GSM9392771: Co_Tp_48_8K; Homo sapiens; RNA-Seq
RNA was isolated from orbital fibroblast cells using the RNeasy Plus Mini kit (Cat#74134, Qiagen) according to manufacturer instructions. Messenger RNA (mRNA) was purified from total RNA using poly-T oligo–attached magnetic beads to isolate polyadenylated transcripts. The purified mRNA was then fragmented into short fragments, which served as templates for first-strand cDNA synthesis using random hexamer primers. The second-strand cDNA was synthesized using dTTP, generating non-directional libraries. Following cDNA synthesis, fragments were subjected to end repair, A-tailing, and adapter ligation. Ligated products were then size-selected, PCR-amplified, and purified to obtain the final sequencing libraries. Library quality and quantity were assessed using a Qubit fluorometer for concentration measurement, real-time PCR for accurate quantification, and a Bioanalyzer for insert size distribution. Final libraries were pooled according to effective concentration and sequenced on an Illumina platform to generate paired-end reads.