GSM9377048: Bulk RNA-seq of CD69-overexpressing NB4 sample 1; Homo sapiens; ssRNA-seq
Total RNA was extracted using TRIzol Reagent (Thermo Fisher, Cat#15596018) following the manufacturer's instructions. Briefly, cells were lysed, extracted with chloroform, precipitated with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water. RNA concentration and purity were measured using NanoDrop ND-1000. RNA integrity was evaluated using Agilent Bioanalyzer 2100; samples with concentration >50 ng/μL, RIN >7.0, and total RNA >1 μg were used for library preparation. PolyA+ mRNA was enriched using Dynabeads Oligo(dT) (Thermo Fisher, Cat#25-61005) with two rounds of purification. mRNA was fragmented using NEBNext Magnesium RNA Fragmentation Module (NEB, Cat#E6150S) at 94°C for 5–7 minutes. First-strand cDNA synthesis was performed using SuperScript II Reverse Transcriptase (Invitrogen, Cat#1896649). Second-strand synthesis was performed using DNA Polymerase I (NEB, Cat#M0209) and RNase H (NEB, Cat#M0297) with dUTP incorporation (Thermo Fisher, Cat#R0133) to produce strand-specific libraries. DNA fragments were end-repaired, A-tailed, ligated to adapters, size-selected using magnetic beads to obtain ~300 bp ± 50 bp fragments, and treated with UDG (NEB, Cat#M0280). Libraries were amplified using PCR (95°C 3 min; 8 cycles of 98°C 15 s, 60°C 15 s, 72°C 30 s; final extension 72°C 5 min). Libraries were quantified and sequenced on an Illumina NovaSeq 6000 (PE150).