Technical Replicates and Assigning Rev Primers

[Mehmet Hazirci]

Hi All,

I have two CRISPR KO screens that I am conducting, which I would like to compare to eachother (resistant vs sensitive cells). For each screen, I would like to sequence at 4 time points, ie.  start, end, and 2 inbetween. 

Also, for each time point, I have taken 4x pellets with sufficient coverage (over 500x), so I have 4 technical replicates at each time point. 

Should these technical replicates be pooled pre-PCR amplification and amplified with the same R primer? Or should they be assigned different R primers, and pooled post PCR-purification and pre-gel extraction.

If second option, 4x technical replicates with 4x technical replicates each = 16 R primers needed, and there is not that many provided. 

An alternative option, which I think would probably be best, is to use one R primer for each technical replicate, and pool together a collection of 8 technical replicates and then repeat this 4 times and sequence in 4 different runs, ie. Run 1 has Screen 1 T0 x4, Screen 2 T0 x4, Run 2 has Screen 1 T1 x4, Screen 2 T1 x4, etc. 

I'd be extremely grateful for any advice. 

Best wishes,

Mehmet 

[Julia Joung]

Hi Mehmet,

I would recommend sequencing the technical replicates separately. Usually I only do 2 replicates for a CRISPR screen because there is redundancy in guides/gene. Please see below a list of 16 reverse primer barcodes you can use:

TCGCCTTG
ATAGCGTC
GAAGAAGT
ATTCTAGG
CGTTACCA
GTCTGATG
TTACGCAC
TTGAATAG
TCCTTGGT
ACAGGTAT
AGGTAAGG
AACAATGG
ACTGTATC
AGGTCGCA
AGGTTATC
CAACTCTC

Best,

Julia

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[Mehmet Hazirci]

Hi Julia,

Thanks for your advice. Just to clarify, do you mean have technical replicates with the same R primer, and sequenced separately? So one mix of DNA that will be run on agarose and gel extracted will contain both screens, multiple timepoints, but only from one replicate? And obviously, all samples in the same sequencing run will have different R primers. 

Is there a limit to the number of different R primer amplified samples one can have in the same gel-extracted sample? Could I put 16 different samples into one mixture (up to 2 ug) and gel extract it and send it off as one sequencing run? 

Thanks.

Best wishes,

Mehmet

[Julia Joung]

Hi Mehmet,

Technical replicates should have different reverse primers. After running PCR amplification, you can pool the reactions into a single gel extraction.

Best,

Julia

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