daylinmorgan
commented
4 months ago Hi Claudia, some time ago we had attempted to utilize the 10X capture in our experiments and the two times we attempted to use it our barcode capture rates were significantly lower than with the Crop-seq derived vector. In addition, we have also started utilizing Parse Biosciences which is compatible with our vector.
Since we don't have any plans to use it internally I opted to remove it from the protocol to avoid any confusion.
The only real modifications that should be necessary are as described in the old version which use different oligos to generate the library and BbsI rather than BsmBI
3.1.1. Perform an extension reaction to generate double-stranded insert gRNA barcode DNA. Mix 10 𝜇L NEB 5X Q5 Reaction Buffer, 1 𝜇L of 10 mM dNTPs, 2 𝜇L 100𝜇M 10X-PrimeF-BgL-BbsI, 1 𝜇L 100𝜇M 10X-RevExt-BgL-BbsI and 0.5 𝜇L to create a 50 𝜇L reaction. 3.1.4. Digest 5-10 𝜇g of 10X Capture vector backbone in a reaction containing 20 𝜇L Digestion Buffer 3.1, 8 𝜇L BbsI, and nuclease-fee water to 200 𝜇L.
In addition to these it may be necessary to modify the CM-FWD-S1/CM-REV-S1 oligos from the updated protocol but you can verify this against the vector itself.
Hello,
Firstly, thank you for publishing this protocol; it's an incredible resource. I am currently undertaking the cloning required for the 10x-variation for an upcoming experiment, but I have noticed that with the update of the website, the relevant materials and methods have been removed. I'm just wondering if you have encountered any issues with the 10x variation and if I should change my approach, or if it is fine for me to follow the previous version of the protocol.
Best wishes, Claudia