Table 1.

Troubleshooting Guide for C. elegans dissociation and CUT&RUN.

Problem	Possible Cause	Solution
Insufficient numbers of embryos are obtained in step 11 of Basic Protocol 1	Worm strain used gives fewer progeny than N2; Temperature used differs from 20 °C Too many embryos washed off the plate in steps 4–5 of Basic Protocol 1 along with the adult population to be discarded	Increase the number of parental worms picked per plate in step 1 of Basic Protocol 1; Increase the number of plates prepared in step 1 of Basic Protocol 1; For slow-growing strains, increase the incubation time in step 3 of Basic Protocol 1 until plates are full of gravid adults. If embryos are being washed off the plates prematurely in steps 4–5 of Basic Protocol 1, save the adult population in a sterile tube, and allow the adult worms to settle to the bottom of the tube for ~30 seconds. Transfer the top ~90% of the supernatant to a new tube – this will contain primarily embryos and L1s.
Embryos stick to the pipette tips during embryo collection in Basic Protocol 1	Insufficient bacteria on the plate in step 6; Use of M9 instead of M9 + 0.05% (v/v) Tween in step 8 of Basic Protocol 1	Ensure that when embryos being pipetted, they are coated in either bacteria or M9 + Tween to avoid sticking. Tween percentage can be increased up to 0.1% (v/v) if necessary.
Worms are not well synchronized after step 13 of Basic Protocol 1	Adults and larvae were present in the embryo suspension seeded on plates in step 12 of Basic Protocol 1	Repeat step 5 of Basic Protocol 1 to wash all adults and larvae off the plates. Check under the microscope to ensure that only embryos (and some L1s) are present on the plates before washing off embryos. Check 15-cm plates after seeding them to ensure that only embryos (and some L1s) were seeded. Pick off any stray adult worms or older larvae that were seeded.
Worms are not well dissociated after Basic Protocol 2	Genotype or age of worms are more difficult to dissociate than N2 Day 1 adults	Optimize worm dissociation conditions – check dissociation efficiency under the microscope every 25 dounces for your strain or age of interest or increase cuticle disruption time. Follow Basic Protocol 2 Steps 14–17 to find the minimum amount of dissociation that leads to uniform permeability.
Worms break apart too quickly during Basic Protocol 2	Genotype or age of worms are less difficult to dissociate than N2 Day 1 adults	Optimize worm dissociation conditions – check dissociation efficiency under the microscope every 25 dounces for your strain or age of interest or decrease cuticle disruption time. Follow Basic Protocol 2 Steps 14–17 to find the minimum amount of dissociation that leads to uniform permeability.
ConA Beads clump during CUT&RUN in Basic Protocol 3	Cells lyse during the reaction Excessive cellular material on the beads (>>3,000 worms)	If beads clump and background in sequencing is high, optimize worm dissociation conditions – consider douncing fewer times or reducing cuticle disruption time if cells become damaged. Reduce worms used for each reaction.
Worms visibly stick to pipette tips during steps involving mixing by pipetting in Basic Protocol 3	BSA was left out of Wash Buffer or is at too low a concentration	Ensure BSA was included in the Wash Buffer. If it was included, remake fresh 10% (w/v) BSA and try again, or increase BSA concentration in the Wash Buffer.
Worms are lost during CUT&RUN wash steps (as evidenced by worm chunks seen in the pipette tip before discarding liquid during wash steps) in Basic Protocol 3	Too much liquid being withdrawn during wash steps; Too many worms used per CUT&RUN reaction	Remove less liquid during wash steps (keep up to 20 μL or slightly more if necessary). Carefully watch the worms on the magnet stand during each wash step, to remove the buffer with minimal worm loss. If it is unclear whether worms are being lost, save the beads containing cells and worm chunks that would normally be discarded at step 26 of Basic Protocol 3. Freeze the beads at -20 °C and return when convenient to count the real number of worms used per reaction.
The ice/water bath does not reach temperatures as low as 0 °C in Basic Protocol 3	Not enough salty water present in the ice/water bath	Add more NaCl and water to the ice/water bath. Ensure that the final mixture of salt, ice, and water is very wet. Keep ice/water bath in the 4 °C room with the top covered.
No or low (undetectable by Qubit) DNA detected before library prep (although this does not necessarily indicate a problem)	Low protein abundance; Low worm number; procedural failure	Proceed to library prep anyways and run a positive control for a histone mark in parallel to ensure the procedure worked.
No or low (<1 ng/μL) DNA detected after library prep	Too few worms used for the protein of interest Too few PCR cycles in library prep Failed library prep; Failed CUT&RUN	If low cell numbers were used and DNA is detectable, perform Bioanalyzer to check for size distribution and continue to sequence if possible; Increase worm number and use a positive control. Increase PCR cycle number, if using a low number of cycles, to increase DNA yield. If positive control fails, carefully check reagents and protocol.
High adaptor or primer content after library prep	Low DNA input	Increase DNA input to library prep; Reduce primer or adaptor concentration when preparing the library; Clean libraries with 1.2X Ampure XP beads an additional time to remove residual primers and adaptors.
A large proportion of amplified CUT&RUN library fragments (>10%) contains genomic DNA	Unsuccessful size selection while cleaning up PCR-amplified library in steps 22–24 of Basic Protocol 4 Carryover of worms or worm chunks to the DNA purification stage in steps 26–27 of Basic Protocol 3 Cell damage leading to excessive DNA leakage from cells in Step 24 of Basic Protocol 3	Repeat 0.5X size selection as in Steps 22–24 of Basic Protocol 4. Ensure that worms are not improperly carried over with the released CUT&RUN DNA fragments in steps 26–27 of Basic Protocol 3. If problem occurs persistently and sequencing data are low quality, carefully check cell dissociation as in the troubleshooting section for high background in sequencing.
Excessive PCR duplicates (>65% duplicated sequences) in sequencing results	Too many PCR cycles used in library preparation	As long as sufficient DNA quantity is produced by library preparation, reduce PCR cycles used in library prep. If this is not possible, start Basic Protocol 2 with more worms to obtain more DNA and then lower the cycle number in library prep.
Low alignment rate to the C. elegans genome	Low worm cell number relative to contaminating gut bacteria Antibody also has targets in bacteria present in the C. elegans gut	Increase worm number; Increase washes to remove bacteria when initially washing worms from plates Ensure that the antibody does not target bacterial proteins. This can be done by using the immunofluorescent staining method described in Critical Parameters with worm chunks, and checking if any of the bacteria from the animal’s gut (which can be observed as tiny, moving particles) also stain with the antibody.
No or low signal in sequencing (i.e. no or few peaks called by MACS2)	Antibody does not specifically bind the target of interest in CUT&RUN conditions or requires a higher concentration Protein abundance is low Antibody is not easily bound by pAG-MNase	Optimize antibody binding conditions with immunofluorescence; Try additional antibodies for the same target. Add more worms. Add a secondary antibody for primary antibodies not easily bound by pAG-MNase.
High background in sequencing (i.e. lots of noise present between peak regions when viewed on IGV)	Cell lysis during the reaction Nonspecific antibody binding; Insufficient antibody Excessive cleavage by pAG-MNase	Carefully check cell dissociation. Check if cells survive well overnight by counting on a hemocytometer. If cells are lost or lyse by the next day, decrease the harshness of the dissociation protocol (less time in Cuticle Disruption Buffer, fewer dounces, etc.). Optimize antibody binding conditions by immunofluorescence; Ensure antibody incubation and pAG-MNase binding steps are at 4 °C continuously. Ensure pAG-MNase cleavage step is at 0 °C throughout the reaction.