Polysome Profiling without Gradient Makers or Fractionation Systems

1. Preparation of 7% – 47% sucrose gradients

NOTE: The linear range of the sucrose gradient can be modified to achieve better separation depending on the cell type used. This protocol is optimized for polysome profiles for S. cerevisiae.

1. Prepare stock solutions of 7% and 47% sucrose in sucrose gradient buffer (20 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl₂, and 1 mM DTT). Filter sterilize the sucrose stock solutions through a 0.22 μm filter and store at 4 °C.
2. Prepare 14 mL of 17%, 27%, and 37% sucrose solutions by dispensing and mixing the 7% and 47% sucrose stock solutions in the manner described in Table 1.
3. Place six polypropylene centrifuge tubes (14 x 89 mm) into a full view test tube rack. Ensure enough space between the tubes so that actions with one tube do not disturb the others.
4. Attach a long needle to a 3 mL syringe. For this protocol, use a 9 in, 22 G needle with a blunt tip (Figure 1), but any needle long enough to reach the bottom of the centrifuge tube will suffice.
   NOTE: Perform a test fill and dispensation to ensure that the syringe can hold the sucrose solution without any dripping prior to setting up the gradients.
5. Add 2 mL of the 7% sucrose to the bottom of each centrifuge tube.
6. Add 2 mL of the 17% sucrose beneath the 7% solution by positioning the needle tip within the immediate vicinity of the tube bottom and dispensing the solution slowly and carefully.
7. Repeat with 2 mL each of the 27%, 37%, and 47% sucrose solutions. Ensure that each layer is distinguishable from one another by a line marking the separation of densities (Figure 2).
   NOTE: If the gradients are not going to be used within 48 hours then at this point, prior to their settlement into a continuous gradient, flash freeze the tubes with layered sucrose solutions in liquid nitrogen and store in a -80 °C freezer for long-term storage.
8. Store gradients at 4 °C overnight to allow gradients to settle into a continuous, increasing percentage of sucrose. It takes 8-12 h for the layered sucrose solutions to settle into a linear sucrose gradient. Linear gradients are stable for up to 48 h. Overnight storage at 4 °C provides sufficient time for thawing and settlement into a linear gradient for using frozen, layered sucrose solutions. Use a densitometer to assess the gradient quality.
   NOTE: It is critical to store the gradients in a stable place where they will not be disturbed, as any movements or vibrations will disrupt the gradient.

2. Preparation of yeast cell extracts

1. Inoculate the yeast strain of interest into 50 mL of yeast extract peptone dextrose (YPD) media and grow overnight at 30 °C in a shaking incubator to stationary phase.
   NOTE: The temperature may vary depending upon yeast strain requirements.
2. Transfer 10 mL of stationary phase culture into 1 L of fresh YPD media. Incubate the cells with vigorous shaking at 30 °C (or other suitable temperature) until the culture reaches the mid-exponential growth phase (OD₆₀₀ = 0.4-0.6).
3. At the mid-exponential growth phase, add cycloheximide to the culture at a final concentration of 0.1 mg/mL. Incubate on ice for 5 min.
4. Harvest the cells by centrifugation at 3,000 x g for 10 min at 4 °C.
   NOTE: At this point, cells can be frozen and stored at -80 °C.
5. Resuspend the cells in chilled 700 μL of polysome extraction buffer (20 mM Tris-HCl pH 7.4, 60 mM KCl, 10 mM MgCl2, 1 mM DTT, 1% Triton X-100, 0.1 mg/mL of cycloheximide, 0.2 mg/mL of heparin). Add 100 units of RNase Inhibitor and transfer it to a 1.5 mL centrifuge tube.
6. Add ~400 μL of pre-chilled glass beads with a size range of 425-600 µm to the centrifuge tube. Disrupt the yeast cells by vigorous agitation in a bead-beater for 5 min.
7. Clarify the lysate by centrifugation at 8,000 x g for 5 min at 4 °C.
8. Determine the concentration of the RNA in the clarified lysate by measuring the absorbance at 260 nm with a spectrophotometer or using a fluorescence-based RNA detection system.
   NOTE: For very accurate RNA concentration measurements, use fluorescence-based RNA detection kits.
9. Ensure that the RNA concentration is 0.5-1 µg/μL. If the RNA concentration is too low, reduce the volume of polysome extraction buffer used to resuspend the cells in future experiments.
   ​NOTE: Load the lysate onto the gradients immediately after lysis and RNA quantitation. If necessary, flash freeze the lysates in liquid nitrogen and store at -80 °C for a few days.

3. Centrifugation of gradients

1. Carefully load the lysate onto the top of the gradients. Place the pipette tip against the inner wall, at the top of the polypropylene tube. Gently angle the tube and slowly dispense the lysate onto the top of the gradient by dribbling against the wall. Take great care not to disrupt or disturb the gradient when loading the lysate.
   NOTE: The amount of lysate loaded will vary per cell type. The content of the RNA will also vary. It may be necessary to perform a number of experiments to determine the amount of lysate needed to generate an optimal polysome profile. For yeast, 300 μg of RNA is a good starting point for optimization.
2. Gently place the tubes into the pre-chilled buckets of a swinging bucket rotor.
   NOTE: Each polypropylene centrifuge tube should have equal volumes of gradient and the amount of the lysate loaded. This can vary from tube to tube. Ensure that the variation does not cause an imbalance error during ultracentrifugation.
3. Centrifuge the gradients at 260,110 x g for 150 min at 4 °C.

4. Fraction and data collection

1. Carefully remove the centrifuge tubes from the swinging bucket rotor and place them in a tube holder.
2. Label the 96-well plates to store the fractions and pre-chill on ice.
   NOTE: Use a 96-well plate suitable for a spectrophotometer that has an optical window down to 230 nm for nucleic acid determinations at 260 nm/280 nm.
3. Collect 100 µL or 200 µL fractions starting from the top of the gradient by carefully inserting a pipet tip into the top of the gradient. Collect fractions until the entire gradient is aliquoted. The number of fractions will depend on total gradient volume.
   NOTE: Collect the fractions in a manner that does not disrupt the rest of the gradient. Ensure that all the fractions have equal volume. In addition to manual fractionation, another low-cost method for fractionation is to use a small peristaltic pump.
4. Transfer each fraction to the 96-well plate to reach the bottom of the centrifuge tube. Keep the collected fractions on the ice at all times.
5. Measure the absorbance of each fraction at 254 nm with a spectrophotometer. Use the 7% and 47% sucrose solutions as blanks.
   NOTE: When measuring the absorbance of fractions, bear in mind that for most spectrometers and colorimeters, the most effective absorbance range is 0.1-1. If the absorbance measurements are out of range (≥1.0), the fractions have too much material. Dilute the sample, recollect the data, and then account for the dilution factor when plotting the profile.
6. Create the polysome profile by plotting the fraction number versus absorbance.