I was wondering if anyone has already experience with constructing sgRNA libraries. We are interested in doing a knock-out experiment (loss-of-function) with 5 different conditions. I have found this protocol from Joung et al.

In the protocol they have 10 different Fwd primer with no unique barcodes, only the eight Rev primers contains unique barcodes for different samples in KO. Do I understand it correctly, that I can pool together only eight samples in one sequencing run with this protocol?

I would like to better understand the protocol of constructing an ngs library for sgRNA samples. If someone can point me to the right link/page?

Are there more complex library preparation kits out there which can deal with more samples?

  • $\begingroup$ Hi @AssaYeroslaviz, the question you are asking is wet-lab question on CRISPR rather than a bioinformatics question. You could consider posting on Biology SE if this is the consensus here. The design of a CRISPR construct is a Bioinfo SE question, rather than wet-lab protocols in my opinion $\endgroup$
    – Michael
    Commented Aug 5, 2020 at 7:50


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