Table 1.

Troubleshooting guide

Step	Problem	Possible reason	Solution
Step 22	Non-specific band in the TBE-Urea polyacrylamide gel.	Non-specific amplifications during limited-cycle PCR.	Pause the limited-cycle PCR right before the amplification curve reaches plateau. Use the correct melting temperature of the primers for annealing in the PCR reaction.
Step 23B(vii)	Tissue sections detach from the coverslip.	Insufficient drying of tissue sections after cryosectioning.	Allow the tissue section to dry at room temperature for 15 s prior to tissue fixation.
		The 0.01% (wt/vol) poly-L-lysine treatment is insufficient for tissue attachment during the experiment.	Increase the poly-L-lysine concentration and incubation time.
Step 33B(ii)	High background of oligo-conjugated WGA. (Fig. 8a)	The concentration of DBCO-PEG5-NHS ester is too high.	Perform a titration of the DBCO-PEG5-NHS ester to find the optimal concentration.
		Insufficient purification of DBCO-conjugated WGA.	Perform spin column-based buffer exchange with DPBS 3 times to remove potential residual DBCO.
Step 33	DNA FISH signals are too weak. (Fig. 8a)	Insufficient chromatin tracing primary probe concentration.	Increase the chromatin tracing primary probe concentration. Perform a titration of primary probes when working with new samples or probe sets.
		Insufficient primary probe incubation time.	Prolong the primary probe incubation time (up to 48 hrs).
		Insufficient tissue permeabilization.	Treat the sample with 0.5% (vol/vol) Triton X-100 for 30 min or longer.
		Insufficient denaturation of the genomic DNA.	Increase the heat denaturation time from 3 min to 5 min.
Step 33	DNA FISH background signals are too high.	Chromatin tracing primary probe concentration is too high.	Decrease the chromatin tracing primary probe concentration. Perform a titration of primary probes when working with new samples or probe sets.
		Autofluorescence is too strong in the sample.	Perform 1 mg/mL sodium borohydride treatment to the sample for 10 min to reduce autofluorescence.
Step 33B	RNA FISH signals are too weak or no RNA FISH signals. (Fig. 8a)	The RNA species are degraded during the experiment.	Use RNase-free reagents and equipment during the experiment. Apply RNase inhibitors as required in the protocol.
		Insufficient primary probe concentration.	Increase the primary probe concentration during tracing. Perform a titration of primary probes when working with new samples or probe sets.
		Insufficient primary probe incubation.	Prolong the primary probe incubation time (up to 48 hrs)
		Insufficient sample permeabilization.	Treat the sample with 0.5% (vol/vol) Triton X-100 for 30 min or longer during sample permeabilization.
Step 33B	RNA FISH background signals are too high.	Primary probe concentration is too high.	Decrease the primary probe concentration. Perform a titration of primary probes when working with new samples or probe sets.
		Autofluorescence is too strong in the sample.	Perform 1 mg/mL sodium borohydride treatment to the sample for 10 min to reduce autofluorescence.
Step 33B	RNA single-molecule FISH signals are patchy without distinct single-molecule foci.	Certain/many RNA species with high expression levels are included in the MERFISH library or target RNA species are localized within spatial clusters.	Check the FPKM values and identities of the target RNA species when designing probes.
Step 33B(i)	Poor antibody staining quality for anti-fibrillarin antibody. (Fig. 8a)	Insufficient sample permeabilization.	Treat the sample with 0.5% (vol/vol) Triton X-100 for 30 min or longer during sample permeabilization.
		Primary antibody concentration of the anti-fibrillarin antibody is too low.	Incubate the sample with higher anti-fibrillarin primary antibody concentration. A 1:100 dilution is typically used for immunofluorescence staining.
Step 38	Poor DAPI staining quality.	Insufficient washing of DAPI stain.	Use 2x SSC to wash away non-specific DAPI stain.
Steps 34, 35	Fiducial bead signals get weaker during sequential imaging.	Fiducial beads gradually get photobleached during the sequential hybridization procedure.	Increase the 488-nm channel laser intensity around hyb20 when performing more than 20 rounds of secondary probe hybridizations.
Steps 34, 35	Sample drift is too large during the sequential hybridizations.	Flow speed is too high.	Decrease the flow speed.
		Tissue sections dislodge from the coverslip.	Increase the poly-L-lysine concentration and incubation time for coverslip treatment prior to tissue section attachment.
Steps 34, 35	Focus lock is lost during the sequential hybridizations.	Air bubble in the flow system.	Tighten the chamber before buffer flow.
		The selected FOVs are too far away from each other.	All selected FOVs are recommended to be within a 2 mm × 5 mm area.
Step 44	Detection efficiency is too low for chromatin tracing or MERFISH.	Start positions of the z-stack images are too low or too high.	Adjust the start positions of the z-stack images to make sure the z-range covers a layer of cells.
		DNA FISH hybridization conditions are not ideal.	Adjust the DNA primary probe concentration, DNA primary probe incubation time, cell permeabilization, and genome heat denaturation conditions.
		RNA FISH hybridization conditions are not ideal.	Adjust the RNA primary probe concentration, RNA primary probe incubation time, and cell permeabilization. Apply murine RNase inhibitors at required steps.
Steps 46, 48	Poor cell segmentation or nuclei segmentation.	Poor labeling quality for the cell membrane (WGA).	Adjust the WGA-oligo conjugation conditions by titrating the WGA and DBCO-PEG5-NHS ester molar ratios and adjusting the purification conditions of DBCO-conjugated WGA.
		Poor labeling quality for the nuclei (DAPI).	Adjust the DAPI incubation and washing conditions.
		Default parameters for cell segmentation and nuclei segmentation are not ideal for the specific sample.	Adjust the parameters in the “parameters_default.m” file until the algorithms are ideal for the specific sample.