How to deplete RNA without RiboZero or Terminator exonuclease?
Hello everyone!
Thank you for reading my question! Please excuse my little experience in the topic, I would like to have some advice on the RNA-seq library preparation. We extracted the RNA from bacterial cultures and we had good results using a kit. However, we used the Turbo DNase from Ambion and followed it with the clean-up using the RNAeasy mini kit. Then we used the Terminator exonuclease (because we do not have access to the RiboZero kit) to deplete larger RNA fragments and constructed the libraries with the ScriptSeq kit. Of course our results were not good and the libraries were degraded. Any advice on how to deplete the RNA without using Ribozero? I am planning to remove the RNA with the on-column protocol from the RNAeasy mini kit from Qiagen. The point is that I am not sure if I can directly proceed to the library construction without depleting. My lab cannot afford the RiboZero kit at the moment and I have been strongly advised against using the terminator exonuclease, hence I need some alternative protocol. Thanks a lot for your advice!!
Thank you for reading my question! Please excuse my little experience in the topic, I would like to have some advice on the RNA-seq library preparation. We extracted the RNA from bacterial cultures and we had good results using a kit. However, we used the Turbo DNase from Ambion and followed it with the clean-up using the RNAeasy mini kit. Then we used the Terminator exonuclease (because we do not have access to the RiboZero kit) to deplete larger RNA fragments and constructed the libraries with the ScriptSeq kit. Of course our results were not good and the libraries were degraded. Any advice on how to deplete the RNA without using Ribozero? I am planning to remove the RNA with the on-column protocol from the RNAeasy mini kit from Qiagen. The point is that I am not sure if I can directly proceed to the library construction without depleting. My lab cannot afford the RiboZero kit at the moment and I have been strongly advised against using the terminator exonuclease, hence I need some alternative protocol. Thanks a lot for your advice!!