 μL of
lithium-TE.
4. Put 450 μL of lithium-TE-PEG in an Eppendorf tube.
132
Xavier Pichon et al.