TITLE: Generating multiple stage-matched C. elegans hybrids and parental strains simultaneously
AUTHORS: Avery Davis Bell, Francisco Valencia, Annalise Paaby

[DESCRIPTION]
Here we provide a protocol for generating RNA from synchronized C. elegans F1s from crosses of multiple wild strains to a common reference strain (for example, for allele specific expression analysis). This protocol, which comprises 9 sequential days (6 active days) followed by RNA extraction, is optimized for synchronization of parental and offspring worms, retention of embryos, and timing. Others could easily adapt this protocol for crossing designs other than the specified multiple wild strains x common reference. This protocol accommodates 8 concurrent crosses and one extra parent with 3-4 active worm pickers on Days 3 and 6, and should accommodate increased or decreased numbers of crosses with commensurate personnel adjustments.






[STEPS]
SECTION: Worm maintenance prior to start of experiment
1. In general, follow standard worm culture procedures (Stiernagle 2006). Grow worms at 18°C or cooler if any strains have known or suspected mortal germline phenotypes (Frezal et al. 2018)
SECTION: Worm maintenance prior to start of experiment
2. Grow healthy cultures of each strain that will be a parental strain. For the reference strain that will be crossed to all wild strains, make sure to have many plates growing (e.g., 15 growing plates).
SECTION: Worm maintenance prior to start of experiment
3. For all strains that will be crossed to the common reference strain, start and propagate male-heavy cultures (to have enough males to mate)
SECTION: Worm maintenance prior to start of experiment
3.1. Set up mating plates with 20-30 hermaphrodites and as many males as you can find (as few as 3 and up to 45) from the wild strain
SECTION: Worm maintenance prior to start of experiment
3.2. Repeat setting up mating plates until you can visibly see a lot of males; then, these male cultures can be propagated as normal (e.g., through chunking)
SECTION: Worm maintenance prior to start of experiment
4. For all strains, keep the original hermaphrodite-heavy culture going
SECTION: Cross set up
5. Day 1. Bleach synchronize all parental strains and re-plate embryos.
SECTION: Cross set up
5.1. Inspect plates, only use plates that are not starved.
SECTION: Cross set up
5.2. Wash 2-3 plates of the wild strains and 10-15 plates of the reference strain to a conical 15 mL tube using water.
SECTION: Cross set up
5.3. Centrifuge tubes (2000 rpm for 2 minutes) and remove the supernatant careful of not disturbing the worm pellet.
SECTION: Cross set up
5.4. Resuspend pellet with 7 mL of water and add 3 mL of a 2:1 Bleach:NaOH (5M) bleaching solution. Gently shake tubes constantly for 5-7 minutes. During this time, inspect tubes using a stereoscope. When carcasses of the worms are largely dissolved (often around 7 minutes), centrifuge tubes as described before.
SECTION: Cross set up
5.5. Remove the bleaching solution from the tube and resuspend the pellet in 13 mL of water. Shake tubes vigorously and centrifuge as before.
SECTION: Cross set up
5.6. Repeat previous washing step one more time. Remove washing solution (water).
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5.7. Resuspend embryos in water and transfer to NGM/Agarose plates as necessary.
SECTION: Cross set up
5.8. Make sure to have several growing plates per strain to distribute across temperatures.
SECTION: Cross set up
6. Grow reference-strain worms on growing plates at multiple temperatures (e.g., 18°C, 19°C, 20°C, and room temperature) to ensure worms at the proper stage of development will be available throughout a wide window when generating mating plates. Grow wild worm cultures at a couple of these temperatures.
SECTION: Cross set up
7. Day 3 (starting 48 hours after bleach syncing):  set up mating plates.
SECTION: Cross set up
7.1. Add 60 L4 hermaprodites from the reference strain to each of 5 mating plates per wild strain.
SECTION: Cross set up
7.2. Screen these plates to ensure no males are on them.
SECTION: Cross set up
7.3. Add 40 L4 males from the wild strain to the 5 mating plates for that strain.
SECTION: Cross set up
7.4. Add 80 L4 hermaphrodites from each of the wild strains to each of 3 mating plates for each strain. (The selfed offspring of these worms will be the matched ‘parent’ worms for the F1 crosses)
SECTION: Cross set up
8. Leave worms to mate for 48 hours at 20°C
SECTION: Synchronizing experimental generation embryos
9. Day 5 after allowing 48 hours of mating: wash all worms, bacteria, and embryos from one cross (or parental selfing) into a single 15mL conical tube, being careful to collect all embryos.
