RNA-Seq of homo sapiens: adult male intervertebral disc
RNA was extracted from the samples using a TRIzol kit (Thermo Fisher, USA), and mRNA was isolated from total RNA using magnetic beads containing oligo-dT. The captured mRNA was fragmented and reverse transcribed to synthesize cDNA, which was subsequently subjected to end repair via the addition of an A base at the 3' end. Subsequently, the cDNA fragments were ligated to the sequencing adaptor, and the incomplete ligation products and the adaptor self-ligation products were removed. The sequencing library was obtained by PCR amplification using primers complementary to the adaptor sequence and purification with magnetic beads. Qubit was used to determine the library concentration, while an Agilent fragment analyser was used to determine the library fragment length to ensure library quality. Libraries were sequenced by PE150 (150 bp paired-end) using an Illumina NovaSeq 6000 sequencing platform.