10. Incubators: For yeast cultures (25–30
C); for smFISH hybridization ad washes (37
C).
11. Yeast strain: S. cerevisiae BY4741 (MATa; his3Δ1; leu2Δ0;
met15Δ0; ura3Δ0).
12. Synthetic complete medium (SC medium): 6.7 g/L Yeast
nitrogen base (YNB) with ammonium sulfate, 2 g/L SC complete mix (e.g., Sunrise Science Products), 20 g/L D-(+) glucose >99.5%. Sterilize in the autoclave.
13. 32% (w/v) Paraformaldehyde. Store at room temperature
(RT) and protect from light.
14. 3 M D-sorbitol: Filter sterilize and store at 4
C.
15. Buffer B: 1.2 M Sorbitol, 100 mM potassium phosphate buffer
[pH 7.5]. Store at 4
C.
16. 200 mM Vanadyl ribonucleoside complex (VRC) stock solution: Dissolve the powder in the vial at 65
C for 10 min and
store aliquots at À20
C.
17. Lyticase solution: Resuspend 25 K units in 1 mL 50% glycerol
and 1Â PBS. Store aliquots at À20
C.
18. Spheroplast buffer: 1.2 M Sorbitol, 100 mM KHPO4
[pH 7.5], 20 mM VRC, 20 mM β-mercaptoethanol, Lyticase
(25 U per OD of cells).
19. TE buffer: 10 mM Tris-Cl [pH 8], 1 mM ethylenediaminetetraacetic acid (EDTA) [pH 8].
20. smFISH probe mix:
21. ASH1-Q670 probe set: smFISH probes mixed in an equimolar
ratio and hybridizing to different positions along the ASH1
mRNA (see Note 1) (cagcagataatgcatgcagt, gctatttgcatgagaatccg, atcggttgtgatattgttcc, aatacttcctaggacttgct, aattggcgacacattgagcg, ccaagtatttcgttagccaa, gctggtgcagtatttgattt, gtaatccatatgatgtggcg, aattctggtgaattgcctgg, atctgagataagcttgcctt,
gacccatagttcaatggatt, acaatggtagtcgaggttgt, aagagacggacgatagcctg, agtgatggtaggctttgttg, gggtaataattgcagatgcc, gtgctgcgttttcttgtaaa, acgttttgatgtatcaggga, tgggtaagatcagttttcca,
gtcttgatagtttgtatcct, ccacttcttcgcgtatttta, gttgggtatacttaatggct,
caattccttgccgtaattga, aacttggacgacctagtcga, atgattccttagacggggaa, gaggagtaatactccatgca, tattggttggtggactcatc, aaaaagaggcctctactcct, atcgttattgctggatttcc, ttccacgtaattgtctttga, gcatttggcatgggaaatga, actgttcgtctttttgtgac, gtctctattcgcaagcaatt, tgttcttagatggtttcctt, gtcttgcagtgttaccgaaa, ggtgaggatgatcttgatct,
gataattgggtgaccttggg, gggagagtcgagagcaaatc, atttgatgatcttcgagggc, tacttccctttttcgttatg, tgtgacgagtgggtgatgag, gggcgaatcactcgaatgac,
gttacatagctgatcttgct,
aataagcaacggtacccttc,
ctctactgtctcagttatgt, cgcggcgtgtcgaatgaaaa). Resuspend the
54
Evelina Tutucci and Robert H. Singer
C); for smFISH hybridization ad washes (37
C).
11. Yeast strain: S. cerevisiae BY4741 (MATa; his3Δ1; leu2Δ0;
met15Δ0; ura3Δ0).
12. Synthetic complete medium (SC medium): 6.7 g/L Yeast
nitrogen base (YNB) with ammonium sulfate, 2 g/L SC complete mix (e.g., Sunrise Science Products), 20 g/L D-(+) glucose >99.5%. Sterilize in the autoclave.
13. 32% (w/v) Paraformaldehyde. Store at room temperature
(RT) and protect from light.
14. 3 M D-sorbitol: Filter sterilize and store at 4
C.
15. Buffer B: 1.2 M Sorbitol, 100 mM potassium phosphate buffer
[pH 7.5]. Store at 4
C.
16. 200 mM Vanadyl ribonucleoside complex (VRC) stock solution: Dissolve the powder in the vial at 65
C for 10 min and
store aliquots at À20
C.
17. Lyticase solution: Resuspend 25 K units in 1 mL 50% glycerol
and 1Â PBS. Store aliquots at À20
C.
18. Spheroplast buffer: 1.2 M Sorbitol, 100 mM KHPO4
[pH 7.5], 20 mM VRC, 20 mM β-mercaptoethanol, Lyticase
(25 U per OD of cells).
19. TE buffer: 10 mM Tris-Cl [pH 8], 1 mM ethylenediaminetetraacetic acid (EDTA) [pH 8].
20. smFISH probe mix:
21. ASH1-Q670 probe set: smFISH probes mixed in an equimolar
ratio and hybridizing to different positions along the ASH1
mRNA (see Note 1) (cagcagataatgcatgcagt, gctatttgcatgagaatccg, atcggttgtgatattgttcc, aatacttcctaggacttgct, aattggcgacacattgagcg, ccaagtatttcgttagccaa, gctggtgcagtatttgattt, gtaatccatatgatgtggcg, aattctggtgaattgcctgg, atctgagataagcttgcctt,
gacccatagttcaatggatt, acaatggtagtcgaggttgt, aagagacggacgatagcctg, agtgatggtaggctttgttg, gggtaataattgcagatgcc, gtgctgcgttttcttgtaaa, acgttttgatgtatcaggga, tgggtaagatcagttttcca,
gtcttgatagtttgtatcct, ccacttcttcgcgtatttta, gttgggtatacttaatggct,
caattccttgccgtaattga, aacttggacgacctagtcga, atgattccttagacggggaa, gaggagtaatactccatgca, tattggttggtggactcatc, aaaaagaggcctctactcct, atcgttattgctggatttcc, ttccacgtaattgtctttga, gcatttggcatgggaaatga, actgttcgtctttttgtgac, gtctctattcgcaagcaatt, tgttcttagatggtttcctt, gtcttgcagtgttaccgaaa, ggtgaggatgatcttgatct,
gataattgggtgaccttggg, gggagagtcgagagcaaatc, atttgatgatcttcgagggc, tacttccctttttcgttatg, tgtgacgagtgggtgatgag, gggcgaatcactcgaatgac,
gttacatagctgatcttgct,
aataagcaacggtacccttc,
ctctactgtctcagttatgt, cgcggcgtgtcgaatgaaaa). Resuspend the
54
Evelina Tutucci and Robert H. Singer