title: GSM9443538: IhESCs, NC_1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using a standard protocol, and its integrity was assessed via the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA); high-quality RNA samples were selected, with 1 µg of total RNA per sample used as input material, and messenger RNA (mRNA) was specifically purified from total RNA using poly-T oligo-attached magnetic beads for subsequent library construction. Two types of libraries were constructed: for the NEB library, purified mRNA was fragmented, first-strand cDNA was synthesized with random hexamer primers followed by second-strand cDNA synthesis, and the library was generated after end repair, A-tailing, adapter ligation, size selection, amplification, and purification; for the strand-specific library, the second-strand cDNA was synthesized with dUTP instead of dTTP, and an additional USER enzyme digestion step was included before amplification, with both libraries quantified by Qubit and real-time PCR and their size distribution detected by the Bioanalyzer for quality control.