Spermatozoal RNA elements in idiopathic infertile males undergoing fertility treatment
The SeqPlex RNA Amplification Kit (Sigma Aldrich) was used to reverse-transcribe the total RNA and amplify the resulting double-stranded cDNA through primer-mediated amplification, according to manufacturer instructions. Primers were subsequently eliminated, and DNA was purified using the Zymo DNA Clean & Concentrator-5 (Zymo Research). The NEBNext Ultra II DNA Library Prep Kit (New England BioLabs) was used to prepare and amplify barcoded libraries from purified cDNA. Individual large RNA libraries underwent bead-based size-selection with SPRI beads (Beckman Coulter). Sequencing data was collected using the NovaSeq 6000 Sequencing System (Illumina). An S2 NovaSeq flow cell was used, with a read length of 100bp, paired-end.