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 count_spacers.py 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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