TITLE: An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae
AUTHORS: John B. Ridenour, Rafal Donczew

[DESCRIPTION]
In this protocol, we describe an end-to-end workflow for rapidly degrading a target protein using the AID system and quantifying newly synthesized mRNA using SLAM-seq in Saccharomyces cerevisiae. We describe methods for targeted protein degradation, 4tU incorporation, rapid fixation, RNA purification, RNA alkylation, and 3' mRNA-seq library preparation. Although the individual methods described in this protocol are not novel per se, this workflow provides a complete resource for turnkey implementation of these methods, which will benefit others working with S. cerevisiae. In addition, we believe this workflow is readily adaptable to other systems, including industrial or pathogenic fungi, and will benefit the larger research community.

REFERENCES

Alalam H, Zepeda-Martínez JA, Sunnerhagen P (2022). Global SLAM-Seq for accurate mRNA decay determination
and identification of NMD targets. RNA, 28(6), 905-915. https://doi.org/10.1261/rna.079077.121

Aslanzadeh V, Huang Y, Sanguinetti G, Beggs JD (2018). Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast. Genome research, 28(2), 203-213. https://doi.org/10.1101/gr.225615.117

Barrass JD, Reid JE, Huang Y, Hector RD, Sanguinetti G, Beggs JD, Granneman S (2015). Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling. Genome biology, 16(1), 1-17. https://doi.org/10.1186/s13059-015-0848-1

Donczew, R, Hahn, S (2021). BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae. Elife, 10, e69619. https://doi.org/10.7554/eLife.69619

Donczew, R., Warfield, L., Pacheco, D., Erijman, A., & Hahn, S. (2020). Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA. Elife, 9, e50109. https://doi.org/10.7554/eLife.50109

Foley JW, Zhu C, Jolivet P, Zhu SX, Lu P, Meaney MJ, West RB (2019). Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ. Genome research, 29(11), 1816-1825. https://doi.org/10.1101/gr.234807.118

Glenn TC, Nilsen RA, Kieran TJ, Sanders JG, Bayona-Vásquez NJ, Finger JW, Pierson TW, Bentley KE, Hoffberg SL, Louha S, Garcia-De Leon FJ, Del Rio Portilla MA, Reed KD, Anderson JL, Meece JK, Aggrey SE, Rekaya R, Alabady M, Belanger M, Winker K, Faircloth BC (2019). Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext). PeerJ, 7. https://doi.org/10.7717/peerj.7755

Herzog VA, Reichholf B, Neumann T, Rescheneder P, Bhat P, Burkard TR, Wlotzka W, von Haeseler A, Zuber J, Ameres SL (2017). Thiol-linked alkylation of RNA to assess expression dynamics. Nature Methods, 14(12), 1198-1204. https://doi.org/10.1038/nmeth.4435

Kara N, Krueger F, Rugg-Gunn P, Houseley, J. (2021). Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq. PLOS Biology, 19(3). https://doi.org/10.1371/journal.pbio.3000886

Koš M, Tollervey D (2010). Yeast pre-rRNA processing and modification occur cotranscriptionally. Molecular Cell,
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Veeranagouda Y, Remaury A, Guillemot JC, Didier M (2019). RNA Fragmentation and Sequencing (RF‐Seq): Cost‐Effective, Time‐Efficient, and High‐Throughput 3′ mRNA Sequencing Library Construction in a Single Tube. Current Protocols in Molecular Biology, 129(1). https://doi.org/10.1002/cpmb.109

[GUIDELINES]
Standard laboratory guidelines and practices should be followed when performing this protocol. To ensure accurate size selection, researchers should use careful and consistent pipetting when performing this protocol. 

[STEPS]
SECTION: Yeast growth
1. Streak appropriate S. cerevisiae strain on YPD agar. Incubate culture at 30°C for 2-3 days.

 

SECTION: Yeast growth
2. Transfer an individual colony to 5 ml of YPD. Incubate culture at 30°C with shaking at 220 rpm overnight.
SECTION: Yeast growth
3. Dilute overnight culture to an OD600 of 0.2 in 40 ml of YPD. Incubate culture at 30°C with shaking at 220 rpm.
SECTION: Yeast growth
4. As the 40 ml culture approaches an OD600 of 0.3-0.4, be prepared to proceed with IAA treatment, 4tU labeling, sampling, and rapid fixation.
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
5. In preparation for IAA treatment, 4tU labeling, sampling, and rapid fixation, cool centrifuge to 30°C and prepare 50 ml conical tubes, 2.0 ml microcentrifuge tubes, and 1.5 ml microcentrifuge tubes.

