I have gotten some RNAseq data and I'm starting to process it. It was carried 3’mRNA library preparation and thus this is a single-end sequencing experiment. It was then sequenced with Illumina Nextseq5000. I'm now trying to understand what I need to do and have a couple of basic questions:
- As this is a 3' sequencing, does this mean that the strandedness of my fastQs is always reverse?
- Despite this, the PCR step still includes both forward and reverse primers. Why is this so considering this is a single-stranded ?
- For each file I have the following, but am confused as to exactly what these mean:
- If this had been a paired-end sequencing experiment, I would've gotten two fastQ files per sample. How do you associate them with forward, reverse, or unstranded sequencing?
Thanks a lot in advance