title: Control
design: Following RNA quality verification, rRNA was depleted from total RNA via probe-based removal to enrich mRNA, which was then randomly fragmented using fragmentation buffer. Using a strand-specific protocol (Parkhomchuk et al., 2009), first-strand cDNA synthesis was performed using fragmented mRNA as template, random hexamer primers, and M-MuLV Reverse Transcriptase. After RNase H-mediated RNA degradation, second-strand cDNA was synthesized with DNA Polymerase I using dNTPs containing dUTP in place of dTTP. Purified cDNA underwent end-repair, A-tailing, and ligated to sequencing adapters. Fragments of 370420 bp were size-selected using AMPure XP beads. The dUTP-incorporated second strand was specifically digested by USER enzyme (uracil-specific excision), leaving first-strand cDNA for PCR amplification. The library was finally purified with AMPure XP beads.
library_name: FRRP230252759-1r