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
6. Add 5 ml of 100% methanol to a 50 ml conical tube for each sample to be collected. Prechill prepared and labeled tubes on dry ice. Two 50 ml conical tubes are needed for each 40 ml culture prepared in Step 4.
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
7. When the 40 ml culture reaches an OD600 of 0.4-0.5, split the culture into three 10 ml aliquots in 50 ml conical tubes and return them to the incubator.

Each 10 ml culture represents one of three treatments:
 

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
8. Equilibrate cultures at 30°C for 5 min with shaking at 220 rpm.
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
9. During equilibration (or just prior to splitting the culture), prepare a 0.2 M IAA (3-indoleacetic acid) solution in DMSO.

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
10. Add 50 μl of 0.2 M IAA solution or DMSO to the appropriate 10 ml culture. Mix the culture vigorously and return it to the incubator. The final concentration of IAA is 1 mM.
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
11. Incubate cultures at 30°C for 25 min with shaking at 220 rpm.

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
12. During IAA treatment, prepare a 2 M 4tU (4-thiouracil) solution in DMSO.

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
13. Add 25 μl of 2 M 4tU solution or DMSO to the appropriate 10 ml culture. 4tU will precipitate when added to the culture; mix the culture vigorously and return it to the incubator. The final concentration of 4tU is 5 mM. It is essential that cultures are mixed vigorously and uniformly to ensure 4tU is dissolved and homogeneously distributed.

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
14. Incubate cultures at 30°C for 4 min with shaking at 220 rpm.

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
15. Immediately after 4tU treatment, decant each 10 ml culture directly into 5 ml of 100% methanol in a 50 ml conical tube prechilled on dry ice (prepared in Step 6). Loosely cap the slurry and gently mix by swirling to ensure homogenization. Keep the fixed sample on dry ice. The final concentration of methanol is approximately 33%. 

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
16. Gently mix the fixed sample and transfer 250 μl to a 1.5 ml microcentrifuge tube. Store the 250 μl aliquot at 4°C for cell counting. Estimate cell number using OD600 or count cells using a Neubauer chamber.

 
 
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
17. Gently mix the fixed sample and transfer 1.5 ml to a labeled 2.0 ml microcentrifuge tube for western blotting. Centrifuge the sample at max speed for 1 min to pellet cells, discard the supernatant, and freeze cell pellets at -20°C. 

 
 
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
18. Centrifuge the remaining slurry (approximately 13 ml) at 3,000 g and 4°C for 10 min to pellet cells.
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
19. Carefully decant supernatant and invert the 50 ml conical tube on a paper towel to remove residual supernatant
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
20. Thoroughly resuspend cells in an appropriate volume of DNA/RNA Shield by pipetting. Adjust samples to a uniform cell number using the following formula to calculate the appropriate volume of buffer for each sample: 

Volume of buffer needed = (final OD600 of sample/0.70)*400.
SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
21. Ensure cells are thoroughly resuspended and transfer the cell suspension to a labeled 1.5 ml microcentrifuge tube.
SECTION: RNA alkylation and ethanol precipitation
46. Prepare the following reaction mixture in a 1.5 ml microcentrifuge tube and mix well by pipetting:

Thiol modification reaction mixture
 

 

SECTION: 3´ mRNA sequencing library preparation
59. RNA fragmentation and cDNA synthesis.

 

SECTION: 3´ mRNA sequencing library preparation
60. RNA removal and single-stranded DNA purification.
SECTION: 3´ mRNA sequencing library preparation
61. Barcoding and enrichment PCR and library purification.
SECTION: 3´ mRNA sequencing library preparation
59.1. Thaw iodoacetamide-treated RNA samples on ice. Quantify RNA using an RNA-specific fluorometry method (i.e., Qubit RNA BR or HS assay kit).
SECTION: 3´ mRNA sequencing library preparation
59.2. Add 200 ng of RNA (total RNA) to a 0.2 ml PCR tube or strip on ice. Adjust the volume of each sample to 5 μl with nuclease-free water.
SECTION: 3´ mRNA sequencing library preparation
59.3. Prepare the following reaction mixture in a 0.2 ml PCR tube (or 1.5 ml microcentrifuge tube) on ice and mix well by pipetting:

Fragmentation reaction mixture
 

SECTION: 3´ mRNA sequencing library preparation
59.4. Prepare the following reaction mixture in a 0.2 ml PCR tube (or 1.5 ml microcentrifuge tube) on ice and mix well by pipetting:

Reverse transcription (RT) reaction mixture
 

SECTION: 3´ mRNA sequencing library preparation
59.5. Bring RNA and fragmentation reaction mixture to room temperature for 2-5 minutes and add 3 μl of the fragmentation reaction mixture to each sample. Mix well by pipetting.
SECTION: 3´ mRNA sequencing library preparation
59.6. Incubate samples at 94°C for exactly 4 min in a thermocycler with heated lid to fragment the RNA. Cool samples to 42°C in a thermocycler. Centrifuge briefly at room temperature to collect samples. Return samples to a thermocycler at 42°C. Fragmentation time can be optimized to generate libraries of different insert sizes.
SECTION: 3´ mRNA sequencing library preparation
59.7. Bring the RT reaction mixture to room temperature for 2-5 minutes and add 2 μl RT reaction mixture to each sample (8 μl fragmented, primer-annealed RNA). Mix well by pipetting. Centrifuge briefly at room temperature to collect samples. Return samples to thermocycler at 42°C.
SECTION: 3´ mRNA sequencing library preparation
59.8. Heat samples to 42°C for 60 min followed by 70°C for 10 min in a thermocycler with heated lid. Cool samples to 37°C in a thermocycler. Proceed immediately to RNA removal (Step 60).
SECTION: 3´ mRNA sequencing library preparation
60.1. Add 1 μl RNase H to each sample. Mix well by pipetting. Centrifuge briefly at room temperature to collect samples.
SECTION: 3´ mRNA sequencing library preparation
60.2. Heat samples to 37°C for 15 min in a thermocycler with heated lid. Proceed immediately to speedbead purification.
SECTION: 3´ mRNA sequencing library preparation
60.3. Purify the single-stranded DNA (ssDNA). Add 39 μl of DNA buffer and 1 volume (50 μl) of speedbeads to the 0.2 ml PCR tube containing the ssDNA and proceed as described in the general method for speedbead purification (Step 65).

 

SECTION: 3´ mRNA sequencing library preparation
60.4. Elute the ssDNA in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C. 
SECTION: 3´ mRNA sequencing library preparation
61.1. Transfer 10 μl of purified ssDNA to a 0.2 ml PCR tube on ice.
SECTION: 3´ mRNA sequencing library preparation
61.2. Add 2.5 μl of 5 μM i5/i7 primer mix to the 0.2 ml PCR tube containing 10 μl of DNA on ice.
SECTION: 3´ mRNA sequencing library preparation
61.3. Add 12.5 μl of 2X Ex Premier master mix to the 0.2 ml PCR tube containing 12.5 μl of DNA and primer mix on ice. Mix thoroughly by pipetting.
SECTION: 3´ mRNA sequencing library preparation
61.4. Barcode and enrich the library using PCR. Perform initial denaturation at 98°C for 45 s, followed by 12-18 cycles of amplification (98°C for 10 s, 60°C for 15 s, and 68°C for 15 s), and bring reactions to 20°C.

 

SECTION: 3´ mRNA sequencing library preparation
61.5. Purify the library. Add 25 μl of DNA buffer and 0.8 volumes (40 μl) of speedbeads to the 0.2 ml PCR tube containing the amplified library.

SECTION: 3´ mRNA sequencing library preparation
61.13. Store the purified sequencing library at -20°C.
SECTION: General method for speedbead purification
62. Equilibrate speedbeads to room temperature and mix thoroughly.
SECTION: General method for speedbead purification
63. Carefully add indicated amount of speedbeads to the sample.
SECTION: General method for speedbead purification
64. Mix well by pipetting up and down a minimum of 10 times. Alternatively, vortex samples on setting 4 for 3-5 seconds. If samples require centrifugation after mixing, stop the centrifuge before beads settle.
SECTION: General method for speedbead purification
65. Incubate samples at room temperature for 5 min. Prolonged incubation or incubation at low temperature will increase binding of small nucleic acids (e.g., adapter dimers).
SECTION: General method for speedbead purification
66. Place samples on magnetic stand for 2-5 min (or until solution is completely clear) to collect beads. Keep samples on magnetic stand for Steps 66-70. 
SECTION: General method for speedbead purification
67. Carefully remove and discard cleared supernatant without disturbing the bead pellet.
SECTION: General method for speedbead purification
68. Add 200 μl of 80% ethanol (freshly prepared with MilliQ water) and incubate for 30 s. Carefully remove ethanol and discard without disturbing the bead pellet.
SECTION: General method for speedbead purification
69. Repeat Step 68 once. Carefully inspect samples and remove remaining ethanol using a 10 μl pipet.
SECTION: General method for speedbead purification
70. Air dry samples for 2 min with lid open. Do not over-dry samples. Speedbeads (and other SPRI products) will clump and can not be fully resuspended during elution if samples are over-dried.
SECTION: General method for speedbead purification
71. Remove samples from the magnetic stand. Add the indicated volume of appropriate solution for elution and mix well by pipetting or vortexing (see Step 64).
SECTION: General method for speedbead purification
72. Incubate mixture at room temperature for 2-5 min.
SECTION: General method for speedbead purification
73. Place samples on a magnetic stand for 2 min (or until solution is completely clear) to collect beads.
SECTION: General method for speedbead purification
74. Transfer cleared supernatant (volume of solution added for elution less 1 μl) to a fresh PCR tube.
SECTION: RNA alkylation and ethanol precipitation
43. Thaw RNA samples on ice. Quantify RNA using a Nanodrop.

