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, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, 3´ mRNA-seq library construction, and data analysis. 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, this workflow is modular and readily adaptable to other systems, including industrial, pathogenic, or other model fungi, which 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

Alexander RD, Barrass JD, Dichtl B, Kos M, Obtulowicz T, Robert MC, Koper M, Karkusiewicz I, Mariconti L, Tollervey D, Dichtl B, Kufel J, Bertrand E, Beggs JD. (2010). RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3′-end processing in Saccharomyces cerevisiae. RNA, 16(12), 2570-2580. https://doi.org/10.1261/rna.2162610

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, Beggs JD (2019). Extremely rapid and specific metabolic labelling of RNA in vivo with 4-thiouracil (Ers4tu). JoVE (Journal of Visualized Experiments), 150, e59952. https://doi.org/10.3791/59952

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

Chappleboim,A, Joseph-Strauss D, Gershon O, Friedman N. (2022). Transcription feedback dynamics in the wake of cytoplasmic mRNA degradation shutdown. Nucleic Acids Research, 50(10), 5864-5880. https://doi.org/10.1093/nar/gkac411

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

Neumann T, Herzog VA, Muhar M, von Haeseler A, Zuber J, Ameres SL, Rescheneder P (2019). Quantification of experimentally induced nucleotide conversions in high-throughput sequencing datasets. BMC Bioinformatics, 20, 1-16. https://doi.org/10.1186/s12859-019-2849-7

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: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
5. Add 5 ml of 100% methanol to a 50 ml conical tube for each sample to be collected. Keep tubes on dry ice. Three tubes are needed for each 40 ml culture.
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
6. 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: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
7. Equilibrate cultures at 30°C for 5 min with shaking at 220 rpm.
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
8. During equilibration (or just prior to splitting the culture), prepare a 0.2 M IAA solution in DMSO. IAA is freshly prepared. To prepare a 0.2 M IAA solution, add 220 mg of IAA to 6 ml of DMSO and vortex until dissolved. Keep the IAA solution at room temperature covered with foil.

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

 

SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
11. During IAA treatment, prepare a 2 M 4tU solution in DMSO. 4tU is freshly prepared. To prepare a 2 M 4tU solution, add 260 mg of 4tU to 1 ml of DMSO and vortex until dissolved. Keep the 4tU solution at room temperature covered with foil.

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

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

 

SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
14. CRITICAL STEP: Immediately after 4tU treatment, decant each 10 ml culture directly into 5 ml of 100% methanol in a 50 ml conical tube on dry ice (prepared in Step 5). 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: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
15. 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: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
16. Gently mix the fixed sample and transfer 1.5 ml to a 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: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
17. Centrifuge the remaining sample at 3,000 g and 4°C for 10 min to pellet cells.
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
18. Carefully decant the supernatant and briefly invert the 50 ml conical tube on a paper towel to remove residual supernatant.
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
19. Adjust samples to a uniform cell number in DNA/RNA Shield. Use the following formula to calculate the appropriate volume of buffer for each sample: volume of buffer = (final OD600 of sample/0.70)*400. Thoroughly resuspend cells by pipetting. 
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
20. Ensure cells are thoroughly resuspended and transfer 400 μl of the cell suspension to a 1.5 ml microcentrifuge tube.
SECTION: RNA alkylation and ethanol precipitation
59. Prepare the following reaction mixture in a 1.5 ml microcentrifuge tube and mix well by pipetting:

Alkylation reaction mixture
 

 

SECTION: 3´ mRNA sequencing library construction
72. RNA fragmentation and cDNA synthesis.

 

