SAM lncRNA library -low titers

Thanks again for all your help.

I have been trying to produce lentivirus from SAM LncRNA library (purchased from Addgene) for a while now, however, I am getting very very low titers.

I followed your Nature protocols paper for all procedures to the dot. My library sgRNA representation was tested by NGS and passed all the criteria mentioned in your protocol.

I have previously produced lentiviruses from two other libraries from your lab with similar reagents and cell lines without any issues. I did all the obvious troubleshooting steps, sequenced the ZeoR gene, replaced all the regents and cells etc. I have know received new DNA from addgene, just to make sure something did not go wrong during the first shipment, and I am going to re-amplify. However, I was wondering if you have any ideas or specific tips for this library before I do that.


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Fuchsia Kelpieover 3 years ago

It's really strange to hear that you are having trouble producing lentivirus from the SAM LncRNA library, especially when you followed everything in the Nature protocols paper.

When I produced lentivirus from this library, I did not do anything special to increase titer. You could try producing lentivirus with lipofectamine 3000 (the protocol is in a few different threads on this forum), which we have found to boost viral titer. Do you do a transfection/lentivirus production control in parallel to producing the SAM LncRNA library? That might help narrow down the suspects. Addgene is distributing the same library that I deposited, so the Addgene stock should not have any issues.

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