Protocol for nuclear dissociation of the adult C. elegans for single-nucleus RNA sequencing and its application for mapping environmental responses

Summary Caenorhabditis elegans is a valuable model to study organ, tissue, and cell-type responses to external cues. However, the nematode comprises multiple syncytial tissues with spatial coordinates corresponding to distinct nuclear transcriptomes. Here, we present a single-nucleus RNA sequencing (snRNA-seq) protocol that aims to overcome difficulties encountered with single-cell RNA sequencing in C. elegans. We describe steps for isolating C. elegans nuclei for downstream applications including snRNA-seq applied to the context of alcohol exposure. For complete details on the use and execution of this protocol, please refer to Truong et al. (2023).1

1. Set up 10-15 60 mm OP50 seeded NGM plates (see the note below) with 5-6 L4 animals per plate.2. After 5 days at 20 C, when the next generation of adults with embryos are present but not starved, proceed to ''rapid bleach to synchronize animals for exposure''.
Note: For OP50 growth conditions and seeding protocol see Stiernagle, 2006 9 .Ensure plates will not starve due to high plating density.
Note: Animal plating densities may vary based on what strain is being grown and the amount of OP50 seeded on each plate.
Note: Ensure 4,000 animals per condition can be harvested.

OP50 + M9 preparation
Timing: 24 h 3. Pick a single colony of OP50 bacteria into a sterile 1 L Erlenmeyer flask containing 100 mL Luria Broth and culture overnight shaking at 37 C. 4. Transfer the culture evenly between 3 sterile and pre-weighed 50 mL tubes. 5. Spin down the culture at 4000 3 g for 5 min and remove the supernatant.6. Re-weigh each tube and calculate the weight of the bacterial pellet.7. Dilute the pellet with M9 (store at room temperature, up to a year) to a stock concentration of 100 mg/mL OP50 + M9.

Nuclei dissociation reagent preparation
Timing: 1 h 8.The day of ''nuclei dissociation''.a. Label all necessary tubes.b.Dilute DAPI (or Hoechst) stain (stock concentration of 2.5 mg/mL DAPI, store at 4 C up to a month).c.Make Modified FA buffer (modified from Jonge et al., 2020 10 ).Buffer can be stored at 4 C for up to 2 weeks; however, you must add protease and RNase inhibitors fresh.d.Make RNase free 13 PBS by diluting 103 PBS with commercially available DEPC-treated water (see ''key resources table'') to 13 PBS, ensuring a volume of 2 mL per condition.e. Make 1 mL of RNase free water containing protease inhibitor tablet(s) (according to manufacturer's instructions).Keep on ice, use the same day.

STEP-BY-STEP METHOD DETAILS
Rapid bleach to synchronize animals for exposure This step will synchronize animals in preparation for ethanol exposure.At this stage, the animals should be gravid adults and the plates should also contain hundreds of eggs (Figure 1).
1. Wash gravid adults and eggs from plates set up during ''animal synchronization'' step with 2-3 mL of M9 reused between 3 plates (e.g., wash plate 1 with M9, transfer M9+embryos to plate 2, repeat with plate 3) into a 15 mL tube.Ensure all animals and eggs are removed from the plates.2. Centrifuge at 3,000 g for 1 min and remove supernatant containing bacteria (some bacteria will be pelleted, this is normal).3. Wash with 1 mL of M9 centrifuging at 3,000 g for 1 min to remove excess bacteria (optional but recommended).4.After the wash, aspirate down to the worm and egg pellet.5. Add 1 mL of bleach solution (see ''bleach solution'' for preparation) and vortex for 2 min.
Note: Animal carcasses will still be visible in addition to eggs.
6. Centrifuge at 3,000 g for 1 min, remove bleach solution taking care not to remove the pellet.7. Add 1 mL of M9, vortex to mix and centrifuge at 3,000 g for 1 min (repeat 3 times).8.After the final wash, aspirate down to about 100 mL volume.9. Mix vigorously with a 1,000 mL pipette and distribute eggs into a corner of an OP50 seeded NGM plate.
Note: Each tube has enough eggs for multiple 60 mm NGM+OP50 plates.Ensure plates are not overcrowded, roughly 500 eggs per 60 mm NGM+OP50 plate.
10.You will need 10 plates per condition for the exposure (e.g., if doing control, 0.05% ethanol, and 0.5% ethanol conditions, you will need 30 synchronized plates.)11.These animals will reach the L4 stage in roughly 50-52 h at 20 C.