SECTION: 3´ mRNA sequencing library construction
73. RNA removal and single-stranded DNA purification.
SECTION: 3´ mRNA sequencing library construction
74. Barcoding and enrichment PCR and library purification.
SECTION: 3´ mRNA sequencing library construction
72.1. Thaw alkylated 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 construction
72.2. Add 200 ng of 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 construction
72.3. 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 construction
72.4. 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 construction
72.5. Bring the 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 construction
72.6. CRITICAL STEP: 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. Fragmentation time can be optimized to generate libraries of different insert sizes.
SECTION: 3´ mRNA sequencing library construction
72.8. Remove samples from the thermocycler, briefly centrifuge, and add 2 μl of the RT reaction mixture to each sample. Mix well by pipetting.
SECTION: 3´ mRNA sequencing library construction
72.10. 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 73.1).
SECTION: 3´ mRNA sequencing library construction
73.1. Add 1 μl of RNase H to each sample and mix well by pipetting. Centrifuge briefly at room temperature to collect samples.
SECTION: 3´ mRNA sequencing library construction
73.2. Heat samples to 37°C for 15 min in a thermocycler with heated lid. Proceed directly to speedbead purification.
SECTION: 3´ mRNA sequencing library construction
73.3. Purify the single-stranded DNA (ssDNA). Add 39 μl of DNA buffer and 1 volume (50 μl) of speedbeads to each sample and proceed as described in the general method for speedbead purification (Step 76).

 

SECTION: 3´ mRNA sequencing library construction
73.4. Elute DNA in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C. 
SECTION: 3´ mRNA sequencing library construction
74.1. Transfer 10 μl of purified DNA to a 0.2 ml PCR tube on ice.
SECTION: 3´ mRNA sequencing library construction
74.2. Add 2.5 μl of an appropriate 5 μM i5/i7 primer mix to each sample on ice.
SECTION: 3´ mRNA sequencing library construction
74.3. Add 12.5 μl of 2X Ex Premier master mix to each sample on ice. Mix thoroughly by pipetting.
SECTION: 3´ mRNA sequencing library construction
74.4. 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 the reactions to 20°C.

 

SECTION: 3´ mRNA sequencing library construction
74.5. 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 construction
74.13. PAUSE POINT: Store purified libraries at -20°C (up to four weeks) or -80°C until proceeding.
SECTION: RNA alkylation and ethanol precipitation
56. Thaw RNA samples on ice. Quantify RNA using a Nanodrop.

 

SECTION: RNA alkylation and ethanol precipitation
57. 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
58. Prepare a 100 mM iodoacetamide solution in DMSO. Iodoacetamide is freshly prepared. To prepare a 100 mM iodoacetamide solution, add 9.25 mg of iodoacetamide to 500 μl of DMSO (1 mg of iodoacetamide to 54 μl of DMSO). Keep the iodoacetamide solution at room temperature covered with foil. Do not store unused iodoacetamide.

SECTION: RNA alkylation and ethanol precipitation
70. Resuspend RNA in 30 μl of nuclease-free water.
SECTION: RNA alkylation and ethanol precipitation
60. 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 57). The final concentration of iodoacetamide is 10 mM.
SECTION: RNA alkylation and ethanol precipitation
61. Gently mix the reaction and incubate in a thermomixer at 900 rpm and 50°C for 15 min in the dark.
SECTION: RNA alkylation and ethanol precipitation
62. Add 1 μl of 1 M DTT to stop the reaction. Briefly vortex to mix. Exposure to light is acceptable following the addition of DTT.
SECTION: RNA alkylation and ethanol precipitation
63. 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
64. 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
65. Centrifuge samples for 30 min at 21,000 g and 4°C to pellet precipitated RNA.
SECTION: RNA alkylation and ethanol precipitation
71. PAUSE POINT: Store alkylated RNA at -20°C (up to four weeks) or -80°C until proceeding.
SECTION: RNA alkylation and ethanol precipitation
68. Carefully decant the supernatant. Take care not to dislodge pellets. Carefully inspect samples and remove remaining ethanol with a 20 μl pipet.
SECTION: RNA alkylation and ethanol precipitation
69. Air dry samples for 5 min with lid open. Do not over dry samples.
SECTION: RNA alkylation and ethanol precipitation
67. 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
66. Carefully decant the supernatant. Take care not to dislodge pellet.
SECTION: RNA purification and DNase I treatment
36. Supplement the following buffers and reagents with DTT (100 mM DTT working stock):

 

 

SECTION: RNA purification and DNase I treatment
37. Thaw samples stored in DNA/RNA Shield at -80°C at room temperature. 
SECTION: RNA purification and DNase I treatment
38.1. OPTION A (without spike-in): Combine 400 μl of RNA Lysis Buffer and 400 μl of cells suspended in DNA/RNA Shield.
SECTION: RNA purification and DNase I treatment
40. 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
41. Repeat Step 40 once.
SECTION: RNA purification and DNase I treatment
43. Centrifuge samples at 16,000 g for 1 min to pellet debris.
SECTION: RNA purification and DNase I treatment
44. CRITICAL STEP: 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
45. Add an equal volume (550 μl) of 100% ethanol to the flow through. Thoroughly mix samples by pipetting.
SECTION: RNA purification and DNase I treatment
46. 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
47. Repeat Step 46 with the remaining mixture volume. 

