. Author manuscript; available in PMC: 2017 Apr 1.

Published in final edited form as: Nat Protoc. 2016 Mar 24;11(4):813–833. doi: 10.1038/nprot.2016.047

TABLE 3.

Troubleshooting table.

Step	Problem	Possible reason	Solution
4, Box 1	Western blot signals of the cytoplasmic marker protein GAPDH in the chromatin fraction	Reduced cell lysis efficiency due to an excess of input per cell fractionation reaction	Reduce the amount of input. The amount of cells per fractionation reaction should not exceed 2× 10⁷ HeLa S3 or HEK293T cells.
		The supernatant after washing the cell nuclei (step 18) is not completely removed.	Completely remove the supernatant after washing the cell nuclei.
4, Box 1	Strong western blot signals for elongating Pol II (CTD Ser5-P and Ser2-P) in the cytoplasmic or nucleoplasmic fractions	Treatment during cell lysis is too harsh leading to dissociation of transcribing Pol II.	Reduce the amount of cell lysis buffer that is added to the cell pellet. Be careful: too little lysis buffer can negatively affect the cell lysis efficiency.
62	No smear of cDNA is observed after the reverse transcription	The DNA linker ligation is inefficient.	Mix the viscous ligation reaction properly (step 26). Replace components of the ligation reaction every 4 months.
		The reverse transcriptase enzyme has lost activity.	Replace the SuperScript III enzyme and other reagents of the RT reaction every 4 months and note the enzyme lot.
68	A brown precipitate forms after adding the RT product to the circularization mix.	The reason is unclear.	Replace the isopropanol precipitation (step 64) by an ethanol precipitation to reduce the amount of salt that co-precipitates with the cDNA. Additionally, reduce the amount of MnCl₂ to 50%.
89	A DNA smear that migrates slower in an 8% TBE gel appears (Fig. 3c, 10 and 12 PCR cycles).	PCR over-amplification	Reduce the number of PCR cycles.
90	Strong PCR band at ~120 nt on an 8% TBE gel (Fig. 3c,d).	PCR product that originates from empty circles. Empty circles arise from unextended RT primers that get circularized by the CircLigase enzyme.	Precisely excise the RT product (step 62) and use less RT primer. Precisely excise the final PCR product at around 150 nt (Fig. 3d). Run the 8% TBE gel longer than 45 min.