Basic polysome analysis protocol for HEK293T cells

Last updated 2024-11-21 by jyelland

Buffers and lysis protocols are derived from R. Green lab's description in Sinha et al., 2020.

Buffers

5X lysis buffer

Component	Stock Concentration	Volume (40 mL)
250 mM HEPES-KOH pH7.4	1 M	10 mL
500 mM KOAC	5 M	4 mL
75 mM MgOAc2	1 M	3 mL
Triton X-100 2.5%	100%	1 mL
Water		22 mL
Total		40 mL

Filter sterilize 5X lysis buffer, and store at 4 °C.

10X gradient buffer

Component	Stock Concentration	Volume (50 mL)
250 mM HEPES-KOH pH7.4	1 M	12.5 mL
1000 mM KOAC	5 M	10 mL
50 mM MgOAc2	1 M	2.5 mL
Water		25 mL
Total		50 mL

Filter sterilize 10X gradient buffer, and store at 4 °C.

1.2X lysis buffer (prepared fresh)

A 20% increase of concentrations of components in the lysis buffer has given somewhat better polysome/monosome ratios.

Component	Volume	Final Conc.
Water	3.1 mL
5X stock	1.2 mL	1.2X
50% glycerol	0.6 mL	5%
1 M DTT	6 μL	1.2 mM
10 mg/mL CHX	60 μL	120 μg/mL
1 M benzamidine	6 μL	1.2 mM
1 mM leupeptin	6 μL	1.2 μM
1 mM pepstatin A	6 μL	1.2 μM
2,000 U/mL Turbo DNAse I	24 μL	9.6 U/mL
200 mM PMSF	30 μL	1.2 mM
Total	5 mL

Dissolve PMSF to 200 mM in isopropyl alcohol. Use caution, as it is extremely toxic! Tare a 15-mL conical on the analytical balance, add powdered PSMF to a conical tube in the fume hood, and re-weigh the closed tube on the analytical balance to determine how much isopropyl alcohol to add.

When making lysis buffer, add PMSF immediately before use, since it is not stable in aqueous solutions.

Commercial protease inhibitor tablets can be used as an alternative to homemade stocks.

1X gradient buffers (prepared fresh)

45 mL of each solution is sufficient for six gradients. Adjust sucrose as necessary for different density gradients (these volumes are for 10%-50%).

Component	10% sucrose	50% sucrose
10X stock	4.5	4.5
1 M DTT	4.5 μL	4.5 μL
10 mg/mL CHX	450 μL	450 μL
60% sucrose	7.5	37.5
Water	32.7	2.7
Total	45 mL	45 mL

DPBS + cycloheximide

Prepare fresh ~30 minutes before harvest, by adding 100 μL 10 mg/mL CHX to 10 mL DPBS. Incubate at 37 °C in the TC room dry bath.

Procedure

Pre-chill rotor and buckets

At least one day before centrifugation, chill SW 41 Ti rotor and buckets in a cold room.

Prepare gradients

1. Prepare 1X gradient buffers in 50-mL conical tubes on ice.
2. Obtain 89 mm Beckman polyallomer $^{a}$ or Sorvall tubes, one for each sample.
3. Use the side of the aluminum marker block to draw a line at the higher of two possible positions (to be used in conjunction with short tube caps).
4. With a 50-mL syringe attached to one metal canula, draw ~40 mL 10% sucrose and deposit to the fill line of each tube.
5. With a 50-mL syringe attached to the other metal canula, draw ~40 mL 50% sucrose and deposit underneath the 10% sucrose, to the bottom of the tube, filling to ~2 mm from the top of the tube.
6. Attach short black caps to tubes, starting opposite the cap hole, to avoid bubble formation.
7. Turn on and level the Biocomp gradient maker and select the correct "sucrose 10-50% long w/v" program. The parameters are: 3:40, 80°, 21 rpm. $^{b}$
8. Place tubes in the carousel and run the gradient program.
9. Remove tubes and either proceed directly with sample loading, or store at 4 °C until ready for centrifugation.

Notes

$^{a}$ The polyallomer tubes seem to be less likely to crush during centrifugation. Anecdotally, crushing is also avoided by pre-chilling the buckets and rotor overnight, and keeping them cold.

$^{b}$ Running this longer program has given me a better gradient, compared to the original program set to 1:50. Maybe this is due to the viscosity of cold sucrose (the gradient maker programs are designed for use at ambient temperature).

Lyse cells

1. Grow ≥10-cm dish(es) of HEK293T cells to ≥70% confluence.
2. Two hours before cell harvest or drug treatment, exchange the cell culture media for fresh, warm media.
3. Prepare PBS + CHX and warm to 37 °C.
4. Prepare 1X lysis buffer on ice and add PMSF just before ready to harvest.
5. When ready to harvest cells, (1) Prepare a sufficient number of cell scrapers and 5-mL serological pipets, (2) Pre-chill Eppendorf tubes on ice, one for each sample, (3) Pre-chill benchtop centrifuge to 4 °C.
6. Aspirate media quickly with vacuum line.
7. Immediately and gently rinse cells with 2.5-5 mL warm DPBS + CHX, and quickly aspirate.
8. Immediately add 250 μL ice-cold lysis buffer dropwise to the (10-cm) plate. Swirl lysis buffer to distribute, and firmly scrape cell material to a pool at the bottom of the plate. For a 15-cm plate, use 400 μL lysis buffer and follow the same procedure.
9. Pipet several times to mix, and transfer to ice-cold Eppendorf tube.
10. Leave on ice for 10-15 minutes to allow complete lysis.
11. While tubes are on ice, pre-evacuate and chill the ultracentrifuge.

Clarification

1. Invert tubes several times to promote gentle lysis.
2. Centrifuge lysate at 8,000 rcf, for 5 minutes, at 4 °C. Pre-chill new Eppendorf tubes on ice.
3. Transfer clarified lysate to fresh, ice-cold Eppendorf tubes.

Normalization (optional) and loading

It is not always necessary to normalize lysate loading, since the recorded traces are easily normalized during analysis. I would do now so only if I suspected large differences between the RNA content of my lysates.

1. Measure lysate RNA content via Qubit or Nanodrop (A260). Dilute lysate 1:10 before measuring, and blank with 1:10 diluted lysis buffer.
2. Gently place gradient tubes in rotor buckets.
3. Load equivalent amounts of RNA, up to 300 μg (and preferably ≥100 μg), on each gradient by gently pipetting the necessary volume directly onto the top of the gradient. If you need to load more than ~200 μL, remove the same volume from the top of the gradient to prevent spillage after cap closure.
4. Weigh opposing tubes, including buckets and caps, and balance to within 0.1 g using lysis buffer.

Centrifugation

1. Close buckets with caps and firmly tighten.
2. Hang buckets in their corresponding position on the rotor. Load all buckets, even if no tube is present, adn gently rock bucket with a slight twisting motion, to be sure that it is properly seated on the axle.
3. Open centrifuge, seat the rotor on the spindle, and close the door.
4. Centrifuge for 1 hour and 45 minutes (105 minutes) at 40,000 rpm, 4 °C.

Fractionation

Startup

1. Close the drain valve (flip toggle switch up), and fill the rinse reservoir with distilled water.
2. Turn piston gradient fractionator on, followed by fraction collector.
3. Attach a silicone tip to the plunger by screwing it onto the taped threads.
4. Press "SCAN" on fractionator.
5. Start BioComp software on ThinkPad and follow initialization prompts. Be sure to choose the correct scan procedure, i.e. select whether you want to collect absorbance, fluorescence, or both readings.
6. Set the number of fractions to be collected in the "SCAN SETUP" page. You can choose either a specific number of fractions, or set the desired volume per fraction. You must collect a minimum of eight fractions, in order for the fraction volume to fit into a 1.5-mL Eppendorf tube.
7. Run rinse water through the system for ~10 seconds, then follow the prompts to blank the absorbance/fluorescence reading.

Fractionating and collecting gradients

1. If collecting samples, prepare a sufficient number of Eppendorf tubes in the fraction collector rack. If not collecting samples, place a weigh boat in the fraction collector tray to catch the flowthrough.
2. Unscrew the bucket cap, gently remove the centrifugation tube with a pair of forceps, and press the collar over the top of the tube to hold it in place.
3. Seat the collar in the pedestal and lock it in place with a 45-degree turn, then place and lock the pedestal in its position under the plunger.
4. Start the scan and collection by pressing F1. It can be helpful to auto-scale the Y-axis during the initial part of the scan, then manually set the Y-axis limits to baseline and just above the top of the 80S peak (which can be quite low).
5. If collecting a lot of fractions, you might either label samples with different colors, or label fractions with a designation like "101" for "Sample 1, Fraction 1", etc.
6. Keep fractions on ice after collecting, to preserve RNA integrity.
7. Follow the water and air rinse prompts between scans, to avoid cross-contamination between samples.

Cleanup

1. Rinse the system with 15 mL water from the rinse reservoir.
2. Fill the small rinsing syringe with distilled water. Remove the plunger silicone tip and attach the rinsing syringe adapter. Rinse the plunger with the attached syringe full of water.
3. Open the brass air valve slightly and run air through the system for ≥10 seconds. Close the air valve.
4. Empty the water rinse reservoir, and remove it from the fractionator. Then attach the empty 50-mL air syringe with its plunger drawn all the way out.
5. Use the syringe to force air through the system. When the plunger is all the way down, open the drain valve (toggle switch down) and draw the plunger back to the top of the syringe.
6. Close the drain valve and repeat step 5 twice more.
7. Detach the air syringe, reattach the rinse reservoir, and leave the system with the drain valve open.
8. Empty the waste flask.
9. Thoroughly rinse the silicone tip with deionized water to remove all traces of sucrose.
10. Close the software, and shut down the fraction collector, followed by the fractionator.

Rotor and buckets

1. Rinse buckets with deionized water, and dry overnight.
2. Store buckets loosely capped. To preserve O-ring integrity, don't store them fully tightened.
3. Check O-ring condition before storing and, if necessary, lubricate them with vacuum grease or replace them.
4. Lubricate the bucket cap threads with a thin layer of Spinkote every few months, or as needed.