C1 (CRX1465951; CRA022407)
mRNA enrichment was performed on total RNA using magnetic beads with oligo dT to capture mRNA with polyA tails. The enriched RNA was then fragmented using a fragmentation buffer, followed by reverse transcription with random N6 primers to synthesize the first strand, and subsequently generating double-stranded cDNA. The ends of the double-stranded DNA were repaired to form blunt ends, phosphorylated at the 5' ends, and an "A" overhang was added to the 3' ends. These ends were then ligated to bubble-like adapters with a complementary "T" overhang at the 3' end. The ligation products were amplified by PCR using specific primers. The PCR products were denatured into single strands, and a bridge primer was used to cyclize the single-stranded DNA into a single-stranded circular DNA library.