SECTION: RNA purification and DNase I treatment
48. Add 400 μl of RNA Wash Buffer to the column and centrifuge at 16,000 g for 30 s. Discard flow through.
SECTION: RNA purification and DNase I treatment
49. Gently mix DNase I reaction mixture (prepared in Step 42) by inversion and add 80 μl directly to the column matrix. Incubate the reaction at room temperature for 15 min.
SECTION: RNA purification and DNase I treatment
50. Add 400 μl of RNA Prep Buffer to the column and centrifuge at 16,000 g for 30 s. Discard flow through.
SECTION: RNA purification and DNase I treatment
51. Add 700 μl of RNA Wash Buffer to the column and centrifuge at 16,000 g for 30 s. Discard flow through.
SECTION: RNA purification and DNase I treatment
52. Add 400 μl of RNA Wash Buffer 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
53. Carefully transfer the column to a labeled nuclease-free 1.5 ml microcentrifuge tube.
SECTION: RNA purification and DNase I treatment
54. Add 50 μl of nuclease-free water prewarmed to 50°C directly to the 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
55. PAUSE POINT: Store purified RNA at -20°C (up to four weeks) or -80°C until proceeding.
SECTION: RNA purification and DNase I treatment
42. 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: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
21. PAUSE POINT: Flash freeze samples on dry ice or in liquid nitrogen and store at -80°C until proceeding.
SECTION: 3´ mRNA sequencing library construction
73.5. Transfer 20 μl of the cleared supernatant to a fresh PCR tube.
SECTION: 3´ mRNA sequencing library construction
73.6. PAUSE POINT: Store purified DNA at -20°C (up to eight weeks) or -80°C until proceeding.
SECTION: 3´ mRNA sequencing library construction
74.10. 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 76).
SECTION: 3´ mRNA sequencing library construction
74.11. Elute the library in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C. 
SECTION: 3´ mRNA sequencing library construction
74.12. Transfer 20 μl of the cleared supernatant to a fresh PCR tube.
SECTION: 3´ mRNA sequencing library construction
74.6. 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 construction
74.8. 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 construction
74.9. 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 construction
74.7. Incubate samples at room temperature for 5 min.
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
1. Streak appropriate S. cerevisiae strain on YPD agar. Incubate culture at 30°C for 2-3 days.

 

SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
22. OPTIONAL STEP: If experiments require spike-in, prepare 4tU-treated S. pombe cells as described in Steps 22-35. Streak appropriate S. pombe strain on YE agar. Incubate culture at 30°C for 3-4 days.

 

SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
25. When the 250 ml culture reaches an OD600 of 0.6, cool centrifuge to 4°C and prepare 50 ml conical tubes and 1.5 ml microcentrifuge tubes.
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
4. When the 40 ml culture reaches an OD600 of 0.3-0.4, cool centrifuge to 4°C and prepare 50 ml conical tubes, 2.0 ml microcentrifuge tubes, and 1.5 ml microcentrifuge tubes.
SECTION: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
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: Saccharomyces cerevisiae growth, IAA treatment, 4tU labeling, and rapid fixation
2. Transfer an individual colony to 5 ml of YPD. Incubate culture at 30°C with shaking at 220 rpm overnight.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
23. Transfer an individual colony to 5 ml of YE. Incubate culture at 30°C with shaking at 220 rpm overnight.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
24. Dilute overnight culture to an OD600 of 0.2 in 250 ml of YE. Incubate culture at 30°C with shaking at 220 rpm.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
26. Add 125 ml of 100% methanol to a 1 l flask for each culture to be collected. Keep flask on dry ice. 
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
28. CRITICAL STEP: When the culture reaches an OD600 of 1.0, add 625 μl of 2 M 4tU solution. 4tU will precipitate when added to the culture; immediately and vigorously mix the culture and return it to the incubator.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
27. When the culture reaches an OD600 of 0.8, prepare a 2 M 4tU solution in DMSO as described in Step 11.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
30. CRITICAL STEP:  Immediately after 4tU treatment, decant the 250 ml culture directly into 125 ml of 100% methanol in a 1 l flask on dry ice (prepared in Step 26). Gently mix the slurry by swirling to ensure homogenization. Keep the fixed sample on dry ice.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
29. Incubate the culture at 30°C for 4 min.