 

SECTION: RNA alkylation and ethanol precipitation
44. Add 5 μg of total RNA to a 1.5 ml microcentrifuge tube on ice. Adjust the volume of each sample to 20 μl with nuclease-free water.
SECTION: RNA alkylation and ethanol precipitation
45. Prepare a 100 mM iodoacetamide solution in DMSO.

 

SECTION: RNA alkylation and ethanol precipitation
57. Resuspend RNA in 30 μl of nuclease-free water.
SECTION: RNA alkylation and ethanol precipitation
47. Add 30 μl of reaction mixture to the 1.5 ml microcentrifuge tube containing 5 μg of total RNA in 20 μl nuclease-free water (prepared in Step 44).
SECTION: RNA alkylation and ethanol precipitation
48. Gently mix reaction and incubate in a thermomixer at 900 rpm and 50°C for 15 min in the dark.
SECTION: RNA alkylation and ethanol precipitation
49. Add 1 μl of 1 M DTT to stop the reaction. Briefly vortex to mix. Exposure to light is acceptable following the addition of 1 M DTT.
SECTION: RNA alkylation and ethanol precipitation
50. Add 1 μl of glycogen (20 mg/ml) and 5 μl of 3 M sodium acetate (pH 5.2) to each sample. Briefly vortex to mix.
SECTION: RNA alkylation and ethanol precipitation
51. Add 125 μl of 100% ethanol to each sample. Briefly vortex to mix and incubate at -80°C for 30 min.
SECTION: RNA alkylation and ethanol precipitation
52. Centrifuge samples for 30 min at 21,000 g and 4°C to pellet precipitated RNA.
SECTION: RNA alkylation and ethanol precipitation
58. Store iodoacetamide-treated RNA samples at -80°C or proceed directly with library preparation.
SECTION: RNA alkylation and ethanol precipitation
55. Carefully decant the supernatant. Take care not to dislodge pellet. Carefully inspect samples and remove remaining ethanol with a 20 μl pipet.
SECTION: RNA alkylation and ethanol precipitation
56. Air dry samples for 5 min with lid open. Do not over dry samples.
SECTION: RNA alkylation and ethanol precipitation
54. Add 1 ml of 80% ethanol to each sample. Centrifuge samples for 10 min at 21,000 g and 4°C to wash pellet.
SECTION: RNA alkylation and ethanol precipitation
53. Carefully decant the supernatant. Take care not to dislodge pellet.
SECTION: RNA purification and DNase I treatment
23. Supplement the following buffers and reagents with DTT (100 mM DTT working stock):

 

 