Timing: 48 h
This step describes a liquid culture ethanol exposure paradigm for snRNA-seq applications.
12. Label three 15 mL tubes as ''1'', ''2'' and ''3''.13.Place 4 mL of M9 into each of the three tubes.14.Wash plates containing L4 animals with M9 using a 1,000 mL pipette (use roughly 2 mL of M9 per 3-4 plates), being careful not to remove dead carcasses from the previous bleaching step, and place into tube ''1''.15.Allow the animals to settle for around 5 min, then transfer the pellet (1 mL) into tube ''2''.16.Allow the animals to settle again, and transfer into tube ''3''.17.Allow animals to settle for around 5 min (Figure 2A), and divide each pellet evenly between two 15 mL tubes using a glass Pasteur pipette to prevent animals sticking (e.g., 2 pellets into four 15 mL tubes in total).18.To each tube, add 1 mL of 100 mg/mL OP50 + M9, and fill with M9 to a final volume of 10 mL (Figure 2B).
Note: You should end up with a total of four 15 mL tubes containing animals in 10 mg/mL OP50 + M9.
19. Quickly vortex each tube and take 5 mL of liquid placed onto a glass slide (average over 3 samples).Ensure that there is no more than an average of 4 worms per 5 mL sampled (Figure 2C).
Note: If the density is too high, dilute by adding additional 10 mg/mL OP50 + M9 until in the proper range.If the density is too low, allow the animals to settle and remove liquid, ensuring you have enough volume to expose all conditions.
CRITICAL: Animals at too high a density will starve.
CRITICAL: Make dilutions of ethanol fresh at room temperature, do not store.Ethanol 200 proof should be stored sealed at room temperature only up to a month after opening.

21.
To each 15 mL tube, add up to 5 mL of previously prepared worms in 10 mg/mL OP50 + M9 leaving room to add ethanol at the proper concentration.CRITICAL: Ensure worms have not settled, as this will create an uneven distribution.
Pause point: Animals will incubate for 24 h.

After 24 h a. Near a
Bunsen burner open the tubes to aerate for 5 min.b.Add 500 mL of 100 mg/mL OP50 + M9 into each tube to prevent starvation.c.Continue to rotate at 20 C for an additional 24 h (48 h total).
Pause point: Animals will incubate for 24 h.

Gravid bleach of exposed P0 for the F1 generation
Timing: 1 h This step will synchronize animals in preparation for nuclear isolation (Figure 3).
24.After removing 15 mL tubes from the rotator, allow animals to settle for 5 min.Wash L4 animals off of plates using M9 and place in 15 mL conical tubes, allowing to settle by gravity (A).The pellet is then evenly distributed among two 15 mL conical tubes and filled up to 10 mL with M9 + 10 mg/mL OP50 (B).Three 5 mL samples from each well mixed tube are taken to attain a worm count/ tube (C).Each exposure condition is then added to its own 15 mL tube and placed on a rotator (D).

25.
Transfer each pellet into a separate 1.5 mL tube (one tube per condition) with a low bind pipette or glass Pasteur pipette (Figure 3A).
Note: Make sure to keep each condition separate.
26. Wash with 1 mL M9 to remove excess bacteria, spinning tubes at 3,000 g for 1 min.27.After the wash, add 1 mL bleaching solution and vortex for 6-6.5 min.Eggs should be present, and bodies should be dissolved after beaching (Figure 3B).The longer duration of the bleaching step helps manage the larger animal input.
CRITICAL: After bleaching, ensure carcasses are not present.Be careful not to bleach for too long as this will kill the eggs that are recovered.
Note: Each tube has enough eggs for multiple OP50 NGM plates.Ensure plates are not overcrowded.
Note: If culturing worms to the F3 generation, some F1 plates from each condition will need to be put aside to obtain the F2 generation, and similarly, some F2 plates will need to be set aside to obtain the F3 generation.This step allows animals in the F2 generation to be synchronized without the need for a gravid adult bleach.This step can be repeated with the F2 adults and L1s to obtain a synchronized F3 population if necessary.
31.Filter 7-10 60 mm plates per condition using a 10 mm filter in a sterile Swinnex filter holder, using M9 to wash the animals through into a 50 mL tube (Figures 4A and 4B).a. Wash animals from NGM plates with M9.
Note: Visually inspect each plate to ensure L1s are present before washing off the animals.
b. Run through 10 mm filter and follow with 3 times 1 mL M9 rinses into a 50 mL tube.c.Only L1s will be washed through the filter.d.Allow L1s to settle (about 5 min).e. Place animals onto fresh OP50 NGM 60 mm plates, taking care not to overcrowd the plates (500 animals/plate, 10 plates per condition).32.When the F2 animals are egg laying adults (96 h, 20 C), with L1s visible on the plates repeat ''Step 31'' to obtain a synchronized F3 generation.
Note: Ensure all conditions remain separate and when plating L1s ensure plates are not overcrowded.
Note: Pre-wetting the filter with M9 will ensure the filter stays in the filter holder properly.

