TITLE: Syngenta divergent strain screen protocol
AUTHORS: Ida Barlow, Adam Mcdermott-Rouse, Luigi Feriani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for screening the 12 divergent </span><span style = "font-style:italic;">C. elegans</span><span> strains with 100 Syngenta pesticide drugs at 3 concentrations and imaging under baseline and bluelight conditions using the Hydra (Loopbio) imaging rigs. The twelve strains were imaged over two days of tracking and on each day of tracking six strains were imaged across all drugs and all concentrations with 3 replicates for all conditions. This protocol should be repeated at least 3 times.</span></div></div>

[STEPS]
?. [Pick L4 worms for bleaching (-9 days from first day of tracking)]
Pick 10 x L4s for 6 strains onto 3 x 90mm plates (pre-seeded with OP50) for each strain
?. [Pick L4 worms for bleaching and pour 96WPs (-7 days)]
Pick 10 x L4s for 6 strains onto 3 x 90mm plates (pre-seeded with OP50) for each strain
?. [Pick L4 worms for bleaching and pour 96WPs (-7 days)]
Prepare 2 x 1.5L low peptone NGM and autoclave
?. [Pick L4 worms for bleaching and pour 96WPs (-7 days)]
Once agar has cooled to around  , add the salts and dispense agar into 140 x square well 96 well plates using VIAFILL dispenser. Dispense   per well. Once cooled, store agar side up in an airtight container at
65 °C
200 µl
4 °C
?. [Dry plates, bleach worms (-5 days)]
Bleach worms prepared for day 1 of tracking
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?. [Dry plates, bleach worms and refeed L1 (-3 days)]
Bleach worms prepared for day 2 of tracking
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?. [Dry plates, bleach worms (-5 days)]
Dry 20 x 150mm plates in cabinet dryer (setting 2) or 3 hours
?. [Dry plates, bleach worms and refeed L1 (-3 days)]
Dry 20 x 150mm plates in cabinet dryer (setting 2) or 3 hours
?. [Dry plates, bleach worms and refeed L1 (-3 days)]
Seed 20 x 150mm plates with OP50 and leave to dry overnight at room temperature
?. [Dry plates, bleach worms and refeed L1 (-3 days)]
At 17:00, 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 3 x 150mm plates per strain.Allow to grow at
20 °C
?. [Dry 96 well plates  (-2 days)]
Take 65-70 poured 96 well plates from the cold room, and weigh three random plates without their lids
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
Pre-label 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 L01_s01_01 where:L01-04 - library plate number (out of 4 library plates)sh01-03 - shuffle number (out of the 3 shuffled stock plates)01-13 - imaging run number for that day
?. [Dry plates, bleach worms (-5 days)]
Seed 20 x 150mm plates with OP50 and leave to dry overnight at room temperature
?. [Dry 96 well plates  (-2 days)]
Place in cabinet dryer (setting 1.5-2) and allow to dry for 2-3 hours with lids off
?. [Dry 96 well plates  (-2 days)]
leave overnight at room temperature with lid
?. [Dry 96 well plates  (-2 days)]
Weigh 3 random plates and verify that at least 3-5% reduction in weight
?. [Refeed L1s for day 2 of tracking (-1 days for day 1 of tracking)]
At 17:00, spin L1s for day 2 of tracking at 2500rpm. Remove supernatent and using glass pipette, drop 4 small droplets around the edges of the plate (off food) onto 3 x 150mm plates per strain.Allow to grow at
20 °C
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
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
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
Pre-label the appropriate (11) skirted 96 well plates with library plate IDs (L01-L04_sh01-sh03) to make up diluted drug plates
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
Dispense water into each well of the prelabeled dilution plates using multichannel pipette and reagent reservoir
19.6 µl
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
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
1.4 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
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
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
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
Using VIAFLO in custom program BC_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
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
Using VIAFILL dispenser, seed all the imaging plates with  per well
5 µl
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
Prepare 1:10 dilution of OP50 in M9 in a small bottle: OP50 M9
5 mL
45 mL
?. [Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)]
Place lids on each plate and leave drugged and seeded imaging plates overnight at room temperatue in the dark (with box ontop)
?. [Day 1 imaging]
Wash worms off 150mm paltes with M9 buffer using pasteur pipette into 15ml falcons
?. [Day 1 imaging]
Spin at 1500rpm for 2 minutes to pellet the worms
?. [Day 1 imaging]
Remove supernatenet and fill with M9
?. [Day 1 imaging]
Repeat steps 28-29
?. [Day 1 imaging]
After final wash, fill falcon with M9 and transfer contents from 15ml falcon to 2 x 50ml and fill up 30ml with M9
?. [Day 1 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 strains to dispense into each plate.
Each strain will sequentially be dispensed into 10.5 imaging plates.
?. [Day 1 imaging]
Allow liquid to dry off by placing imaging plates in  incubator with lid off for 30 minutes, then replace lids and keep in 20oC incubator
20 °C
?. [Day 1 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 11:00middle wormsorter time 10:30cave time 12:00Imaging start time 12:30
?. [Day 1 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)
?. [Prepare imaging plates for day 2 of imaging (on same day as day 1 of imaging)]
Take 65-70 poured 96 well plates from the cold room, and weigh three random plates without their lids
?. [Prepare imaging plates for day 2 of imaging (on same day as day 1 of imaging)]
Place in cabinet dryer (setting 1.5-2) and allow to dry for 2-3 hours with lids off
?. [Prepare imaging plates for day 2 of imaging (on same day as day 1 of imaging)]
Weigh 3 random plates and verify that at least 3-5% reduction in weight
?. [Prepare imaging plates for day 2 of imaging (on same day as day 1 of imaging)]
leave overnight at room temperature with lid
?. [Perpare drug imaging plates for day 2 of imaging (+1 from day 1 of imaging)]
Repeat steps 16-25 for the other 6 strains
?. [Day 2 imaging]
Repeat steps 27-35