 

SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
33. Thoroughly resuspend the cells in each 50 ml conical tube in 540 μl of DNA/RNA Shield by pipetting and pool cell suspensions in a fresh 50 ml conical tube on ice. Samples are adjusted to the equivalent of an OD600 of 7.0.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
32. Carefully decant supernatant and briefly invert 50 ml conical tubes on a paper towel to remove residual supernatant.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
31. Distribute 30 ml aliquots of the culture slurry to 50 ml conical tubes and centrifuge at 3,000 g and 4°C for 10 min to pellet cells.
SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
34. Ensure cells are thoroughly resuspended and distribute 500 μl aliquots of the cell suspension to 1.5 ml microcentrifuge tubes on ice.
SECTION: RNA purification and DNase I treatment
39.2. OPTION B (with spike-in): Transfer 760 μl of S. cerevisiae cells suspended in DNA/RNA Shield:RNA Lysis Buffer and 40 μl of S. pombe cells suspended in DNA/RNA Shield:RNA Lysis Buffer to a ZR Bashingbead Lysis Tube.
SECTION: RNA purification and DNase I treatment
38.2. OPTION B (with spike-in): Combine 360 μl of RNA Lysis Buffer and 400 μl of cells suspended in DNA/RNA Shield, and combine 500 μl of RNA Lysis Buffer and 500 μl of S. pombe cells suspended in DNA/RNA Shield.

 

SECTION: Data analysis
95. Run fastp to extract unique molecular identifiers (UMIs) using the following commands:
 

 


SECTION: Data analysis
96. Run bbduk from the BBMap package to trim adapter sequences and polyA tails using the following commands:
 

SECTION: Data analysis
98. Run slamdunk all from the SLAM-DUNK package to align data and quantify total reads and reads that have ≥1 T>C conversions using the following commands:
 

 

SECTION: Data analysis
100. Run slamdunk count from the SLAM-DUNK package to quantify reads that have ≥2 T>C conversions using the following commands:
 

SECTION: Data analysis
101. Run the following alleyoop commands from the SLAM-DUNK package to summarize and merge slamdunk output:  
 

SECTION: Data analysis
89. Download and install the following software and dependencies:

fastp (version 0.23.2)
BBMAP (version 39.06)
Python (version 3.10.2)
SLAM-DUNK (version 0.4.3)
NextGenMap (version 0.5.5)
SAMtools (version 1.18)
VarScan 2 (version 2.4.5)
R (version 4.1.2-mkl)
Fastqc (version 0.12.1)
Multiqc (version 1.21)
 
 

SECTION: Data analysis
91. Download the reference BED file attached below. This BED file will be used to define counting windows in SLAM-DUNK.

 

