TITLE: Salmaso Lab TRAP Adaptation
AUTHORS: Stephanie Simard

[DESCRIPTION]
This protocol details the procedure of Salmaso Lab TRAP Adaptation.

[BEFORE_START]
Day 3: mRNA Extraction & Quantification:

Before beginning; turn on the incubator to 37 °C; and the dry bath to Level 3-5 on the “low” setting. Place the sulfolane bottle on top of the dry bath to warm and liquify. 


[GUIDELINES]

 


The maximum number of IP that may be performed using a single 1.5 mL tube is limited to 1mL of reagent; this works out to a MAXIMUM of 6 IPs per tube, this protocol should be limited to using a maximum of 20 IPs per 3-Day protocol (limits of the equipment).


The main goals of the protocol are as follows:

Preparing the magnetic beads for incubation with your sample. This is achieved through washing the beads and then coating them in Protein L and then incubating the coated beads with anti-GFP
Homogenizing your tissues and preparing them to be incubated with anti-GFP coated magnetic beads
Extracting the mRNA that has been extracted from your tissues currently bound to the anti-GFP magnetic beads
Use RNAse Zap wipes throughout (Ambion AM#9786)


[STEPS]
SECTION: Day 1: Magnetic Bead Preparation
1. 
 

Gather the magnetic beads; pipet the solution up and down to mix the beads.
 

SECTION: Day 1: Magnetic Bead Preparation
2. Extract 360 µL of the magnetic beads into a 1.5mL low-bind tube [60 µL per IP]. Place the tube into the tube carriage and place onto the magnet. Wait for the beads to settle on the walls of the tube.

SECTION: Day 1: Magnetic Bead Preparation
3. Wash beads 3 times: Discard the excess liquid from the tube, wash with 1 mL of 1x PBS. Remove the tube carriage and shake to suspend magnetic beads in 1x PBS. Replace the magnet, discard the excess 1x PBS and repeat 2 more times. 

SECTION: Day 1: Magnetic Bead Preparation
4. Collect the beads on the magnet and remove all excess liquid. Incubate the beads with 144 µL of Protein L (1 µg/µL) [24 µL per IP]. Fill the tube with 1x PBS for a total volume of 1 mL, in this case 496 µL of 1x PBS. 

                (360 µL beads + 144 µL Protein L + 496 µL 1 x PBS = 1000 µL, 1 mL)

SECTION: Day 1: Magnetic Bead Preparation
5. Place the tube in the rotating rotisserie and spin slowly for ~60 min at Room temperature.
 



SECTION: Day 1: Magnetic Bead Preparation
6. While beads rotate, prepare 10 mL of 3% BSA in 1x PBS (weight/volume).

SECTION: Day 1: Magnetic Bead Preparation
6.1. Measure out 300 mg of solid BSA and add to a 15mL conical tube.

SECTION: Day 1: Magnetic Bead Preparation
6.2. Top up conical tube to 10 mL with 1x PBS, mix vigorously until BSA is dissolved.

SECTION: Day 1: Magnetic Bead Preparation
7. Also prepare 10 mL of FINAL 0.15 Molarity (M) KCl Wash Buffer in a 15mL conical tube by adding:

SECTION: Day 1: Magnetic Bead Preparation
7.1. 5 µL 1 Molarity (M) DTT.
 


SECTION: Day 1: Magnetic Bead Preparation
7.2. 10 µL activated 1000x CHX (100% MeOH, see dissection protocol).

SECTION: Day 1: Magnetic Bead Preparation
8. After beads have been incubated with Protein L, collect the beads using the magnet and discard excess liquid. Wash the beads with the 3% BSA solution 5 times.

 

SECTION: Day 1: Magnetic Bead Preparation
9. After the final wash, resuspend the beads with 175 µL of 0.15 Molarity (M) KCl and 47 µL of anti-GFP solution. Rotate 1440 min (~1440 min) in a walk-in cold room, or fridge (4 °C).

SECTION: Day 2: Tissue Homogenization and Bead Incubation
10. 
 