Nuclei dissociation
Timing: 2 h After animals have been synchronized at the adult stage, the following steps will allow dissociation of individual nuclei from whole worms for downstream 103 Genomics sequencing.
33. Ensure all reagents are prepared ahead of time (Figure 5A).iii.New nuclei.iv.Filtered nuclei.v. 103 Genomics submission (marked with each condition).vi.96 well plate for BD Celesta cell analyzer equipped with a 405 nm Laser.e. Sterilize the work area and treat with RNase Zap.
Note: Each exposure condition should get its own set of tubes; never mix nuclei from different exposure conditions!34.Wash worms from plates with M9 (roughly 2 mL per 3-4 plates) into 15 mL tubes.
a. Try to limit bacteria transfer while washing the plates.b.If too much bacteria transfers, allow the worms to settle (5 min).c.Transfer the pellet to a new tube and with 5 mL fresh M9. d.Repeat ''Steps 34b-c'' 3 times.
Note: Bacteria will negatively affect sequencing results; ensure bacteria are fully removed from the animals before continuing.
35.Allow the worms to settle into a pellet (5 min), and transfer to a low bind 1.5 mL tube using a glass Pasteur pipette.36.Centrifuge at 1,300 g for 1 min, remove M9 and wash with 1 mL M9 (Repeat for 3 total washes).
a. Be careful not to disturb the worm pellet.37. Starve animals for 30 min on a rotator in 1 mL fresh M9.
a.This step will evacuate the gut of bacteria improving sample quality.38.Allow the worms to settle for 10 min to obtain a 30 mL pellet.39.Place 450 mL Modified FA buffer into a Wheaton Dounce homogenizer (Figure 5B).40.Transfer a 30 mL worm pellet into a Dounce homogenizer containing 450 mL of Modified FA buffer (Figure 5C).41.Homogenize each sample with an up and down corkscrew motion (10 strokes).
a. Be careful not to spill sample and try to limit the creation of excess bubbles (Figure 5D).42.Transfer Modified FA buffer with nuclei from the Dounce homogenizer to a fresh low bind 1.5 mL tube and centrifuge at 100 g for 1 min at 4 C (Figure 5E).a.The supernatant will contain nuclei.b.The pellet will contain debris and waste.43.Remove the supernatant and place it into 1.5 mL tube labeled ''Pooled Nuclei''.44.Resuspend the pellet with 350 mL Modified FA buffer in the same 1.5 mL tube.45.Homogenize the resuspended pellet with a sterile, RNase free pestle (plastic) in the 1.5 mL tube, using 10 corkscrew motions.46.Centrifuge at 100 g for 1 min to pellet debris.47.Remove the supernatant and place it into ''Pooled Nuclei'' tube.48.Resuspend the pellet again in 350 mL Modified FA buffer in the same 1.5 mL tube.49.Homogenize with a plastic pestle with 10 corkscrew motions.50.Centrifuge at 100 g for 1 min to pellet any remaining debris.51.Transfer the supernatant into ''Pooled Nuclei'' 1.5 mL tube.52.Pellet the nuclei by centrifuging at 500g for 4 min.
Note: Ensure that different conditions remain separate.Note: After this step nuclei will be in the pellet.53.Remove the supernatant (modified FA buffer), making sure to leave the nuclei pellet behind.
Note: If the pellet isn't visible, aspirate supernatant down to 0.1 mL.54.Resuspend the nuclei pellet using 1 mL of 1% BSA in 13 PBS, pH 7.4 with RNase inhibitor.55.Centrifuge the nuclei at 500g for 1 min.56.Remove supernatant and resuspend the pellet to a total volume of 900 mL with 1% BSA in 13 PBS pH 7.4 with RNase inhibitor.
Note: Resuspend in less volume if the pellet is barely visible (700 mL total volume).
57. Filter with a Flowmi 40 mm filter to remove extra debris and clumped nuclei (Figure 5F).a. Transfer to a final tube for 103 Genomics sequencing.58.Use a BD Celesta cell analyzer with 96 well plate and 405 nm laser compatibility or similar cell analyzer for the final nuclei counts.a. From each ''Final Nuclei'' tube, remove 450 mL of filtered nuclei for DAPI staining.Make sure that the nuclei are well mixed to ensure you are not over or under sampling the nuclei count.
CRITICAL: The remaining nuclei samples will be sent in for 103 Genomics snRNAsequencing.Ensure they are kept on ice to prevent damage or degradation.
b. Stain with DAPI by adding 2 mL of 2.5 mg/mL DAPI (final concentration 11 mg/mL).
Note: While flow cytometry tends to over-estimate nuclei, this is our recommended methodology for consistent counts.Additionally, we have found cell counters to be inconsistent with C. elegans nuclei due to the heterogeneous nature of their size and shape.
c. Prepare a 96 well plate, with each condition in technical triplicates.i. Load 150 mL of sample per well.
Note: An accurate count is essential for proper submission to 103 Genomics and accurate sequencing results.
d. Use the total count number for your 103 Genomics submission.

