TITLE: RNAi Library screen
AUTHORS: e.warren

[DESCRIPTION]
Protocol for screening a RNAi knockdown library in for behavioural and phenotypic effects in C. elegans

[STEPS]
SECTION: Perparing C. elegans
1. Prepare in advance
Prepare ten 90 mm NGM agar plates for each replicate of the screen for worm maintenance.  

To prepare NGM follow the steps in the protocol for "Making normal NGM" and pour 35 ml per plate.
 

SECTION: Perparing C. elegans
3. 11 days before tracking
Chunk worms onto ten seeded 90 mm NGM agar plates.
SECTION: Perparing C. elegans
2. Seed each NGM agar plate with 1 ml of OP50 bacteria, and allow to dry.
SECTION: Prepare tracking plates
4. 11 days before tracking
Prepare 75 tracking plates (WHAT7701-1651, PK100 : 512-1093) containing NGM agar, prepared as above, with the addition of 1 mM IPTG, 100 µg/ml carbenicillin, 10 µg/ml tetracycline, 1x nystatin (60 Units/ml). 
Dispense 200 µl per well using the protocol "Dispensing agar into multiwell plates".
 

SECTION: Prepare tracking plates
4.1. Dry the tracking plates in a drying oven (with lids removed) until they lose 2.5 to 3.5% of their total weight. 
SECTION: Bleach synchronising C. elegans
5. 8 days before tracking
Bleach synchronise worms from ten 90 mm plates according to the protocol "Bleach synchronisation of C. elegans". 
Leave the eggs in M9 buffer in 15 ml faclon tubes on a rotator at 20 dgerees for two days.
 

SECTION: Grow up RNAi library
6. We are using a version of the Ahringer RNAi library containing 6198 C. elegans genes with orthologues in humans. 
This library is stored as frozen glycerol stocks in seventy three 96 well plates.

8 days before tracking
Grow up the RNAi library. 

 

 

SECTION: Grow up RNAi library
9. Place the LB plates containing the replicated library into large plastic boxes containing moistened paper towel, and incubate for 15 hours at 37 degrees.

SECTION: Grow up RNAi library
10. Record OD600 values of 10 of the library plates using a plate reader (Tecan, Spark) to evaluate library growth. 
Visually check the whole library and manually record any wells which have not grown.
SECTION: Seeding tracking plates
11. 7 days before tracking 
Seed the tracking plates with the bacterial cultures using the Integra VIAFLO. 

Using the Integra VIAFLO with GRIPTIPs mix each well of the library by pipetting 12.5 µl up and down 6 times, before transferring 10 µl from the library to the tracking plates twice.


 


SECTION: Seeding tracking plates
12. Dry the tracking plates in a biosafety cabinet with lids removed for 3 hours.
SECTION: Dispensing C. elegans to tracking plates
14. 6 days before tracking
Dispense C. elegans onto the tracking plates using the protocol "Dispensing C. elegans to 96 well tracking plate using Integra VIAFILL". 

We aimed to dispense an average of 4 worms per well, but will have some unavoidable variation between wells.
 


 


SECTION: Seeding tracking plates
13. Stack the tracking plates plates (upside down) in a sealed plastic box containing damp paper towel and incubate at 20 degrees.
SECTION: Dispensing C. elegans to tracking plates
15. Leave plates to dry for 1 hour in a biosafety cabinet with lids removed.

SECTION: Addition of FUDR
17. 4 days before tracking, 
53 hours after dispensing worms, add FUDR to each well of the tracking plates to prevent hatching of progeny.

DIspense 10 µl of 20X FUDR to each well using the Integra VIAFILL with the small 8 channel cassette.
SECTION: Addition of FUDR
18. Leave plates to dry for 1 hour in a biosafety cabinet with lids removed.
 

SECTION: Tracking using Hydra rigs
20. On the day of tracking

Move tracking plates into the tracking room at 12 pm, one hour before tracking commences to aclimatise to temerature and humidity.
SECTION: Tracking using Hydra rigs
21. Record worm behaviour using the syngenta script (5 mins prestim; 6 mins bluelight with 60 sec OFF, [10sec ON, 90sec OFF] x 3 times; 5 mins postsim).
SECTION: Tracking using Hydra rigs
21.1. Run the following script
~/scripts$ python run_syngenta_experiment_v2.py -f 291122_rnaifullscreen_run1 – r 01 02 03 04 05 

-f : date_file_ run number :
-r : specifies which rigs to start recording on

 

SECTION: Tracking using Hydra rigs
22. Move all files to the NAS and then transfer files from the NAS to the group drive on the network for processing. 
SECTION: Grow up RNAi library
7. Dispense LB broth (containing 100 µg/ml Carbenicillin, and 10 µg/ml Tetracycline), into seventy four 96 well plates (numbered 1-74) using the Integra VIAFILL.
SECTION: Grow up RNAi library
8. Replicate all wells of the RNAi library into corresponsing plates of LB (with carbenicillin and tetrecycline).
SECTION: Grow up RNAi library
8.1. Use a metal 96 pin replicator (Boekel) to transfer glycerol stock from stock plates to the LB culture plates. 

To do this remove plates a few at a time from the -80 on dry ice.
Allow each plate around 1 minute to deforst slightly.
Firmly press the replicator into the stock plate, making sure to pick up bacteria from each well.
Transfer replicator to corresponding LB culture plate.
SECTION: Grow up RNAi library
8.2. Between replicating each plate, the replicator is dipped in 100% ethanol and flame sterilised. This process is repeated twice to ensure full sterilisation.

One negative control plate is inncoluated after double flame sterilisation of the replicator.
 

SECTION: Dispensing C. elegans to tracking plates
16. Stack the tracking plates plates (upside down) in a sealed plastic box containing damp paper towel and incubate at 20 degrees.
SECTION: Addition of FUDR
19. Stack the tracking plates plates (upside down) in a sealed plastic box containing damp paper towel and incubate at 20 degrees.