Hi Biostars community, I am making my first foray into RNA Sequencing on chicken tissues and I have run into a problem. We have conducted an experiment on isolated AV Cushion cells and performed an RNA Seq to gather some exploratory data. We prepped rna using the RNEasy plus kit, which has a genomic dna removal column, and at the end we judged the RNA Quality to be good (RINs all were >9.7). As far as I know, library preparation and sequencing went off went off without a hitch (my collaborators performed a polyA enrichment and then sent the samples for sequencing on an Illumin Novaseq 6000 platform). Alignment of my reads to the Galgal6 genome using STAR worked ok (~63% of reads were unique paired aligned reads, and about 37% were not mapped). However, when I looked at the coverage breakdown, only 3% of the reads mapped fully within an exon (about 18% mapped partly within an exon, 43.15% mapped fully within an intron, and 33.65% was mapping to intergenic DNA). Does anyone have an idea why we are getting so few of our reads mapping to exons? Can anyone who has done chicken RNASeq before share their thoughts/experience? Potentially a problem with polyA enrichment maybe? I am new to the game and need help!