Hoping the community can provide some insight. I manage a NGS core facility and recently did some miRNA-Seq for a user. The library kit was NEB Next Small RNA and I sequenced on the MiSeq generating 50 bp reads. Now...the user tells me that 3 of the samples are mir-21 Knock-Out mice confirmed by PCR however I get reads mapping to mir-21a in ALL of the samples. It is significantly down-regulated in the Knock-Outs but there are plenty of reads there nonetheless. Does anyone have any explanations for this, I'm at a loss.
If it helps here is the Bioinformatic pipeline I am using:
-CutAdapt to trim low quality reads and adapters -Bowtie to map to GRCm38 -HTSeq to count reads mapping to miRNAs -DESeq2 for differential expression