Hi everyone I have a question about the parameters to use for my SE RNA-Seq samples for the alignment and counting steps of my pipeline.
I am using hisat2 and featureCounts for the alignment and counting programs, upstream of DESEQ2 for detecting differential expression. I know that my reads are reversely stranded, not only from the protocol for library preparation but also because if I run hisat2 with the [--rna-strandedness "F"] option the bam file viewed via igv shows almost all of the reads pointing in the opposite direction as what the annotation file gtf shows.
My question is this: If I run hisat2 [--rna-strandedness "F"] and then featureCounts [-s 2], I get the same results as if I do hisat2 [--rna-strandedness "F"] and then featureCounts [-s 2]. But wouldn't the "R" and -s 2 options counteract each other when used in the same pipeline and thus the reverse complement of the read would not be used for counting?