I am very new to this world of RNA seq data analysis. I performed RNA seq between wildtype and Gene of interest (GOI) Knockout (KO) mutant to determine the effect of GOI in differential gene regulation. I am using Galaxy for the data processing. My experimental pipeline is (a trial and error one) >> Trimmomatic >>Hisat2 >>Htseq count >>DeseQ2 analysis(No intermediate steps in between) . I have one big question that worries me a lot at the moment. In the normalised count data, I could see value 12 for the GOI in GOI KO RNA sample whereas other two replicates show 0 in its respective place for the gene. What could be the chances of it??. It bothers me a lot. please help me.
Are the three samples based on the same clone or are these three independent KOs? How was the KO achieved (CRISPR?). Complete deletion of the gene or a partial deletion of some exons?
Yes, all the replicates belong to the same clone. The knock out is performed by simple replacement of the GOI by hygromycin resistance cassette using homologous recombination method. I am working with Ustilago maydis, a basidiomycete fungus which has high homologous recombination rate.