Number of PCR2 reactions to maintain screen coverage (human gecko v2 library)

[Zena Shourbaji]

Dear all,

I'm running a crispr-cas9 screen using the GeCKO v2.0 pooled human library (Addgene #1000000048). The library is composed of 122 411 sgRNAs. After experiment, we will sort by facs a positive population to extract gDNA for "the 2-step PCR"  and NGS while maintaining a 300x coverage.

 To do that, we are extracting gDNA from at least 36M unsorted cells (control)  and from all the positive cell population we can get (at least 1M). Our positive population should be around 2-5% of the cells for one timepoint, and 20-25% at another timepoint.

 We know that we have to run the first PCR (that amplifies our guides) with all the gDNA that we get. After that, according to STAR protocol using the same library [1] , we should run 1 second-PCR (to add illumina adaptators) per 5000 sgRNAs so at least 24 reactions using 2.5µL of 1^st PCR product.

For their sorted positive population, I assume that they only extracted 6µg of gDNA because they only did 3 x 2µg 1^st PCRs. However, for this condition, they do only 6 second PCR reactions.

Do you know how they determine the number of 2^nd PCR they should run? Shouldn’t we run the same number of PCR2 as in the control?

[1] Qi S, Sivakumar S, Yu H. CRISPR-Cas9 screen in human embryonic stem cells to identify genes required for neural differentiation. STAR Protoc. 2022;3(4):101682.

Thank you for your response,

Zéna

[Xavier De Deken]

Dear all,

First of all, I wish you all an Happy New year 2025 and all the best in your CRISPR Screen experiments.

We are also going to perform a CRISPR screen based on the GeCKO v2.0 pooled human library using the 2-step PCR amplification protocol to add the illumina adapters for the NGS. 

We are wondering also how many PCR2 should be performed during this second step to maintain the coverage of the amplified gDNA during the first round of PCR ? For a 300x coverage, we are planing to isolate 40-30 million of cells, which should give around 200 µg of gDNA that will all be amplified during PCR1 (+/- 100x PCR with 2-3µg).

However, how many PCR2 should we have to perform to maintain the coverage when adding the Illumina adapters as we already amplified the sgRNAs during the PCR1 (x25 cycles corresponding to 33 millions) ?

Thank you in advance for your supports.

Best Regards,

Xavier DE DEKEN