hESCs cultured to 80%-100% confluence in 10-12 wells were digested with 300 μl of collagenase (1 mg/ml) for 30 minutes at 37℃. This was followed by a 5-minute incubation at 37℃ with 500 μl of Trypsin-EDTA and 50 μl of DNase, shaken at 1,000 rpm. The reaction was halted with 50 μl of 100% FBS. The cells were then washed with PBS and filtered through a 40 μm cell strainer to remove clumps, resulting in a single-cell suspension, which were collected into 900 μl of the RNAiso Plus solution (TaKaRa) for RNA extraction. Library preparation is performed using Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China), where poly(A)+ RNA was enriched with oligo(dT) beads, fragmented, and converted to double-stranded cDNA with dUTP incorporation for strand specificity. Following end repair, A-tailing, and adapter ligation, the library was PCR-amplified, circularized, and transformed into DNA nanoballs (DNBs) via rolling circle amplification. These DNBs were then sequenced on BGI's DNBSEQ-G400 platform.