An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae
John B. Ridenour, Rafal Donczew
Abstract
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.
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Steps
Yeast growth
Streak appropriate S. cerevisiae strain on YPD agar. Incubate culture at 30°C for 2-3 days.
Transfer an individual colony to 5 ml of YPD. Incubate culture at 30°C with shaking at 220 rpm overnight.
Dilute overnight culture to an OD600 of 0.2 in 40 ml of YPD. Incubate culture at 30°C with shaking at 220 rpm.
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.
IAA treatment, 4tU labeling, sampling, and rapid fixation
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.
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.
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:
| A | B | C |
|---|---|---|
| Treatment | IAA treatment | 4tU treatment |
| A | DMSO | 4tU |
| B | IAA | 4tU |
| C | DMSO | DMSO |
Equilibrate cultures at 30°C for 5 min with shaking at 220 rpm.
During equilibration (or just prior to splitting the culture), prepare a 0.2 M IAA (3-indoleacetic acid) solution in DMSO.
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.
Incubate cultures at 30°C for 25 min with shaking at 220 rpm.
During IAA treatment, prepare a 2 M 4tU (4-thiouracil) solution in DMSO.
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.
Incubate cultures at 30°C for 4 min with shaking at 220 rpm.
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%.
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.
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.
Centrifuge the remaining slurry (approximately 13 ml) at 3,000 g and 4°C for 10 min to pellet cells.
Carefully decant supernatant and invert the 50 ml conical tube on a paper towel to remove residual supernatant
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.
Ensure cells are thoroughly resuspended and transfer the cell suspension to a labeled 1.5 ml microcentrifuge tube.
Flash freeze samples on dry ice or in liquid nitrogen and store at -80°C until proceeding.
RNA purification and DNase I treatment
Supplement the following buffers and reagents with DTT (100 mM DTT working stock):
| A | B | C | D |
|---|---|---|---|
| Reagent | Reagent vol. per sample | DTT vol. per sample | Final DTT conc. |
| RNA Lysis Buffer | 400 μl | 0.8 μl | 0.2 mM |
| RNA Wash Buffer | 1.5 ml | 1.5 μl | 0.1 mM |
| RNA Prep Buffer | 400 μl | 0.4 μl | 0.1 mM |
| 100% ethanol | 550 μl | 0.55 μl | 0.1 mM |
| Nuclease-free water | 50 μl | 0.5 μl | 1 mM |
Thaw samples stored in DNA/RNA Shield at -80°C at room temperature.
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.
Transfer 800 μl of cells suspended in 1:1 DNA/RNA Shield:RNA Lysis Buffer (supplemented with DTT) to a ZR Bashingbead Lysis Tube.
Process samples in a Mini-Beadbeater-24 at 3800 rpm for 45 s and immediately incubate on ice for 2 min.
Repeat Step 27 once.
While samples are incubating on ice, prepare the following reaction mixture in a 1.5 ml microcentrifuge tube on ice:
DNase I reaction mixture
| A | B | C |
|---|---|---|
| Reagent | 1X reaction vol. (μl) | Master mix vol. (μl) |
| DNA Digestion Buffer | 35 | |
| DNase I, reconstituted (1 u/μl) | 5 | |
| Total | 40 |
Centrifuge samples at 16,000 g for 1 min to pellet debris.
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.
Add an equal volume (550 μl) of 100% ethanol (supplemented with DTT) to the flow through. Thoroughly mix samples by pipetting
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.
Repeat Step 34 with the remaining mixture volume.
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.
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.
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.
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.
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.
Carefully transfer the column to a labeled nuclease-free 1.5 ml microcentrifuge tube.
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.
Store purified RNA samples at -20°C (up to eight weeks) or -80°C until proceeding.
RNA alkylation and ethanol precipitation
Thaw RNA samples on ice. Quantify RNA using a Nanodrop.
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.
