Table 1.

Troubleshooting table.

Step	Problem	Possible Reason	Solution
18	You have not enough cells after the FACS sorting.	S phase cell population in the original sample is low.	Stain more fixed cells Collect more early and late S cells.
73	The concentration of one or two samples is much lower than the others.	Those samples were probably lost during BrdU IP.	Start over with those samples rather than re-amplifying them.
	The concentration of all samples is too low to quantify with the Qubit dsDNA HS assay kit.	Your cells probably did not incorporate BrdU well.	Repeat the library preparation using twice the starting material (DNA from 40,000 cells/library).
			Start over from BrdU labeling of cells using higher concentration of BrdU.
			Use S/G1 method described by Ryba et al.18.
74	Target enrichment is not confirmed.	There may have been errors during BrdU IP or serious cross contamination.	Start over using the backup aliquot from step 24.
		The cell species is not what you think, or PCR condition is not optimum.	Perform control loci PCR using pre-BrdU genomic DNA to see if expected PCR products show up.
82	You have adaptors in your reads.	Adaptors have not been clipped by the sequencing facility.	Remove the adaptors using cutadapt (Martin M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads; see http://cutadapt.readthedocs.io/en/v1.13/index.html for installation and documentation)
82	The quality of the 3’ end of the reads is bad.	Either low complexity of the pool or sequencer issue.	Trim the bad quality part to avoid the loss of reads during mapping (see bowtie2 manual) Contact sequencer operator and/or Illumina tech support for troubleshooting.
82	There are duplicated reads.	Too many PCR cycles have been made during the amplification (Step 58 and 73)	Duplicated reads are removed in the provided pipeline.