Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
13	Difficult to resuspend pellet	Much variability exists in fecal samples. Some precipitations can be more crude than others	Use a pipette tip to gently break apart the pellet first, then gently vortex for a few minutes
14	Low yield (<80 μg total)	Low biomass of microbes in fecal sample. In this case, the RNA extractions will also be low yield. Alternatively, it could be an issue of RNA degradation during sample storage. For example, the RNAlater may not have been mixed well with the sample	Increase the number of extractions per sample. Also, it is acceptable to proceed with 40 μg, cutting the MNase treatment concentration in half as a last resort. Although the libraries will work, you will likely have just enough input for each downstream step
34	Not enough RNA for library preparation	RNA was degraded during rRNA depletion, from too many freeze-thaw cycles, or during the T4 PNK reaction	Backtrack and quantify how much RNA remained after rRNA depletion Try to avoid some pausing points to minimize freeze-thaws, and try to complete the entire protocol in 2 d Ensure that RNase inhibitors are being added diligently
38	Substantial amount of primer dimers	Too much primer was added	We recommend adding 1.5 instead of 2.5 μL of primers as the manufacturer recommends. If need be, this can be reduced to 1 μL Although less ideal, a few rounds of Ampure beads (1.3× ratio of beads to sample) can reduce the dimers in the library