SECTION: Schizosaccharomyces pombe growth, 4tU labeling, and rapid fixation
35. PAUSE POINT: Flash freeze the samples on dry ice or in liquid nitrogen and store at -80°C until proceeding.
SECTION: RNA purification and DNase I treatment
38. Combine cells suspended in DNA/RNA Shield with RNA Lysis Buffer using one of the following options:
SECTION: RNA purification and DNase I treatment
39. Transfer cells suspended in DNA/RNA Shield:RNA Lysis Buffer to a ZR Bashingbead Lysis Tube using one of the following options:
SECTION: RNA purification and DNase I treatment
39.1. OPTION A (without spike-in): Transfer 800 μl of cells suspended in 1:1 DNA/RNA Shield:RNA Lysis Buffer to a ZR Bashingbead Lysis Tube.
SECTION: 3´ mRNA sequencing library construction
72.7. During RNA fragmentation, bring the RT reaction mixture (prepared in Step 72.4) to room temperature for 2-5 minutes. 
SECTION: 3´ mRNA sequencing library construction
72.9. Briefly centrifuge and return samples to a thermocycler at 42°C. 
SECTION: General method for speedbead (SPRI) purification
76. Equilibrate speedbeads to room temperature and mix thoroughly.
SECTION: General method for speedbead (SPRI) purification
78. 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 (SPRI) purification
77. CRITICAL STEP: Carefully add the indicated amount of speedbeads to the sample.
SECTION: General method for speedbead (SPRI) purification
83. Repeat Step 82 once. Carefully inspect samples and remove remaining ethanol using a 10 μl pipet.
SECTION: General method for speedbead (SPRI) purification
82. 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 (SPRI) purification
81. Carefully remove and discard cleared supernatant without disturbing the bead pellet.
SECTION: General method for speedbead (SPRI) purification
80. 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 81-85. 
SECTION: General method for speedbead (SPRI) purification
79. 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 (SPRI) purification
84. CRITICAL STEP: 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 (SPRI) purification
88. Transfer cleared supernatant (volume of solution added for elution less 1 μl) to a fresh PCR tube.
SECTION: General method for speedbead (SPRI) purification
85. Remove samples from the magnetic stand. Add the indicated volume of appropriate solution for elution and mix well by pipetting or vortexing (see Step 78).
SECTION: General method for speedbead (SPRI) purification
87. Place samples on a magnetic stand for 2 min (or until solution is completely clear) to collect beads.
SECTION: General method for speedbead (SPRI) purification
86. Incubate mixture at room temperature for 2-5 min.
SECTION: 3´ mRNA sequencing library construction
75. Library quality control, quantification, pooling, and sequencing.
SECTION: 3´ mRNA sequencing library construction
75.2. Quantify libraries using a DNA-specific fluorometry method (i.e., Qubit dsDNA BR or HS assay kit).
SECTION: 3´ mRNA sequencing library construction
75.1. Assess library quality using an Agilent Bioanalyzer or agarose gel electrophoresis. 
SECTION: Data analysis
90. Download reference genome and annotation files. The reference genome assembly and annotation for S. cerevisiae strain S288C (version R64-3-1, RefSeq Assembly GCF_000146045.2) can be retrieved from the NCBI Datasets repository.

 

SECTION: 3´ mRNA sequencing library construction
75.3. Pool equimolar concentrations (i.e., 5 nM) of each library. The recommended final concentration of the pool is 5 nM.

 

SECTION: Data analysis
102. Run multiqc to compile output from the SLAM-DUNK package using the following commands: 
 

 

SECTION: 3´ mRNA sequencing library construction
75.4. Sequence the library pool on an Illumina instrument (e.g., NovaSeq 6000). The recommended sequencing depth is 10 million reads per sample.
SECTION: Data analysis
94. Run fastqc to assess raw data quality and multiqc to compile the fastqc output using following the commands: 
 

 

SECTION: Data analysis
105. OPTIONAL STEP: If experiments used S. pombe spike-in cells, set variables and make subdirectories using the commands below. Download the reference S. pombe genome assembly. Download the reference S. pombe BED file attached below. Repeat Steps 98-103 to generate spike-in count tables.
 

 

SECTION: Data analysis
92. CRITICAL STEP: Set required variables using the commands below. Directories or files within quotations must be changed appropriately before running. 
 

 

SECTION: Data analysis
93. Make subdirectories to store outfiles created in Steps 94-101 using the following commands: 
 

SECTION: Data analysis
97. Run fastqc to assess preprocessed data quality and multiqc to compile the fastqc output using following the commands: 
 

SECTION: Data analysis
103. Key results from this analysis are the three count tables described below. These files are located in the alleyoop directory (i.e., $DIRECTORY/slamdunk_scer/alleyoop).

merge_totalreadcount.tsv - a table (tab-separated values) providing total read counts for all processed samples 
merge_onetcreadcount.tsv - a table (tab-separated values) providing read counts with ≥1 T>C conversions for all processed samples
merge_twotcreadcount.tsv - a table (tab-separated values) providing read counts with ≥2 T>C conversions for all processed samples

 

SECTION: Data analysis
104. Additional processing, analysis, and plotting can be performed in R (version 4.2.3) (https://www.R-project.org/) and RStudio (version 2023.12.0+369) (http://www.rstudio.com/) based on specific experimental goals.
SECTION: Data analysis
99. Rename the count directory created by slamdunk all using the following command: