Make sucrose gradients (~1hr or 1 day ahead of harvesting depending on method)

1. CRITICALThese instructions are for SW-41 rotor gradients; other rotor instructions will differ

2. Make 40mL of 10% and 50% sucrose in SGB solutions with DTT and cycloheximide and allow to come to room temperature

3. Mark tubes with line for short gradients (optional, if you have the gradient guide)

4. Fill tubes with 10% sucrose in SGB to line (or add 6mL)

5. Underlay 50% sucrose in SGB so that the 50% solution reaches line (or add 6mL)

(use a ~6 inch, ~10 gauge stainless steel blunt syringe needle for this e.g. from VWR 20068-690 or hamilton http://www.hamiltoncompany.com/products/syringes-and-needles/needles/luer-lock-needles/10-gauge-Metal-Hub-NDL-custom-length-0375-to-12-in-point-style-2-3-or-4-6PK)

6. Cap tubes and aspirate excess solution from inside the caps

7. Ensure there are no air bubbles in any of the tubes on the sides or top

8. Save remainder of 10% sucrose in SGB for setting the baseline on the gradient fractionator

9. If using gradient maker (Biocomp Gradient Master 108):

Turn on the power in the back

Use the round bullseye level to check that the plate is flat and adjust with DOWN/UP buttons if not

Push DONE when level

Choose GRAD

Choose LIST

Choose SW₄₁ (assuming you're making SW₄₁ gradients)

Select "Short Sucr 10-50%" for a 10-50% sucrose gradient (use DOWN/UP to find it if not shown)

Push USE

Place the samples on the plate in the center

Push RUN

Allow the program to complete (takes a minute or so; the plate stopping and moving around is normal)

When the plate is flat slide the tube holder off the plate (don't pull it as the release will jostle the gradients)

10. If using horizontal diffusion (I have not tried this, below is from Bartel lab protocol):

Seal the top of the tube with parafilm

Gently tilt tube to horizontal and leave in cold room for 6.5 hrs

After 6.5 hours tilt tube slowly back to vertical

Leave in cold room overnight in SW-41 tube jackets

11. Ensure tubes are balanced to within ~50mg - gently add 10% sucrose solution to rebalance them if not

Note: 1µL ~= 1mg, so if the tubes are off by 50mg add 50µL to the low one

Lyse cells/tissue (below is for mammalian cell culture) (~3hr)

12. Note: input should be ~10⁷ cells or more ideally. Also, this procedure will lyse cytoplasm but not nuclei, which is preferred if possible.

13. CRITICALEnsure cycloheximide is in all solutions from lysis onward.

14. CRITICALStart cooling ultracentrifuge at this point or earlier

15. Treat cells with 100µg/mL cycloheximide by adding to media for 5 minutes before harvesting

16. Aspirate media, transfer to ice, and replace media with ~5mL ice cold PBS + cycloheximide

17. Aspirate PBS and scrape cells into a microfuge tube

18. Spin at 16k g for 30 seconds at 4 deg

19. Aspirate PBS and resuspend in 3 pellet volumes of lysis buffer + cycloheximide (typically this is ~100µL pellet volume and 300µL lysis buffer in my hands).

20. Incubate ten minutes on ice to swell cells

21. Triturate ten times through a 26G needle

22. Spin at 1500g for 5 minutes at 4 deg to pellet nuclei

23. Transfer ~50µL of cytoplasm (supernatant) to a fresh tube to save for total RNA-seq

24. Layer 100µL of cytoplasm on top of a gradient and repeat until out of cytoplasm

25. Save the tube with the pellet (this is the nuclear fraction)

26. If not all tubes were used then add 100µL of water to each

27. Spin at 36,000 RPM for 2 hours at 4 degrees

Extracting cytoplasm and nuclear fractions

28. Add 300µL Trizol to the retained cytoplasmic fraction and to the tube with the nuclear pellet.

29. Triturate each tube ten times through a P-1000. The nuclear pellet is usually very clumpy and sticky.

30. Follow the trizol extraction protocol, after the lysis step (start at step 2).

Fractionating gradients (~1hr)

