Bulk RNA‑seq profiling of CD69‑overexpressing and control NB4 cells

RNA quantity and purity were evaluated using NanoDrop ND-1000, and RNA integrity was confirmed on an Agilent Bioanalyzer 2100, with samples meeting the criteria of concentration >50 ng/µL, total RNA >1 µg, and RIN >7.0. Polyadenylated mRNA was enriched using Dynabeads Oligo(dT) (Thermo Fisher, Cat#25-61005) with two rounds of purification.

Fragmentation was performed using the NEBNext Magnesium RNA Fragmentation Module (NEB, Cat#E6150S) at 94°C for 5–7 minutes. First-strand cDNA was synthesized with SuperScript II (Invitrogen, Cat#1896649), and second-strand synthesis used DNA Polymerase I (NEB, Cat#M0209) and RNase H (NEB, Cat#M0297) incorporating dUTP (Thermo Fisher, Cat#R0133) to generate strand-specific libraries. Libraries were end-repaired, A-tailed, ligated to adapters, and size-selected (~300 bp ± 50 bp), followed by UDG treatment and PCR amplification.