SECTION: RNA purification and DNase I treatment
24. Thaw samples stored in DNA/RNA Shield at -80°C at room temperature. 
SECTION: RNA purification and DNase I treatment
25. Combine 400 μl of RNA Lysis Buffer (supplemented with DTT) and 400 μl of cells suspended in DNA/RNA Shield adjusted as described in Step 20.
SECTION: RNA purification and DNase I treatment
26. Transfer 800 μl of cells suspended in 1:1 DNA/RNA Shield:RNA Lysis Buffer (supplemented with DTT) to a ZR Bashingbead Lysis Tube.
SECTION: RNA purification and DNase I treatment
27. Process samples in a Mini-Beadbeater-24 at 3800 rpm for 45 s and immediately incubate on ice for 2 min.
SECTION: RNA purification and DNase I treatment
28. Repeat Step 27 once.
SECTION: RNA purification and DNase I treatment
30. Centrifuge samples at 16,000 g for 1 min to pellet debris.
SECTION: RNA purification and DNase I treatment
31. Transfer 550 μl of the cleared supernatant to a Zymo-spin IIICG Column in a collection tube and centrifuge at 16,000 g for 30 s. Save the flow through.
SECTION: RNA purification and DNase I treatment
32. Add an equal volume (550 μl) of 100% ethanol (supplemented with DTT) to the flow through. Thoroughly mix samples by pipetting
SECTION: RNA purification and DNase I treatment
33. Transfer 700 μl of the mixture to a Zymo-spin IICR Column in a collection tube and centrifuge at 16,000 g for 30 s. Discard flow through.
SECTION: RNA purification and DNase I treatment
34. Repeat Step 34 with the remaining mixture volume. 
SECTION: RNA purification and DNase I treatment
35. Add 400 μl of RNA Wash Buffer (supplemented with DTT) to the column and centrifuge at 16,000 g for 30 s. Discard flow through.
SECTION: RNA purification and DNase I treatment
36. Gently mix DNase I reaction mixture (prepared in Step 29) by inversion and add 40 μl directly onto column matrix and incubate at room temperature for 15 min. DNase I is easily denatured. Do not vortex reaction mixture.
SECTION: RNA purification and DNase I treatment
37. Add 400 μl of RNA Prep Buffer (supplemented with DTT) to the column and centrifuge at 16,000 g for 30 s. Discard flow through.
SECTION: RNA purification and DNase I treatment
38. Add 700 μl of RNA Wash Buffer (supplemented with DTT) to the column and centrifuge at 16,000 g for 30 s. Discard flow through.
SECTION: RNA purification and DNase I treatment
39. Add 400 μl of RNA Wash Buffer (supplemented with DTT) to the column and centrifuge at 16,000 g for 1 min to ensure complete removal of buffer. 
SECTION: RNA purification and DNase I treatment
40. Carefully transfer the column to a labeled nuclease-free 1.5 ml microcentrifuge tube.
SECTION: RNA purification and DNase I treatment
41. Add 50 μl of nuclease-free water (supplemented with DTT) prewarmed to 50°C directly to column matrix, incubate at room temperature for 2 min, and centrifuge at 16,000 g for 30 s.
SECTION: RNA purification and DNase I treatment
42. Store purified RNA samples at -20°C (up to eight weeks) or -80°C until proceeding.
SECTION: RNA purification and DNase I treatment
29. While samples are incubating on ice, prepare the following reaction mixture in a 1.5 ml microcentrifuge tube on ice:

DNase I reaction mixture
 

 

SECTION: IAA treatment, 4tU labeling, sampling, and rapid fixation
22. Flash freeze samples on dry ice or in liquid nitrogen and store at -80°C until proceeding.
SECTION: 3´ mRNA sequencing library preparation
60.5. Transfer 20 μl of the cleared supernatant to a fresh PCR tube.
SECTION: 3´ mRNA sequencing library preparation
60.6. Store purified ssDNA at -20°C or proceed directly with barcoding and enrichment PCR and library purification.
SECTION: 3´ mRNA sequencing library preparation
61.10. Add 0.8 volumes (24 μl) of DNA binding buffer to the 0.2 ml PCR tube containing the library  and bead suspension. Proceed as described in the general method for speedbead purification (Step 65).
SECTION: 3´ mRNA sequencing library preparation
61.11. Elute the library in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C. 
SECTION: 3´ mRNA sequencing library preparation
61.12. Transfer 20 μl of the cleared supernatant to a fresh PCR tube.
SECTION: 3´ mRNA sequencing library preparation
61.6. Mix well by pipetting up and down a minimum of 10 times. Alternatively, vortex samples on setting 4 for 3-5 seconds. If samples require centrifugation after mixing, stop the centrifuge before beads settle.
SECTION: 3´ mRNA sequencing library preparation
61.8. Place samples on magnetic stand for 2-5 min (or until solution is completely clear) to collect beads. Carefully remove and discard cleared supernatant without disturbing the bead pellet.
SECTION: 3´ mRNA sequencing library preparation
61.9. Remove samples from the magnetic stand. Add 30 µl of DNA buffer and mix well by pipetting or vortexing. Ensure beads are fully resuspend and incubate samples at room temperature for 2 min.
SECTION: 3´ mRNA sequencing library preparation
61.7. Incubate samples at room temperature for 5 min.