If 10ul RNA was used for 3′ adapter ligation, would you suggest using 10ul in 5′ adapter ligation reaction or the whole volume (20ul) from 1st reaction would work too?-Anastasia

Dear Anastasia,

Thank you for your email. While we recommend sticking to 2 uL of RNA input when possible, we found that we can still obtain good library prep results with up to 10 uL of RNA (without changing any other volumes in the 3′ Adapter Ligation Reaction). We recommend that you carry the entire volume of this reaction (10 uL + excess) to each of the next steps. This means that each subsequent reaction will now contain larger volumes than outlined in the manual.

Please let me know if you have any additional questions.