SECTION: Synchronizing experimental generation embryos
9.1. Rinse plates twice with water, then gently swipe tip of glass pipet back and forth across the surface of the plate while expelling water, holding plate at an angle. Goal is to remove all the embryos but not damage them, and not dislodge any agar into the liquid.
SECTION: Synchronizing experimental generation embryos
9.2. Repeat several times, reusing the same water (collecting in bottom edge of plate; very gently but methodically swipe the pipet tip back and forth across the surface of the plate in a comprehensive pattern), until lawn is completely removed by eye.
SECTION: Synchronizing experimental generation embryos
9.3. Check under scope, re-focus on rinsing remaining areas as needed.
SECTION: Synchronizing experimental generation embryos
10. Add water to 14mL (so that all tubes have equal volume)
SECTION: Synchronizing experimental generation embryos
11. Spin 2000rpm x 2 min
SECTION: Synchronizing experimental generation embryos
12. Use vacuum to remove most of liquid, taking care not to disrupt loose pellet (remove as much bacteria as possible – but don’t risk the pellet!).
SECTION: Synchronizing experimental generation embryos
13. Resuspend each tube to 3.5mL with fresh water
SECTION: Synchronizing experimental generation embryos
14. Add 1.5 mL bleaching solution (2:1 bleach:NaOH). Start timer.
SECTION: Synchronizing experimental generation embryos
15. Shake tubes every 30s, examine.
SECTION: Synchronizing experimental generation embryos
16. When adult carcasses fully dissolve (often around 7 minutes), add water to each tube up to 14mL.
SECTION: Synchronizing experimental generation embryos
17. Spin 2000 rpm x 2 min.
SECTION: Synchronizing experimental generation embryos
18. Vacuum and long glass pipet aspirate bleaching solution, getting as close to pellet as is safe.
SECTION: Synchronizing experimental generation embryos
19. Resuspend to 10mL with water and shake
SECTION: Synchronizing experimental generation embryos
20. Spin 2000 rpm x 2 min.
SECTION: Synchronizing experimental generation embryos
21. Vacuum aspirate only to the 1.5mL mark (avoid embryo loss).
SECTION: Synchronizing experimental generation embryos
22. Resuspend with water + 0.25% tween-20 to 15mL
SECTION: Synchronizing experimental generation embryos
23. Spin 2000 rpm x 2 min, being careful to position the tubes within the centrifuge so the tube sides with the interior striations are not on the inside where embryos would collect.
SECTION: Synchronizing experimental generation embryos
24. Vacuum aspirate as close to the pellet as possible.
SECTION: Synchronizing experimental generation embryos
25. Resuspend with water (to wash out detergent)
SECTION: Synchronizing experimental generation embryos
26. Spin 2000 rpm x 2 min
SECTION: Synchronizing experimental generation embryos
27. Vacuum aspirate as close to the pellet as possible
SECTION: Synchronizing experimental generation embryos
28. Resuspend embryo pellet to 10mL with M9 buffer.
SECTION: Synchronizing experimental generation embryos
29. Leave tubes gently rocking at room temperature. Embryos will develop into L1s and arrest.
SECTION: Growing synchronized experimental generation L1s
30. Day 6/7 – 30ish hours after leaving embryos in buffer. Add tween-20 to 0.25% to each tube
SECTION: Growing synchronized experimental generation L1s
30.1. Add 25 μl Tween-20 directly to the synced worms in tubes
SECTION: Growing synchronized experimental generation L1s
30.2. OR, combine 500 μl Tween-20 to 4mL of water and mix well, then add 200uL of this diluted solution to the synced worms in tubes
SECTION: Growing synchronized experimental generation L1s
31. Spin 2000rpm x 2 min
SECTION: Growing synchronized experimental generation L1s
32. Aspirate using vacuum, leaving under 0.5mL volume in tube. The pellet of L1 worms is loose, so take care not to disturb it.