Prepare a 100 mM iodoacetamide solution in DMSO.
Prepare the following reaction mixture in a 1.5 ml microcentrifuge tube and mix well by pipetting:
Thiol modification reaction mixture
| A | B | C |
|---|---|---|
| Reagent | 1X reaction vol. (μl) | Master mix vol. (μl) |
| 100 mM iodoacetamide | 5 | |
| 0.5 M sodium phosphate buffer (pH 8.0) | 5 | |
| DMSO | 20 | |
| Total | 30 |
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).
Gently mix reaction and incubate in a thermomixer at 900 rpm and 50°C for 15 min in the dark.
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.
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.
Add 125 μl of 100% ethanol to each sample. Briefly vortex to mix and incubate at -80°C for 30 min.
Centrifuge samples for 30 min at 21,000 g and 4°C to pellet precipitated RNA.
Carefully decant the supernatant. Take care not to dislodge pellet.
Add 1 ml of 80% ethanol to each sample. Centrifuge samples for 10 min at 21,000 g and 4°C to wash pellet.
Carefully decant the supernatant. Take care not to dislodge pellet. Carefully inspect samples and remove remaining ethanol with a 20 μl pipet.
Air dry samples for 5 min with lid open. Do not over dry samples.
Resuspend RNA in 30 μl of nuclease-free water.
Store iodoacetamide-treated RNA samples at -80°C or proceed directly with library preparation.
3´ mRNA sequencing library preparation
RNA fragmentation and cDNA synthesis.
Thaw iodoacetamide-treated RNA samples on ice. Quantify RNA using an RNA-specific fluorometry method (i.e., Qubit RNA BR or HS assay kit).
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.
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
| A | B | C |
|---|---|---|
| Reagent | 1X reaction vol. (μl) | Master mix vol. (μl) |
| 5X reverse transcription buffer | 2 | |
| 20 mM dNTP mix | 0.5 | |
| 20 μM IRA_UMI_24dTVN oligo | 0.5 | |
| Total | 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:
Reverse transcription (RT) reaction mixture
| A | B | C |
|---|---|---|
| Reagent | 1X reaction vol. (μl) | Master mix vol. (μl) |
| 20 mM DTT | 1 | |
| 20 μM IFA-isoTSO oligo | 0.5 | |
| Reverse transcriptase (200 u/μl) | 0.5 | |
| Total | 2 |
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.
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.
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.
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).
RNA removal and single-stranded DNA purification.
Add 1 μl RNase H to each sample. Mix well by pipetting. Centrifuge briefly at room temperature to collect samples.
Heat samples to 37°C for 15 min in a thermocycler with heated lid. Proceed immediately to speedbead purification.
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).
Elute the ssDNA in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C.
Transfer 20 μl of the cleared supernatant to a fresh PCR tube.
Store purified ssDNA at -20°C or proceed directly with barcoding and enrichment PCR and library purification.
Barcoding and enrichment PCR and library purification.
Transfer 10 μl of purified ssDNA to a 0.2 ml PCR tube on ice.
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).
Elute the library in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C.
Transfer 20 μl of the cleared supernatant to a fresh PCR tube.
Store the purified sequencing library at -20°C.
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.
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.
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.
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.
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.
Incubate samples at room temperature for 5 min.
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.
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.
General method for speedbead purification
Equilibrate speedbeads to room temperature and mix thoroughly.
Carefully add indicated amount of speedbeads to the sample.
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.
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).
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.
Carefully remove and discard cleared supernatant without disturbing the bead pellet.
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.
Repeat Step 68 once. Carefully inspect samples and remove remaining ethanol using a 10 μl pipet.
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.
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).
Incubate mixture at room temperature for 2-5 min.
Place samples on a magnetic stand for 2 min (or until solution is completely clear) to collect beads.
Transfer cleared supernatant (volume of solution added for elution less 1 μl) to a fresh PCR tube.
