Table 2:

Troubleshooting

Step	Problem	Possible reason	Solution
28	Unexpected cell-cycle profile	Hoechst too old. Hoechst undergone too many freeze-thaw cycles.	Prepare a fresh Hoechst solution.
89, 125	Low or no aRNA product on Bioanalyzer (Fig. 3a)	Loss of material during bead purifications.	Remove all ethanol before elution.
113	Increased aRNA product distribution (Fig. 3a)	Inefficient fragmentation	Resuspend fragmentation buffer and make sure whole tube makes contact with the heat block during fragmentation at 94 °C.
143	Low or no library product on Bioanalyzer (Fig. 3b)	Loss of material during bead purifications. Failed library preparation.	Remove all ethanol before elution. Repeat library prep with leftover aRNA. Increase PCR cycles.
143	Low product (Fig. 3b)	High adapter/product ratio inhibits library prep.	Bead purify the aRNA 1–2 times extra Repeat library prep.
Post data processing	Low complexity	Too little material Too many PCR cycles Too deeply sequenced	If possible, increase the number of samples in a library and decrease the number of PCR cycles. Make sure the amount of material included in a sequencing run is proportional to the expected output.
Post data processing	A uniform DamID signal over the whole genome; signal is very similar to the mappable GATC density	Too high expression of the Dam-POI Too long induction of the Dam-POI Leaky induction system for Dam-POI expression	Select a clone with lower expression levels or modify the Dam-POI construct. Optimize the time of induction of the selected clone. Ensure there is no expression of Dam-POI prior to induction.
Post data processing	Little DamID signal, despite good signal in positive control	Too low expression of Dam-POI Too short induction	Select a clone with a higher expression level or modify the Dam-POI construct. Induce expression for a longer time.
Post data processing	Sparse CEL-Seq2 data compared to DamID data	Low transcript content of cells Transcript degradation Errors in CEL-Seq2 primer plate preparation Very efficient preparation and amplification of DamID material	Include positive controls to make sure single-cell transcription data can be obtained from the material (e.g. WT control). Renew CEL-Seq2 primer plate. Optimize CEL-Seq2 primer and DamID adapter concentrations, e.g. reduce DamID adapter concentration.
Post data processing	Low fraction of valid DamID reads	Too low expression of Dam-POI Presence of many random gDNA breaks High adapter contamination	Select clone with optimal expression levels. Include negative control (e.g. WT control). Reduce adapter concentrations or perform additional bead purifications.