Rw5N3h-2
mRNA enrichment from total RNA was performed using Oligo(dT) magnetic beads to isolate polyA-tailed mRNA. The RNA was fragmented, reverse-transcribed with random N6 primers, and converted into double-stranded cDNA. The DNA ends were blunt-ended, 5'-phosphorylated, and A-tailed, followed by ligation to a bubble-shaped adapter with a 3'-T overhang. The ligated products were PCR-amplified, heat-denatured into single strands, and circularized using a bridge primer to generate single-stranded circular DNA libraries. After quality control, qualified libraries were sequenced.