title: GSM8855455: CAR3_DKO_CD58KO; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from samples using Trizol reagent (Thermo Fisher) following the manufacturer's protocol. mRNA was enriched from 5 µg of total RNA using Dynabeads Oligo(dT) (Thermo Fisher) through two rounds of purification. The purified mRNA was fragmented using divalent cations (Magnesium RNA Fragmentation Module, NEB) at 94°C for 5–7 minutes. First-strand cDNA synthesis was performed with SuperScript™ II Reverse Transcriptase (Invitrogen), followed by second-strand synthesis using E. coli DNA polymerase I (NEB), RNase H (NEB), and dUTP Solution (Thermo Fisher) to generate U-labeled double-stranded cDNA. After A-tailing and adapter ligation (dual-indexed adapters), size selection was performed with AMPureXP beads. UDG enzyme (NEB) treatment removed U-labeled strands, and PCR amplification (8 cycles) generated the final cDNA libraries with an average insert size of 300 ± 50 bp. Paired-end sequencing (2 × 150 bp) was conducted on an Illumina Novaseq™ 6000 platform (LC-Bio Technology).