Illumina HiSeq 2000 paired end sequencing; RNA-seq of A431 cell line after gefitinib treatment
RNA-seq of A431 cell line after gefitinib treatment
At 2 h (treated and controls), 6 h and 24 h after treatment, cells were harvested by trypsination and washed in PBS. Cell suspensions were divided into aliquots for whole-cell analysis, subcellular fractionation and RNA sequencing. A431 human cells (DSMZ #ACC-91, tested for, and found free of, Mycoplasma contamination) were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, non-essential amino acids, and sodium pyruvate, all from Invitrogen. Twenty four hours after seeding, the A431 cell cultures (in triplicates) were treated with gefitinib to induce altered gene/protein expression. Controls were left untreated. Total RNA was prepared from A431 cells (aliquots from the samples run on mass spectrometry) in biological triplicates using RNeasy Mini kit (Qiagen). cDNA libraries were constructed according to a modified Illumina RNA-seq library preparation protocol described in http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021910