Can we use MAGeCK for shRNA screening?

Question

[donny.dw]

Can we use MAGeCK for shRNA screening? How can we get the hairpin sequences for each shRNA?

[ATpoint]

Yes, MAGeCK has been developed for genomic screening libraries such as CRISPR or shRNAs. Towards the sequences, you will have to check the paper if it is published data, or ask the person who made the experiment if it is something in-house. In shRNA and CRISPR applications you do not sequence the shRNA (or DNA template) itself but barcodes attached to it, so you won't get that from the sequencing data. Reason is that shRNAs tend to form secondary structures that are difficult to sequence, hence one puts a barcode close to them which is easy to sequence and unique to every shRNA. PCR regime during NGS preparation based on the extracted genomic DNA is then designed to PCR-out the barcode and put sequencing adapters on it. The output of such an experiment is typically a simple count matrix with column being samples and rows being the barcodes which you then can use to find back the shRNA sequence and by this the gene, but for this you need the initial table that links barcodes to shRNA sequences. You cannot, as said, infer this from the data itself.