Broad library size distribution on Bioanalyzer

Symptom

Library shows a broad size distribution on Bioanalyzer with longer insert sizes than expected. This indicates heterogeneous fragment lengths in the final library.

1. 1 Under-fragmentation of the input RNA
2. 2 Insufficient fragmentation time during RNA fragmentation step
3. 3 Suboptimal fragmentation temperature or buffer conditions
4. 4 RNA secondary structures preventing efficient fragmentation

1. 1 Increase RNA fragmentation time to achieve desired size range
2. 2 Verify fragmentation temperature is at recommended setting
3. 3 Optimize fragmentation conditions for specific RNA sample type
4. 4 Perform test fragmentation with different time points to determine optimal conditions
5. 5 Consider RNA denaturation step before fragmentation if secondary structures suspected

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