SECTION: Growing synchronized experimental generation L1s
33. Quick count worms to determine their density. For each tube:
SECTION: Growing synchronized experimental generation L1s
33.1. Add 10μl of mixed worm mixture as a single droplet to an empty 6cM plate
SECTION: Growing synchronized experimental generation L1s
33.2. Roughly count the number of L1s immediately after adding the droplet by eye under the scope. Record this number.
SECTION: Growing synchronized experimental generation L1s
33.3. Calculate the volume of liquid containing 400 worms
SECTION: Growing synchronized experimental generation L1s
34. Add 400 worms to 3 or more  un-syncing plates (6cM large-lawn plates) per strain.
SECTION: Growing synchronized experimental generation L1s
34.1. Spot directly onto the lawn
SECTION: Growing synchronized experimental generation L1s
34.2. Split the 400 worms into multiple smaller spots on the lawn if possible to facilitate drying
SECTION: Growing synchronized experimental generation L1s
34.3. Leave lids off the plates until droplets absorb, typically a few minutes
SECTION: Growing synchronized experimental generation L1s
35. Leave closed plates at 20°C
SECTION: Male removal
36. Day 8. Remove males from at least 3 plates from each strain. F1s will be ~50% males so this is a challenge; parentals only have spontaneous males so just checking them a couple times should suffice.

Males start to be visible ~32 hours after un-syncing, however, they’re very young. Males are much more evident 36+ hours after un-syncing.
SECTION: Worm harvesting
37. Day 9. Prepare liquid nitrogen for flash-freezing worms after harvest.
SECTION: Worm harvesting
38. Identify strains where most hermaphrodites on the plate are gravid and embryos have been laid on the plate, typically 55-60 hours after un-syncing at 20°C. Several strains may become mature young adults like this at once while others lag; all strains should reach this within a few hours on this day.
SECTION: Worm harvesting
38.1. While looking at strains, remove any straggler males that made it through the initial screen.
SECTION: Worm harvesting
38.2. Group strains that are developmentally similar and do the following harvesting in these similar groups.
SECTION: Worm harvesting
39. Rinse all plates from one strain with M9 buffer into 15mL tubes
SECTION: Worm harvesting
39.1. Re-use 1mL of buffer on several plates
SECTION: Worm harvesting
39.2. Make sure each plate gets rinsed at least twice
SECTION: Worm harvesting
39.3. Check to make sure all worms are removed, especially for strains with fewer worms (F1s). Do not scrape up the bacteria or rinse embryos, focus on adult worms.
SECTION: Worm harvesting
40. Spin 2000rpm x 2 min
SECTION: Worm harvesting
41. Remove buffer using vacuum down close to pellet
SECTION: Worm harvesting
42. Resuspend in 13 mL M9 buffer.
SECTION: Worm harvesting
43. Spin 2000rpm x 2 min
SECTION: Worm harvesting
44. Remove buffer using vacuum down close to pellet
SECTION: Worm harvesting
45. Add TRIzol to a total volume of ~1500 μl /1.5mL. (this is probably adding ~1200uL TRIzol, but try to have final volume of worms + trizol consistent across strains).
SECTION: Worm harvesting
46. Transfer equivalent volume of worms in trizol to each of 3 1.5mL centrifuge tubes, ending with ~500μl per tube. Using smallest glass pipette works well.
SECTION: Worm harvesting
47. Quick spin down tubes
SECTION: Worm harvesting
48. Flash freeze tubes in liquid nitrogen
SECTION: Worm harvesting
49. Store at -80°C until ready to proceed with RNA extraction.
SECTION: RNA extraction (following He 2011)
50. Set up for RNA extraction. Full details on reagents and equipment are found in the appropriate sections; this step describes set up for this specific experiment.