Note:
The remaining nuclei without DAPI added will be submitted for 103 Genomics sequencing.
Note: Ensure you have an accurate estimate of your number of nuclei to get the cleanest sequencing results.
Note: You can use a fluorescent microscope to check for clumping by mounting DAPI stained nuclei onto Vectashield or similar mount (3-5 mL of nuclei suspension) (Figure 6).
e.If nuclei samples are clumping, run the samples through an additional Flowmi 40 mm filter.59.Deliver the samples for library preparation and sequencing as soon as possible to ensure the sample quality doesn't degrade (here we used the 103 Genomics platform).

Data processing
Timing: variable i. DIEM to remove droplets with extranuclear contamination. 11i.SOUPx to identify ambient RNA and correct the expression reads for remaining droplets.12iii.Filter out low-quality droplets (droplets with identified genes less than 300 or more than 3,500, UMIs more than 15,000 or mitochondrial reads more than 20%).Threshold should be determined based on visualization of QC plots.c.Canonical correction analysis (CCA) in Seurat v3 13 was utilized to remove batch effect across samples.d.Principal component analysis (PCA) and Louvain clustering 14 were further used to identify clusters.e.We recommend visualizing cell clusters with either t-distributed stochastic neighbor embedding (t-SNE) plot or Uniform Manifold Approximation and Projection (UMAP).The former works better to identify different subpopulation within cluster and latter works better to identify distinct clusters.f.We utilized known biomarkers (e.g., tra-2 for the germline, unc-25 for GABA neurons-specific markers; see  Cross-reference the previous outputs with in situ expression for top transcripts utilizing the Nematode Expression Pattern Database (NEXTDB https://nematode.nig.ac.jp).

LIMITATIONS
This protocol was solely tested in adult hermaphrodite C. elegans.It is probable that the number of input animals required to examine younger stages (i.e., larval stages) would need to be increased from our original protocol.Additionally, the mechanical disruption to the worms can potentially lead to under sampling of certain cell types; however, we were able to observe clusters representing all tissue types.

TROUBLESHOOTING Problem 1
Issue during maintenance of the animals during scaling up of cultures: the animals have starved and there is no more OP50 when in liquid culture or on plates.

Potential solution
Add less animals to each plate or into the liquid culture.Estimate the number of animals you are using to ensure consistency (see step 19).

Problem 2
Animal carcasses are still present after gravid adult bleach.

Potential solution
Double the amount of bleaching solution.If too many animals are in the tube, it can affect the quality of the bleaching output.
Reduce the number of animals being bleached.Extend the bleach time by 30 s; however, bleaching for too long may kill the embryos.

