Table 5.

Troubleshooting table.

Step	Problem	Possible reason	Solution
1	The cell or nuclei pellet is hard to visualize after centrifugation.	Nuclei and certain cell types can be hard to properly pellet. Pellets of 50,000 cells, even for small cell types such as B cells, should be easily visualized.	Add sterile BSA to a final concentration of 0.5% wt/vol or Tween-20 to a final concentration of 0.1% wt/vol to help cells/nuclei pellet properly.
33	There is no amplification of the ATAC-seq libraries.	the incorrect barcode/adapter sequences were used when performing the barcoding step.	Ensure that compatible barcodes were added, as detailed in Supplementary Table 2.
33	There is no amplification of the ATAC-seq libraries.	Excess ethanol from the column may not have been properly removed using a dry spin prior to elution of transposed DNA.	Ensure that the additional dry spin after the second wash step of the DNA Clean and Concentrator-5 kit is performed.
45	The ATAC-seq libraries are outside the standard curve of the NEB Quant kit.	The libraries may be either too dilute or too concentrated.	We make dilutions in the 2000-4000-fold range. Adjust the dilutions of the final libraries as needed.
50	Low-depth sequencing shows a low TSS Enrichment Score.	Low signal-to-background ratios are often caused by unhealthy or otherwise non-ideal input material.	Consider pre-treating cells with DNase or using flow cytometry to sort viable cells.
	There is no nucleosomal periodicity in the Bioanalyzer traces.	Not all ATAC-seq libraries show nucleosomal periodicity on Bioanalyzer.	This may not be a problem. See the section on Quality control of ATAC-seq libraries.