Table 5.

SHAPE-MaP Troubleshooting

Step	Problem	Possible cause	Solution
20A(vii)	Low library yield	Size selection too stringent	Use a 1:1 or 1.2:1 ratio of beads to sample.
20C(iv)	Low dsDNA yield	Poor cDNA yield from reverse transcription	Use more RNA; do not exceed 5 μg.
			Increase the RT primer concentration by 10-fold. For the Randomer Workflow this will result in shorter sequencing libraries.
21	Low library yield	Failed Step 1 or Step 2 PCR	Optimize PCR conditions for each reaction individually before performing them in sequence.
			Some RNAs amplify better with a different ratio of cycles between Steps 1 and 2. Perform PCR using 20 cycles of Step 1 and 10 cycles of Step 2.
22	Extra peaks observed in Small RNA Workflow library	Non-specific primers	Redesign the [RNA-specific] and [RT primer] sequences of Step 1 primers with an online tool such as Primer-BLAST to avoid off-target binding. Test primers by performing Step 1 PCR for 25–30 cycles and verifying product on a gel or Bioanalyzer chip.
		Low Step 1 PCR input	Increase the number of Step 1 cycles to 20, reduce Step 2 PCR to 10 cycles.
		Incomplete conversion of Step 1 product to Step 2 product	Reduce the number of Step 1 cycles or carry less Step 1 product through to Step 2 PCR.
24	Few reads align to RNA of interest	Non-specific primers	Redesign PCR primers (Small RNA and Amplicon Workflows, see above); optimize RNA purification (Randomer Workflow).