Problem 3
Unable to grow enough animals to perform nuclear isolation.
UCLA Eureka fellowship and Burroughs Wellcome Fund Inter-school Training Program in Chronic Diseases, and P.A. is supported by NIEHS R01 ES027487, NIAAA R21 AA024889, NIAAA R01 AA030160, the John Templeton Foundation, and the Burroughs Wellcome Innovation in Regulatory Science Awards.BioRender was used for the graphical abstract and Figure 1.

Figure 1 .
Figure 1.Flowchart overview of exposure, maintenance, and nuclear dissociation

Figure 2 .
Figure2.Liquid culture ethanol exposure setup Wash L4 animals off of plates using M9 and place in 15 mL conical tubes, allowing to settle by gravity (A).The pellet is then evenly distributed among two 15 mL conical tubes and filled up to 10 mL with M9 + 10 mg/mL OP50 (B).Three 5 mL samples from each well mixed tube are taken to attain a worm count/ tube (C).Each exposure condition is then added to its own 15 mL tube and placed on a rotator (D).
30.If you are collecting F1 nuclei from JK560 animals: a.After roughly 50-52 h post bleach, place half of the L4 plates per condition at 25 C for 24 h prior to nuclei dissociation to prevent sperm production.b.Otherwise, keep bleached animals at 20 C until they are Day 1 adults.

Figure 3 .
Figure 3. C. elegans gravid adult bleach for post-exposure synchronization Post exposure animals washed off plates should be at a high density with animals easily visible (A).After bleaching, ensure that no bodies are present to ensure the tightest synchronization.Embryos should still be visible under a dissecting microscope (B).

ProtocolNote:
Save the remaining half of the plates at 20 C until ready to proceed to ''Step 31''.

Figure 4 .
Figure 4. Filtering to isolate and synchronize L1 animals Prewet 40 mm filter to ensure proper position and function (A).Use a pipette to slowly filter animals washed off of plates with M9.Run through M9 will contain L1 animals in the 50 mL tube.

CRITICAL:
Perform all centrifugation steps at 4 C. Perform all steps in RNase free conditions.Keep all samples and reagents on ice.Minimize pipetting and handling time to avoid nuclei lysis or clumping (Figures5A-5F).a. 1.2 mL per condition Modified FA Buffer (store on ice, use the same day).b. 2.0 mL per condition of 13 PBS with 1% BSA and RNase inhibitor (store on ice, use the same day).c.Clean, label with each condition, and pre-chill homogenizers at -20 C. d.Ensure all tubes are labeled and the 103 Genomics sequencing facility is ready to receive samples.For each condition, the labels should be: i. Homogenized sample.ii.Pooled nuclei.

Figure 5 .
Figure 5. Nuclear extraction setupEnsure that sufficient ice is available to keep all reagents cold.Ensure the environment is sterile and RNase free in a 4 C environment (A).Ensure that Modified FA buffer is not able to overflow from the Dounce tissue grinder and keep contents cold to preserve nuclei integrity (B).Add the worm pellet to the Dounce homogenizer (C) and homogenize the samples (D).Centrifuge the homogenized samples (E), the pellet (arrow) contains cellular debris.Finally, filter the supernatant through a Flowmi filter (F).
This step will briefly outline the bioinformatic pipeline utilized to process raw 103 Genomics snRNAseq data to remove low quality datapoints stemming from debris containing droplets and ambient RNA contamination (detailed version is available in Truong et al., 2023 1 ).60. Resources to process raw snRNA-seq data.a. First, snRNA-seq raw reads were: i. Demultiplexed and aligned to the C. elegans ENSEMBL ce10 transcriptome utilizing Cell Ranger, using ''include-introns'' flag to align nucleus reads.ii.Sample quality control was determined through checking Cell Ranger reports such as: saturation, mapping rate and sample clustering.b.To remove debris contamination and RNA contamination we utilized:

Figure 6 .
Figure 6.Ensure nuclei are not clumped and intact with DAPI staining Scale bar = 10 mm.
16 Differential expressed genes (DEGs) were identified based on Monocle15pipeline with batch effect variable involved and corrected with FDR <5%.h.Diferential expressed genes of different clusters were further compared with different ontological gene sets through enrichment analysis (FDR <5%).i.Tissue enrichment.ii.Gee Ontology.iii.Wombase phenotype.16