Plan to extract RNA in batches of no more than 18 samples/batch, with one sample per strain in each batch (likely 1 or 3 batches)
Equipment:
Heat block set to 37°C
Procure dewer of liquid nitrogen for freeze-thawing
Centrifuge at (or set to) room temperature
Centrifuge at (or set to) 4°C
Reagents:
TRIzol (fume hood!)
Chloroform (fume hood!)
Freshly prepared 70% ethanol
Qiagen RNeasy buffer RW1
Qiagen RNeasy buffer RPE (with ethanol added as directed by the kit)
RNAse-free water
Sets of tubes to label:
1.5mL tubes for aqueous phase after TRIzol-chloroform spin
RNeasy spin columns
1.5mL tubes for eluted RNA collection
Clean with RNAse away:
Gloves
Fume hood
Outside of tip boxes, reagents to be used
Freeze thaw area
Centrifuge area
SECTION: RNA extraction (following He 2011)
51. Break up worms
SECTION: RNA extraction (following He 2011)
51.1. Vortex worms (previously stored at -80°C in TRIzol) for 2 minutes
SECTION: RNA extraction (following He 2011)
51.2. Thaw worms at 37°C
SECTION: RNA extraction (following He 2011)
51.3. Quick spin tubes
SECTION: RNA extraction (following He 2011)
51.4. Freeze by submerging tube in liquid nitrogen
SECTION: RNA extraction (following He 2011)
51.5. Thaw at 37°C
SECTION: RNA extraction (following He 2011)
51.6. Repeat the previous 3 steps for a total of 6 freeze thaw cycles. Spin down.
SECTION: RNA extraction (following He 2011)
52. TRIzol-chloroform extraction (do in fume hood)
SECTION: RNA extraction (following He 2011)
52.1. Add 200μl fresh TRIzol to each tube
SECTION: RNA extraction (following He 2011)
52.2. Incubate for 5 minutes at room temperature
SECTION: RNA extraction (following He 2011)
52.3. Add 140μl chloroform to each tube
SECTION: RNA extraction (following He 2011)
52.4. Shake at 1450 rpm for 1 minute (make sure thorough mixing has occurred)
SECTION: RNA extraction (following He 2011)
52.5. Incubate for 2 min at room temperature
SECTION: RNA extraction (following He 2011)
52.6. Spin at 12,000g for 15 minutes at 4°C
SECTION: RNA extraction (following He 2011)
52.7. Remove top aqueous phase to new tubes; this is what will be used downstream
SECTION: RNA extraction (following He 2011)
52.8. Add 1 volume of fresh 70% ethanol to the aqueous phase; mix and spin down
SECTION: RNA extraction (following He 2011)
53. RNA elution using Qiagen columns
SECTION: RNA extraction (following He 2011)
53.1. Transfer 650μl of each sample to a Qiagen RNeasy spin column (1 per sample)
SECTION: RNA extraction (following He 2011)
53.2. Spin at max speed for 30 seconds; discard flow through
SECTION: RNA extraction (following He 2011)
53.3. Add remaining sample to Qiagen RNEasy spin column with 700μl buffer RW1 (from the Qiagen kit)
SECTION: RNA extraction (following He 2011)
53.4. Spin at max speed for 30 seconds; discard flow through
SECTION: RNA extraction (following He 2011)
53.5. Add 500 uL buffer RPE (from Qiagen kit)
SECTION: RNA extraction (following He 2011)
53.6. Spin at max speed for 30 seconds; discard flow through
SECTION: RNA extraction (following He 2011)
53.7. Repeat buffer RPE wash (add, spin, discard – previous two steps)
SECTION: RNA extraction (following He 2011)
53.8. Spin column at max speed for 2 minutes to remove any residual buffer
SECTION: RNA extraction (following He 2011)
53.9. Transfer spin column to new tube (labeled, where RNA will be collected)
SECTION: RNA extraction (following He 2011)
53.10. Add 50uL RNAse-free water to column
SECTION: RNA extraction (following He 2011)
53.11. Incubate for 1 min at room temperature
SECTION: RNA extraction (following He 2011)
53.12. Spin at max speed for one minute, retain eluant
SECTION: RNA extraction (following He 2011)
53.13. Place samples on ice; quantify; store at -80°C