Effect of ALK kinase inhibitor, ceritinib, on the gene expression profile of SUDHL-1 cell line
Effect of ALK kinase inhibitor, ceritinib, on the gene expression profile of SUDHL-1 cell line
Cells were harvested after treatment and washed 1x with PBS prior to RNA extraction. Human NPM1::ALK-positive anaplastic large cell lymphoma (ALCL) SU-DHL-1 cell line was cultured in RPMI 1640 supplemented with 100 units/mL of penicillin, 100 µg/mL of streptomycin (Pen/Strep) and 10% fetal bovine serum (FBS). Cells were grown in standard conditions (37°C and 5% CO2) and routinely checked for mycoplasma contamination. Cells were treated with 50nM of ceritinib (Selleck Chemicals) or vehicle (DMSO) diluted in culture medium for 48 hours. RNA was extracted using the RNeasy plus mini kit (Qiagen) following manufacturer's instruction To construct the libraries, 1 µg of high-quality total RNA sample (RIN>8) was processed using TruSeq Stranded mRNA kit (Illumina) according to manufacturer instructions. Briefly, after purification of poly-A containing mRNA molecules, mRNA molecules were fragmented and reverse- transcribed using random primers. Replacement of dTTP by dUTP during the second strand synthesis permitted to achieve strand specificity. Addition of a single A base to the cDNA was followed by ligation of Illumina adapters.  Libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina Libraries (KapaBiosystems, Wilmington, MA) and library profiles were assessed using the DNA High Sensitivity LabChip kit on an Agilent Bioanalyzer. 