sgRNA alignment for the SAM library

I have downloaded the sgRNA sequences from Addgene website for the human SAM library, however, there is <1% alignment with the sgRNA sequences obtained by NGS of both the treated and untreated samples post-selection/screen. The cells died as a result dual transduction and antibiotic selection (MS2-P65-HSF1 transduction/Hygromycin selection followed by dCas9-VP64-sgRNA transduction/Blasticidin selection), so the screen was carried out using only the dCas9-VP64-sgRNA vector and Blasticidin selection. Please let me know if there is an updated list of sgRNAs or if I should modify my alignment parameters,

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Fuchsia Kelpieover 4 years ago

The human SAM library does not have a 'G' in front of the sgRNA target sequence, so if you are using from Joung Nat Protoc 2017 to do the NGS analysis, you will need to specify that with the flag 'no-g'. Maybe also check if you are seeing the sgRNA backbone in your Fastq files?

I generally do not recommend doing dual selection for the sgRNA library, because it is quite harsh on the cells, and will definitely affect your screening selection. For the 2-vector system, you should make a cell line with the MS2-P65-HSF1, grow up the cell line, and then transduce with dCas9-VP64-sgRNA. I really don't think you should do a screen with only the dCas9-VP64-sgRNA, since you will not obtain robust activation for a lot of genes.

Mauve Yetiover 4 years ago

thank you for your reply, I really appreciate it and it is most helpful. I was not able to generate a stable cell line expressing MS2-P65-HSF1 because I was working with neurons. I had to terminally differentiate cells into neurons, then transduce with MS2-P65-HSF1, select with Hygromycin, allow them to recover (2d in antibiotic-free media), then transduce with dCas9-VP64-sgRNA, select with Blasticidin, allow the cells to recover (2d in antibiotic-free media) and perform selection screen. I was not able to transduce with MS2-P65-HSF1, select with Hygromycin, harvest cells and freeze stocks since the cells are terminally differentiated into neurons. Would you know of a way to overcome this challenge when using 2-vector system with neurons? I would really appreciate any advice,

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