TITLE: Prestwick screen protocol
AUTHORS: Ida Barlow, Thomas O'brien

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for screening the Prestwick </span><span style = "font-style:italic;">C. elegans</span><span> drug library of 240 drugs at 3 concentrations and imaging under baseline and bluelight conditions using the Hydra (Loopbio) imaging rigs. All drugs at all concentrations were imaged with N2 in a single day of tracking. This protocol was repeated 3 times so that there were 12 replicates per drug per concentration.</span></div></div>

[STEPS]
?. [Pick L4 worms for bleaching (-9 days from first day of tracking)]
Pick 10 x N2 L4s onto 8 x 90mm plates (pre-seeded with OP50)
?. [Pour 96WPs (up to 3 days before tracking)]
Prepare 1L low peptone NGM and autoclave
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?. [Pour 96WPs (up to 3 days before tracking)]
Once agar has cooled to around  , add the salts and dispense agar into 40 x square well 96 well plates using VIAFILL dispenser. Dispense   per well. Once cooled, store agar side up in an airtight container at
55 °C
200 µl
4 °C
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?. [Dry plates, bleach worms (-5 days)]
Dry 15 x 150mm plates in cabinet dryer (setting 2) for 3 hours
?. [Dry plates, bleach worms (-5 days)]
Seed 15 x 150mm plates with OP50 and leave to dry overnight at room temperature
?. [Dry plates, bleach worms (-5 days)]
Bleach worms prepared for day 1 of tracking
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?. [Refeed L1s (-3 days)]
At 11:00, spin L1s for day 1 of tracking at 2500rpm. Remove supernatent and using glass pipette, drop 4 small droplets around the edges of the plate (off food) onto 14 x 150mm platesAllow to grow at See above Step (6) for bleach synchronisation of C. elegans.
20 °C
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
In the morning, take 40 poured 96 well plates from the cold room, and weigh three random plates without their lids
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Place in cabinet dryer (setting 2) and allow to dry for 2-3 hours with lids off
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Weigh 3 random plates and verify that at least 3-5% reduction in weight
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Pre-label dried imaging plates (square well) with the imaging run and drug plate information, so that every plate on a single day of imaging has a unique plate id, for example P01_sh01_01 where:P01-09 - library plate number (out of 4 library plates)sh01-04 - shuffle number (out of the 4 shuffled stock plates)01-07 - imaging run number for that day
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Remove the shuffled library plates (stock) plates from the freezer, allow to thaw at room temperature and spin to collect contents at bottom of wells
-20 °C
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Pre-label the skirted 96 well plates with library plate IDs (P01-L09_sh01-sh04) to make up diluted drug plates
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Dispense water into each well of the prelabeled dilution plates using multichannel pipette and reagent reservoir
7 µl
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Using VIAFLO (hedgehog) dispenser on BG_STOCK custom program, premix drug in drug library plates in slot A, and then transfer drug in DMSO to the dilution plates prefilled with water in slot B. Repeat for all drug library plates
0.5 µl
Double check the dispense volumes before making up the diluted platesZ-heights have been configured in this program to prevent pipette and plate crashes
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Using VIAFILL (octopus) dispenser, dispense  water onto 5 x predried imaging plates
5 µl
5 plates at a time to prevent the agar absorbing all the liquid before the drug is dispensed into the water droplet
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Using VIAFLO in custom program BG_AGAR, with correct drug library plate in slot B, transfer  of diluted drug and water mixture onto the correctly labelled imaging plate in slot A.Repeat until all imaging plates have had drug dispensed onto them.
3 µl
Z-heights have been configured in this program to prevent pipette tips from piercing the agar
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Prepare 1:10 dilution of OP50 in M9 in a small bottle: OP50 M9
5 mL
45 mL
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Using VIAFILL dispenser, seed all the imaging plates with  per well
5 µl
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Place lids on each plate and leave drugged and seeded imaging plates overnight at room temperatue in the dark (with box ontop)
?. [Imaging]
Wash worms off half (7) of the 150mm plates with M9 buffer using pasteur pipette into 15ml falcons
?. [Imaging]
Spin at 1500rpm for 2 minutes to pellet the worms
?. [Imaging]
Remove supernatent and fill with M9
?. [Imaging]
Repeat steps 23-24
?. [Imaging]
After final wash, fill falcon with M9 and transfer contents from 15ml falcon to 3 x 50ml and fill up 30ml with M9
?. [Imaging]
Use COPAS wormsorter to dispense 3 worms per well (pure, no double) into 5 imaging plates at a time. Use pre-made YYYYMMDD_wormsorter.csv to plan and determine which plates to dispense onto
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?. [Imaging]
Allow liquid to dry off by placing imaging plates in  incubator with lid off for 30 minutes, then replace lids and keep in  incubator
20 °C
20 °C
?. [Imaging]
Expose worms to drug for 4 hours in total as calculated from the middle wormsorter time and allow worms to acclimate for 30 minutes in the cave prior to imaging
Example:wormsorter start time 10 :00wormsorter end time 10:40middle wormsorter time 10:20cave time 13:50Imaging start time 14:20
?. [Imaging]
Imaging on hydra using protocol script (5 mins prestim; 6 mins bluelight with 60 sec OFF, [10sec ON, 90sec OFF] x 3 times; 5 mins postsim)
python3 ~/scripts/run_syngenta_experiment_v2.py
?. [Imaging]
After 15 plates have been filled with this batch of N2s, repeat steps 23-30 with the other batch of N2s on 150mm plates and record which batch was used in each plate in the wormsorter.csv file.
?. [Dry plates and dispense drugs onto imaging plates usinv VIAFLO (-1 days from imaging)]
Leave plates to dry for approx 15 mins, so all drugs are absorbed into the agar.