 

Prepare 1mL/sample of homogenization buffer from stock solution. Measure into a 15mL conical tube (try to make at least 10 mL at a time; up to 12 mL at a time maximum).
SECTION: Day 2: Tissue Homogenization and Bead Incubation
10.1. Add 1 protease inhibitor tablet per 10 mL (can use 1 tablet up to 12 mL) (Fisher #A32955).
SECTION: Day 2: Tissue Homogenization and Bead Incubation
10.2. 1 µL CHX per 1 mL of homogenization buffer.
SECTION: Day 2: Tissue Homogenization and Bead Incubation
10.3. 100units SUPERase (RNAse inhibitor) per sample (Ambion #AM2694).
SECTION: Day 2: Tissue Homogenization and Bead Incubation
10.4. 5 µL DTT per  10 mL of homogenization buffer (0.5 1824).
SECTION: Day 2: Tissue Homogenization and Bead Incubation
11. Retrieve samples from freezer. Move sample to a new, labelled 5mL tube on ice. Add 1 mL of homogenization buffer to each sample. Let sit on ice (to thaw) for ~ 5 min .

 

SECTION: Day 2: Tissue Homogenization and Bead Incubation
12. In a FUME HOOD, set up homogenization blade or pestle. Gather enough dH2O in a beaker to be able submerge the end of the blade or pestle. Turn on blade to a slow setting and submerge into homogenization buffer, moving the sample up and down until completely homogenized. Sample will be yellow and completely liquid. Clean blade with RNAse wipe + dH2O between each sample.
SECTION: Day 2: Tissue Homogenization and Bead Incubation
13. Transfer homogenized tissues back into a new, labelled 1.5mL low-bind tube. Spin at 20000 x g for 10 min at 4 °C. Collect supernatant, do not disturb the pellet, into a new, labelled 1.5mL low-bind tube.
SECTION: Day 2: Tissue Homogenization and Bead Incubation
15. Spin the samples at 20000 x g for 15 min at 4 °C.
SECTION: Day 2: Tissue Homogenization and Bead Incubation
16. Suspend the beads on the magnet, discard excess liquid and wash 3 times with 1 mL of 0.15 Molarity (M) KCl wash buffer.

 

SECTION: Day 2: Tissue Homogenization and Bead Incubation
17. After the 3rd wash, resuspend the beads using 1080 µL 0.15 Molarity (M) KCl Wash Buffer + 120 µL DHPC [180 µL 0.15 Molarity (M) KCl, 20 µL DHPC per sample].
 

SECTION: Day 2: Tissue Homogenization and Bead Incubation
18. Collect post-mitochondrial (S20), take care to avoid disturbing the pellet, into a new 1.5 tube. Collect 50 µL of the S20 post-mitochondrial supernatant into a new PCR tube, label and freeze at  -80 °C. 
 

SECTION: Day 2: Tissue Homogenization and Bead Incubation
19. Add 200 µL of the bead solution to each sample, place on the rotisserie and let rotate 10 min at 4 °C.

SECTION: Day 2: Tissue Homogenization and Bead Incubation
14. Add 100 µL 10% NP-40 & 115 µL DHPC (use a 1ml syringe because it is very thick/viscous) to each sample, mix with inversion and let sit on ice for 5 min.
SECTION: Day 3: mRNA Extraction and Quantification
20. 
 


Prepare 20 mL of FINAL 0.35 Molarity (M) KCl  in a 50mL conical tube by adding:
SECTION: Day 3: mRNA Extraction and Quantification
20.1. 10 µL 1 Molarity (M) DTT.
 

SECTION: Day 3: mRNA Extraction and Quantification
20.2. 20 µL activated 1000x CHX (in 100% MeOH, see dissection protocol).
SECTION: Day 3: mRNA Extraction and Quantification
21. Retrieve the rotating samples from the rotisserie and collect in the magnet caddie, wait until beads have settled on the magnet. Extract the liquid into a labelled 1.5mL tube, this is the “unbound” fragment used for validation.
 

SECTION: Day 3: mRNA Extraction and Quantification
22. Wash the beads with 200 µL of  0.35 Molarity (M) KCl  Wash Buffer 4 times.
SECTION: Day 3: mRNA Extraction and Quantification
23. During the 4th wash, prepare the lysis solution in the fume hood by mixing 600 µL lysis buffer with 4.2 µL βME  in a 1.5mL tube. 
 

