RNA sequencing of lung tumor cell lines

H1975 and H1299 lung cancer cells were treated with 1 µM of EGFRi Osimertinib (LC Laboratories, USA) for 0 hour, 12 hours, 24 hours, or 24 hours with 500 U/mL recombinant human IFN-γ (R&D Systems, USA) added for the last 12 hours of culture. Cells were lysed, and total RNA was prepared using an RNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s protocol. RNAseq was performed by the Avera Institute for Human Genetics (South Dakota, USA) as follows: total RNA was assessed for degradation on an RNA 6000 Nano chip ran on a 2100 Bioanalyzer (Agilent, USA) where the average RNA integrity score for the sample set was 9.7. Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) following the low sample procedure. Briefly, ribosomal RNA was depleted from total RNA, and the remaining RNA was purified, fragmented appropriately, and primed for cDNA synthesis. Blunt-ended cDNA was generated after first and second strand synthesis. Adenylation of the 3′ blunt-ends was followed by adapter ligation prior to the enrichment of the cDNA fragments. Final library quality control was carried out by evaluating the fragment size on a DNA1000 chip ran on a 2100 BioAnalyzer (Agilent, USA). The average library yielded an insert size of 326 base pairs (bp). The concentration of each library was determined by quantitative PCR by the KAPA Library Quantification Kit for Next Generation Sequencing (KAPA Biosystems, USA) prior to sequencing. Libraries were normalized to 2 nM in 10 mM Tris-Cl, pH8.5 with 0.1% Tween 20 then pooled evenly. The pooled libraries were denatured with 0.05 M NaOH and diluted to 20 pM. Cluster generation of the denatured libraries was performed according to the manufacturer’s specifications (Illumina, USA) using the HiSeq PE Cluster Kit V.2 chemistry and flow cells. Libraries were clustered appropriately with a 1% PhiX spike-in. Sequencing-by-synthesis was performed on a HiSeq2500 using V.2 chemistry with paired-end 101 bp reads resulting in an average of 52.4 million paired-end reads per sample. Sequence read data were processed and converted to FASTQ format for downstream analysis by Illumina BaseSpace analysis software, FASTQ Generation V.1.0.0.