NGS of H358 cells R2
Sequencing libraries were generated, and sample-specific indexes were incorporated. Targeted regions were amplified using specific primers (KRAS_FW: 5 TGTATCAAAGAATGGTCCTGCAC 3; KRAS_RV: GATACACGTCTGCAGTCAACT) including barcodes. PCR amplification was conducted with Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific, #F530S) using the following protocol: 5 min at 95 C; 35 cycles of (30 s at 95 C, 30 s at 60 C, 30 s at 72 C); 5 min at 72 C. PCR products with proper size were selected by 2% agarose gel electrophoresis. Equal amounts of PCR products from each sample were pooled, end-repaired, A-tailed, and ligated with Illumina adapters. Sequencing was performed on a paired-end Illumina platform, generating 250 bp paired-end raw reads. Library quality and quantity were assessed using qPCR. Quantified libraries were pooled and sequenced on the Illumina NovaSeq6000 platform, aiming for a minimum of 30,000 paired-end reads per sample.