(100 µL Lysis buffer, 0.7 µL βME per sample)

SECTION: Day 3: mRNA Extraction and Quantification
24. Resuspend each of the sample beads with 100 µL of the prepared lysis solution, mix well and let incubate at Room temperature for 10 min. Take this time to label out a new set of tubes, as well as the filters from the RNA extraction kit.
 

SECTION: Day 3: mRNA Extraction and Quantification
25. Place the samples back on the magnet and collect the liquid (which now has the mRNA) into a new, labelled, low-bind 1.5mL tube; begin with RNA extraction from the solution.
SECTION: Day 3: mRNA Extraction and Quantification
26. Add 100 µL of the warmed 80% sulfolane to each of the samples and mix by inversion. 
 

SECTION: Day 3: mRNA Extraction and Quantification
27. Make the DNAse soltution by combining 15 µL  reconstituted DNAse with 75 µL  DNAse Digestion Buffer. Set aside for now.

(2.5 µL DNAse, 12.5 µL DNAse Digestion Buffer per sample)
SECTION: Day 3: mRNA Extraction and Quantification
28. Put the whole sample into a labelled filter cup with a collection tube underneath, cap and spin at 12000 x g for 1 min at 4 °C.
 

SECTION: Day 3: mRNA Extraction and Quantification
29. Discard the liquid in the collection tube (filtrate), add 300 µL Low-Salt Wash  to the filter cup, spin at 12000 rpm for 1 min at 4 °C. Discard the filtrate and spin at 12000 x g for 2 min to dry the filter.
SECTION: Day 3: mRNA Extraction and Quantification
30. Add 14.5 µL DNAse + Digestion Buffer directly onto the filter, taking care not to touch the filter of each sample, incubate at 37 °C for 15 min. 
 

SECTION: Day 3: mRNA Extraction and Quantification
31. Add 300 µL High-Salt Wash to each sample, spin at 12000 x g for 1 min at 4 °C. Discard filtrate.
SECTION: Day 3: mRNA Extraction and Quantification
32. Add 300 µL  Low-Salt Wash to each sample, spin at 12000 x g for 1 min at 4 °C. Discard filtrate.
SECTION: Day 3: mRNA Extraction and Quantification
33. Add 300 µL  Low-Salt Wash to each sample, spin at 12000 x g for3 min at 4 °C. Discard filtrate.
SECTION: Day 3: mRNA Extraction and Quantification
33.1. Spin at 12000 x g for 1 min at 4 °C. Discard filtrate and collection tube. Replace with a new collection tube.
SECTION: Day 3: mRNA Extraction and Quantification
34. Add 20 µL heated Elution Buffer directly to the filter of each sample, incubate at  Room temperature for 2 min.
SECTION: Day 3: mRNA Extraction and Quantification
34.1. Spin samples at 12000 x g for 5 min at 4 °C.
SECTION: Day 3: mRNA Extraction and Quantification
35. Collect the resulting filtrate into a cold PCR tube, label as the “Primary Elution” (1°E).
SECTION: Day 3: mRNA Extraction and Quantification
36. Repeat steps 34 & 35, labelling the second set of PCR tubes as “Secondary Elution” (2°E).
SECTION: Day 3: mRNA Extraction and Quantification
37. Bring 1°E & 2°E to the NanoDrop; record the RNA concentrations for each sample and the 260nm/280nm values.
SECTION: Day 2: Tissue Homogenization and Bead Incubation
10.5. Mix the solution well, vigorously and until the protease inhibitor has dissolved completely (this may take a few minutes). Set aside on ice, keep cold.
SECTION: Day 2: Tissue Homogenization and Bead Incubation
15.1. During the 15-minute cycle, begin to prepare the beads to be incubated with the sample. Prepare ~10 mL- 15 mL of 0.15 Molarity (M) KCl Wash Buffer.
SECTION: Day 3: mRNA Extraction and Quantification
38. Freeze the samples at -20 °C for short term use (~2 weeks) or -80 °C for a longer period (good for up to 2 years, depending on storage conditions).