PAT-Seqimmunoprecipitation Protocol

PAT-SeqImmunoprecipitation Protocol

Worm liquid culture

“Anti Anti Solution” (Pen/Step/AmpB) Gibco#15240

(or prepare yourself comparable mixture)

1M CaCl2

1M MgSO4

1M K-Citrate (pH6) 100mL:

21.01g Citric acid, monohydrate

Adjust pH with KOH (~17g) (temp will rise, Adjust pH at~25oC)

Autoclave

Cholesterol solution (1mL/L of S-Medium) 5mg/mL in ~95% Ethanol

Store at -20oC

S-Basal (1L/culture)4000mL :
23.4 gNaCl(0.1M Final)

4gK2HPO4(6mM Final)

24gKH2PO4(44mM Final)

4000mL dH2O

 Autoclave

4mL Cholesterol solution (may precipitate a bit)

S-Medium4000mL : ~3950 mL S-Basal (shake vigorously before using) (1L/3cultures)

12mLMgSO4 1M(3mM Final)

24mLCaCl2 0.5M(3mM Final)

40mLK-Citrate 1M, pH6(10mM Final)

Do not autoclave S-Medium

Optionally add 2x Antibiotic Antimycotic

 Cholesterol will form some precipitate, don’t worry about this

 Shake vigorously before using

pH will be around ~ 6 and osmolarity 370 mOsmol/kg (do not adjust)

Growing worm food:

Grow OP50 in LB-Lennox (5g NaCl per Liter) overnight

Check OD600 should be > 2.5

Spin down at 3000 rpm for 15min at 4oC

Store OP50 pellets at -20oC

Pellet of 1g of culture (OD600 ~2.5) is enough for 50,000 worms for one generation (L1  gravid Adult)

1. Add 250 ml S Medium to a sterilized 1-2 liter flask.
2. Inoculate the S Medium with a concentratedE. coliOP50 pellet made from 2-3 liters of an overnight culture.
3. Wash each of 4 large plates ofC. elegans(just cleared of bacteria) with 5 ml S Medium and add to the 250 ml flask.
4. Put the flask on a shaker at 20°C. Use fairly vigorous shaking so that the culture is well oxygenated.
5. Cultures should be monitored by checking a drop of the culture under the microscope. If the food supply is depleted (the solution is no longer visibly cloudy) add more concentratedE. coliOP50 suspended in S Medium. When there are many adult animals in each drop, the culture is ready to be harvested. This is usually on the 4thor 5thday.
6. Put the flask on ice for 15 minutes to allow the worms to settle.
7. Aspirate most of the liquid from the flask.
8. Transfer the remaining liquid to a 50 ml sterile conical centrifuge tube and spin for at least 2 min at 1150 × g to pellet the worms. Young larvae may take longer than 2 min to pellet.
9. Aspirate the remaining liquid.

The worms harvested from growth in liquid culture are usually longer and thinner than those grown on petri plates, and tend to hold their eggs. The number of adults, larvae and eggs obtained depends on the strain of worms used, the amount of bacteria provided and the length of time the culture is grown. Freshly cleared cultures will yield worms equal to half the weight of the bacteria used

RNA Immunoprecipitation (RIP)

Worm crosslinking and lysis

1. Harvest worms from 200ml liquid media onto empty LB agar plates

2. Crosslink on ice using Stratalinker 2400 with energy setting: 3J/cm2

3. Resuspend worms in a 50ml falcon tube, spin down to remove supernatant and then resuspend in 2mllysis buffer/ml of worms

4. Sonicate lysate using the following

- Program = 1 (5x with 10sec pulses, 50sec rest between pulses)

- Amplitude 20%

5. Spin at top speed at 4°C for 15 min.

6. If there is still debris or DNA floating around,spin one more time at 4°C for 5 min and transfer supernatant again.Keep samples on ice.

7. Take out 100ul of lysate to a new eppendorf tube and extract RNA using Trizol andChloroform reagents, precipitate with isopropanol supplemented with 2ul glycolbluecoprecipitant (NOTE: the gylcoblue carrier is optional but makes the pellet much easierto follow and recover). Resuspend in 30ul nuclease-free H20 and quantify using nanodrop.

M2 FLAG bead preparation and Immunoprecipitation

1. Transfer 100ul M2 FLAG beads to 2ml eppendorf tube.

2. Place on magnet and remove supernatant. Wash 2x with 200ul TBS. Then wash 2x

with 200ul Lysis Buffer. Make sure beads are on ice after washes.

3. Add amount of lysate corresponding to 90ug of RNA to tube with washed beads.

Incubate overnight at 4C on rotary wheel.

Immunoprecipitation and Protein degradation

1. Set thermomixer at 4°C. Make sure refrigerated centrifuge is at 4°C.

2. Make sure TBS buffer (used for washes) is cold.

3. Prepare Proteinase K solution (4mg/ml) in 1ml PK buffer, and incubate at 37C for 20mins to inactivate RNAses.

4. Put IP samples on magnet and remove supernatant (Can be used as supernatant

controls).

5. Wash 3x with 200ul ice-cold TBS. Shake in thermomixer at 4°C for 1min (1000rpm) between washes. Move to a fresh tube.

6. Wash 3x with 200ul ice-cold 1 PK buffer. Shake (4°C, 1min , 1000rpm) between washes.

8. You may take an aliquot for IP control at this point.

9. Add 200ul of proteinase K solution to washed bead pellet.

10. Incubate in thermomixer at 37C for 20 min at 1000 rpm.

11. Add 200 ul of 1x PK buffer + 7 M urea (0.42gr Urea in 1ml total volume of PK

buffer)

12. Incubate in thermomixer at 37C for 20 min at 1000 rpm. Bring Trizol to RT.

RNA Extraction

1. Add 800 ulTrizol, Shake, sit at RT for 5 min.

2. Add 200 ulcholorform. Shake for 30sec, sit at RT for 2min.

3. Spin at 12000g 1for 5 min at 4C.

4. Transfer sup to 1.5 ml tube with 550 ul chloroform. Shake, spin for 5 min at top speed at RT.

5. Transfer sup to 1.5 ml tube with 550 ul isopropanol, 2 ul glycogen and precipitate >1hr -20C or O/N.

6. Spin top speed for 10 min at 4C. Wash with 700 ul cold 75% EtOH. Spin top speed for 5 min at 4oC.

7. Remove sup, let sit at RT for 5-10 min. Spin down briefly if necessary to collect allethanol.

8. Add 30 ul nuclease-free H20, incubate at 42C for 5-10min to dissolve pellet.

9. Follow this with DNAse treatment (according to NEB-or other vendor- protocol) toremove potential gDNA contaminants.

Reagents

PBS:

10x, #70011, tissue culture grade, no Mg2+, no Ca2+, NOT DPBS that contains K

Proteinase K:
Invitrogen #25530-015

Lysis Buffer (10 ml per IP):

(from Zhang L. et al., Mol Cell, 2007)

Add 1 protease inhibitor tablet to every 10 ml

Tris-buffered Saline (TBS): 1ml per IP

50mM TrisHCl, 150mM NaCl

1x Proteinase K (PK) Buffer: 2ml per IP