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Ribosomal RNA (rRNA) was removed from total RNA using DNA probes designed to hybridize with rRNA, followed by RNase H digestion of RNA-DNA hybrids and DNase treatment to degrade the DNA probes. The remaining RNA was fragmented using a fragmentation buffer, and random N6 primers were used for reverse transcription to synthesize the first strand of cDNA, followed by second-strand synthesis. Libraries with an average insert size of ~300 bp were prepared by blunting the double-stranded DNA ends, ligating adapters, and amplifying the ligated products using specific primers. The amplified products were denatured into single strands and circularized using a bridge primer to generate single-stranded circular DNA libraries.