Table 1.

Troubleshooting

Step	Problem	Possible Reasons	Solutions
1	It is not feasible to obtain 108 cells.	The cells are not expandable or are difficult to expand up to 108 cells.	108 cells are recommended for convenience to ensure sufficient recovery of nuclei. However, it is possible to use fewer cells – we have successfully conducted nuclei preparations with ~10 million cells using smaller volumes of Buffer N and Sucrose Cushions. However, if the cell numbers are less than that, then it may be necessary to skip the sucrose cushion purification of nuclei, which may marginally reduce efficiency of digestion and IP specificity.
7	The pellet is not whiter in color.	The cells may not have lysed.	To confirm lysis, it is advisable to observe the cells under a microscope with trypan exclusion staining. If the cells are confirmed to not have fully lysed, it is possible your cell line is somewhat resistant to 0.3% NP-40, in which case the amount of NP-40 in the Lysis Buffer can be increased.
8	The pellet will not resuspend in Buffer N.	Some of the nuclei may have lysed, releasing the chromatin.	If possible, it is highly recommended to start the procedure over with new cells and to use less NP-40 in the Lysis Buffer and carefully adhere to the listed amount of time that cells and crude nuclei are exposed to detergent containing buffer. If starting over is not possible due to a lack of cells, then in principle, you can go directly to chromatin digestion and HAP chromatography. This is challenging because optimizing MNase digestion conditions without knowing the amount of chromatin in the pellet is difficult, but if the cells are irreplaceable or difficult to reobtain, then it is possible. A small sample can be titrated with MNase empirically, if there is enough sample for this to occur.
8	There is no nuclei pellet after the purification by Sucrose Cushion.	The cells do not have intact nuclei (e.g. granulocytes), the nuclei do not pellet through the Sucrose Cushion at 500g, too few cells were used at the beginning of the protocol, or too many cells were lost in the nuclei purification process.	If your cells do not have intact nuclei (e.g. granulocytes), then nuclei purification by Sucrose Cushion is not possible. In this case, you should proceed immediately from cell lysis to quantification and MNase digestion without a Sucrose Cushion purification. If your cells do have intact nuclei, then it is possible that your nuclei do not pellet through the Sucrose Cushion at 500g. In such circumstances, you can try a second spin up to 1300g to pellet nuclei. A white film atop the sucrose cushion is diagnostic of the flux of nuclei crossing the boundary being too high, such that nuclei encounter one another and stick. In this case, remove the whole buffer layer and the film above the cushion with as little cushion as possible, dilute 2x further in Buffer N and reapply to a new cushion, also recovering any nuclei that did pellet. If a pellet still does not appear, then it is likely that you started with too few cells, or you have lost too many cells to yield a sizeable pellet. In this case, it is recommended that you start over and either use more cells or skip Sucrose Cushion purification and instead move straight to chromatin quantification (Steps 9–11).
12	During UV-vis quantitation, the chromatin remains too viscous to pipette even after 10–15 minutes of sonication.	The chromatin has not completely solubilized and remains too intact to pipette easily.	Sonicate for longer, in increments of 5 minutes. Depending on the age and quality of the water bath sonicator, sonicating enough to permit sufficient pipetting can take quite some time. You can also dilute all your aliquots with additional 2 M NaCl and sonicate longer. If using a water bath sonicator other than the Branson 2800 Ultrasonic Cleaner, then optimization of sonication conditions and time may be needed.
13	The chromatin concentrations are vastly different across the three replicates.	Some nuclei may have settled as you were taking aliquots, so some aliquots may not be representative of the sample as a whole. Alternatively, the chromatin in some samples is still not sufficiently solubilized.	Try to repeat the process of UV-vis sample preparation (Steps 10–13), resuspending and mixing nuclei well (but gently) while taking aliquots. Alternatively, it is possible that your chromatin in some samples is still too viscous, which is preventing it from being easily pipetted. In this case, continue sonicating for longer (see above).
13	The amount of chromatin is not detectable by Nanodrop.	The chromatin concentration of the aliquot may be below the sensitivity of the Nanodrop instrument (~5ng/μL), particularly if working with a very small number of nuclei (~1 million or fewer).	You can prepare aliquots again by diluting with less 2 M NaCl (or using a smaller volume of 5 M NaCl to maintain the same level of salt), you can pellet and resuspend nuclei in a smaller volume of Buffer N, or you can use a more sensitive DNA quantification tool (e.g. Quant-iT PicoGreen dsDNA Assay Kit). If none of these are possible for you, then you can “guesstimate” the concentration of chromatin. From our experience, there are about 1μg of crude mammalian chromatin per 60k-100k nuclei (assuming 3–3.5 Gbp per genome). Needless to say, this method is not the most accurate and should be reserved only for situations in which no other alternative presents itself.
15	The antibody does not bind Protein G well, per antibody characterization by the manufacturer or based on the antibody isoform.	Some isotypes of antibodies do not bind Protein G well.	Try using Protein A magnetic beads instead.
57	Digested chromatin is not a mononucleosomal population.	The MNase may not have the activity that is anticipated, the nuclei may be clumped, or excess debris in the nuclei solutions is preventing accurate quantification or complete digestion.	If there is a >5% population of di- or oligonucleosomes, repeat the protocol but titrating several up to several fold increases in of MNase used or employ a freshly thawed aliquot of MNase from stock solution. Conversely, if there is a large population of DNA fragments smaller than ~150 bp corresponding to mononucleosome fragments, the digestion has progressed too far (often characteristic ~115 bp or ~80 bp). If the DNA fragment population displays both over and underdigestion, then the nuclei are not sufficiently uniform-- mix nuclei well to ensure that the nuclei are not forming clumps during MNase digestion. If even mononucleosomal digestion remains a problem, begin nuclei preparation with fresh cells, but use two sucrose cushions sequentially to improve their homogeneity.
57	Nucleosomes are appearing in HAP wash flow-throughs.	The pH may be incorrect, or there may be too much phosphate in the wash buffers.	Ensure that the buffers have a pH of 7.2 and ensure that the phosphate concentrations are reasonably accurate. This may require preparing fresh HAP Buffers.
64	High Cq values (>32 cycles) are observed in both the IP and the Input.	Insufficient primer and probe were added to the qPCR reaction. Alternatively, the primer and probe may not span a well-positioned nucleosome.	Remake your qPCR 20x Primer-Probe Master Mix to rule out the possibility that there was insufficient primer and probe. If you still observe high Cq values, it is very likely that your primer and probe set are inappropriate. Try creating and using a different primer and probe set. Primers and probes should ideally line up with a well-positioned nucleosome, which would appear as regions of buildup in MNase-seq datasets, if available for your cell type.
64	Enrichment of the target standards for the IP is low.	The antibody may not have saturated the Protein G magnetic beads, or it may be an inherently low-enrichment antibody in these conditions.	Ensure that the amount of antibody used was enough to saturate the beads, per the recommendation of the manufacturer of the beads; if this was not the case, then increase the amount of antibody used. For the beads we recommend, this is approximately 6 μg of antibody per 25 μL of beads. Additionally, if the IP showed high specificity (see Step 64), then it may be possible to increase enrichment by reducing the number of washes (e.g. one wash with ChIP Buffer 2 rather than two washes, no washes with ChIP Buffer 3 rather than one wash). As a practical matter, increasing the target enrichment percentage of an IP is difficult and often not worth the trouble. If possible, obtain a new antibody or scale up the reaction with more antibody and more chromatin instead. If, as measured by sensitive DNA quantification methods (e.g. Quant-iT PicoGreen dsDNA Assay), you are still obtaining more than 10 ng of DNA in the IP, then you are certainly recovering enough DNA even for next-generation sequencing library preparation, and the enrichment is not worth worrying about.
66	Specificity of the antibody is low/the antibody binds substantial off-target modifications.	The antibody is of poor quality.	Try adding washes with ChIP Buffer 2 and 3, but understand that this will come at the expense of enrichment. If at all possible, get a different antibody.
79	There is little to no DNA found in my sample, as measured by PicoGreen.	Nucleases may have contaminated the samples. Alternatively, the antibody used has low enrichment for the target.	Redoing the experiment with fresh, nuclease-free buffers may work if the sample was contaminated with nucleases. However, it is much more likely that your antibody has low enrichment for target. If there is at least 5 ng of DNA, then your sequencing will probably be fine. If there are 1–5 ng of DNA, then your sequencing may be slightly overamplified but may be workable. With less than 1 ng of DNA, it is likely that the samples will be dramatically overamplified. If you have low DNA yield, it is recommended that you either obtain a better antibody or scale up the reaction with more antibody and more chromatin.
98	There is not enough DNA found in my library, as measured by PicoGreen.	The library preparation has likely failed.	If all of your library preparations have failed, then it is likely that a step was done incorrectly or that your kit has spoiled. In this case, the preparation can be redone if enough sample DNA remains, or the entire experiment can be redone. If only one library preparation has failed, then if enough DNA remains, it can be redone, but if the initial DNA concentration was so low that not enough remains, that is likely a major reason the library preparation has failed.
100–107	The file was not found (or file could not be opened).	The path pointing to the file may be incorrect, or you may not have the necessary permissions to open that file.	Ensure that the path pointing to the file is correct. For example, if the file is not in the directory that you are currently working in, then you need to list the full path of the file in question. Also ensure that you have the necessary permissions to open that file.
100–107	A command has returned an error: [command]: command not found	The program being run may not be properly added to the PATH.	Ensure that the program being run is appropriately added to the PATH. To do this, first find the path of the program installation (different for each program, but generally listed in the online documentation for each). Then run the command: nano ~/.bashrc Scroll to the end of the file using the “PgDn” key. Enter the following at the end of the file (without the brackets): export PATH=$PATH:[full path to program folder] Then press Ctrl+x to close the file, press “Y” and press enter to save the file. Finally, run the command: source ~/.bashrc
106	The specificity or calibration is not what was expected or a divide-by-zero error was reported.	You may be using an incorrect calibration table.	Ensure that you are using a custom calibration table for your set of barcodes and corresponding nucleosomal marks rather than the calibration table we have provided strictly as a formatting example.
107	The terminal window cleared and displayed unusual characters.	Your calibration table may not be a properly formatted plain text tab-delimited file.	Please ensure that the calibration table being used is a custom calibration table for your specific application that is provided in a plain text tab-delimited file rather than an Excel file.