4.10. RNA Extraction, cDNA Library Construction and Sequencing

SGC-7901 cells were treated with SPPH for 36 h, collected, washed thrice with ice-cold PBS, and stored at −80 °C before extraction. RNA extraction was performed using Trizol Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). Untreated SGC-7901 cells under normal growing conditions were used as a control. The 23S and 16S rRNA were depleted by MicrobExpress Kit (Ambion, Austin, TX, USA). Genomic DNA was removed by using Amplification-grade DNase 1 (Invitrogen, Life Technologies, Carlsbad, CA, USA). RNA integrity was verified using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany). Then, the RNA was sheared and reverse transcribed with random primers to obtain cDNA to conduct library construction. The quality of constructed library was measured using Agilent 2100 Bioanalyzer. Qualified libraries were subsequently sequenced using BGISEQ-500 platform at BGI (Shenzhen, China). Two biological repetitions of SPPH treatment and control were performed in RNA-seq. The transcriptome raw sequencing datasets are available from Sequence Read Archive database in NCBI, and their accession number is SRP128982.