31. CRITICAL30 minutes prior to spin ending, turn on all frationation equipment

32. CRITICALKeep tubes on ice while fractionating

33. Flow the leftover 10% sucrose in SGB solution through the fractionator and press "auto-baseline" on the detector to zero the absorbance trace

34. Insert a tube into the fractionator

35. Puncture the bottom with a ~16G needle connected to a syringe pump or a peristaltic pump and pump at ~2mL/min

36. Manually collect fractions corresponding to the monosome, and all polysome peaks as high as can be separated by hand (typically ~8 ribosomes) and then collect the remainder, all in microfuge tubes.

37. This is an example of how I would fractionate the polysome profile:

38. Ensure no tube has more than 500µL.

39. Add 50µL of 3M NaOAc pH 5.2 to each tube

40. Add 2.5µL of GlycoBlue to each tube

41. Mix well

42. Add 1mL 100% EtOH to each tube

43. Mix well

44. PAUSEStore at -80 for 1 hour to overnight or at -20 overnight

DNase treat fractions (~1.5hr)

45. Pellet precipitations from above at 16k RPM in a microfuge for 30 minutes. Note carefully the location of the tube hinge and point it to the outside to locate the pellet on that side of the tube.

46. Keep tubes on ice when removing from the centrifuge

47. The pellets from this will likely be very small. It is recommended to save the supernatant until RNA is quantified after DNase treatment in the event that re-precipitation to obtain more RNA is desired. Typically quantification is reserved until the end to maximize yield. Pellets in most fractions should be barely visible by eye; poly2, poly3 and poly4 fractions tend to be the most difficult to see. Pellets from the RNP fraction, if collected, will be large and very chalky due to the large amount of protein in these fractions.

48. Aspirate supernatant carefully without disturbing the pellet with a P-1000 - recommended to not get too close to the pellet.

49. Quick-spin the tubes to collect the remainder of the ethanol

50. Aspirate the remainder of the ethanol with a P-10, again avoiding the pellet

51. Uncap the tubes and rest for 5 minutes at room temperature

52. Add 80µL RNase-free water, 10µL 10X RQ1 DNase buffer and 10µL RQ1 DNase

53. Incubate at 37 degrees for 30 minutes

Phenol-chloroform extraction (~1hr) [or optionally use a Directzol kit (Zymo)]

54. Perform these steps at room temperature

55. Add 200µL of 0.3M NaOAc pH 5.2 to each tube and mix

56. Add 300µL of acid phenol:chloroform and vortex for 5 seconds

57. Spin at 16k RPM in cold microfuge for 5 minutes

58. Carefully aspirate the aqueous layer with a P-200 and transfer to a new tube

59. Add 300µL chloroform:isoamyl alcohol and vortex for 10 seconds

60. Spin at 16k RPM in cold microfuge for 5 minutes

61. Carefully aspirate the aqueous layer with a P-200 and transfer to a new tube - avoid transferring any of the organic phase

62. Add 2.5µL glycoblue to each tube and mix well

63. PAUSEAdd 1mL 100% ethanol to each tube and store at -80 for > 1 hour or overnight

Resuspend RNA, quantify, and begin library prep

64. Pellet precipitations from above at 16k RPM in a microfuge for 30 minutes. Note carefully the location of the tube hinge and point it to the outside to locate the pellet on that side of the tube.

65. Keep tubes on ice when removing from the centrifuge

66. Aspirate supernatant carefully without disturbing the pellet with a P-1000 - recommended to not get too close to the pellet.

67. Quick-spin the tubes to collect the remainder of the ethanol

68. Aspirate the remainder of the ethanol with a P-10, again avoiding the pellet

69. Uncap the tubes and rest for 5 minutes at room temperature

70. Resuspend the pellet in 30µL H₂O

71. Measure concentration with nanodrop or qubit (recommended)

72. Begin library prep or use as-is for qPCR/etc.