title: RNA-seq of cells: gastric cancer line
design: RNA Extraction: Total RNA was isolated from human gastric cancer cell line HGC-27 using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Residual genomic DNA was removed by DNase I treatment (RNase-free DNase Set, Qiagen). RNA integrity was verified using an Agilent 2100 Bioanalyzer, with RNA Integrity Number (RIN) > 8.0.Library Preparation: Strand-specific RNA sequencing libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770). Polyadenylated mRNA was enriched using oligo(dT) magnetic beads, followed by fragmentation at 94C for 8 minutes. First-strand cDNA synthesis was performed with random hexamer primers and ProtoScript II Reverse Transcriptase, and second-strand cDNA was synthesized using dUTP for strand marking. Adapters (Illumina TruSeq) were ligated, and libraries were amplified with 12 cycles of PCR.Sequencing Platform: Paired-end sequencing (2150 bp) was conducted on the Illumina HiSeq 2500 platform (High-Output Mode) at BGI Genomics (Shenzhen, China). The average sequencing depth was 40 million reads per sample, with >85% of bases achieving Q30 quality score.Quality Control: Library concentration was measured using a Qubit 4.0 Fluorometer (Thermo Fisher), and insert size distribution was validated by Agilent 4200 TapeStation (average insert size: 300  20 bp). Raw data quality was assessed using FastQC v0.11.9.Experimental Design: Three biological replicates were analyzed for each experimental group to ensure reproducibility.