Ovation pico wta system kit

6-well (3516), 96-well tissue culture (3596), 96-well round bottom (3799), T75 (430641) flasks, and 35 um filter cap FACS tubes (352235) were from Corning. DMEM (11960), Penicillin-Streptomycin (15070, 5000 U/mL), Glutamax (100X, 35050-061), Sodium Pyruvate (11360, 100 mM), and 0.25% trypsin/1 mM EDTA (25200) were from Invitrogen. Fetal Bovine Serum (SH30396.03) was from Hyclone. AO/PI (F23001) and Luna (L12002) slides were from Logos Biosystems. CD44-APC (clone G44-26; #559942) was from BD Biosciences. DMSO (D2650), resazurin (R7017), and DAPI (D8417) were from Sigma. EREG ELISA (SEB945HU) was from Cedarlane. RNeasy Mini Kit (74106) was from Qiagen. RNA 6000 Pico and Nano Kits were from Agilent. Ovation Pico WTA System Kit was from NuGEN. Human Gene ST 2.1 microarrays were from Affymetrix. Clinical-grade cisplatin (Teva Pharmaceuticals; 1mg/mL) was obtained from University of Michigan Hospital Pharmacy. BGJ398 (S2183) was from Selleckchem and dissolved in DMSO to 1.78 mM. DEAB (01705) and Aldefluor Assay Buffer (01702) were from StemCell Technologies. Aldefluor reagent, BAAA-DA, was synthesized by the University of Michigan Vahlteich Medicinal Chemistry Core.

Total RNA was extracted with the RNeasy Plus Micro Kit (Qiagen) and was quantified with the NanoDrop ND-1000 (ThermoFisher Scientific). Quality of the extracted RNA was evaluated with the Agilent 2100 Bioanalyzer system (Agilent Technologies, Massy, France). Forty nanograms of total RNA per sample was amplified with the Ovation^® Pico WTA System Kit (NuGEN, San Carlos, CA), then two micrograms of amplified and purified cDNA was labeled with the SureTag DNA Labeling kit (Agilent Technologies). Cy3-labeled cDNA samples (2.5 μg) were used for 17-hour hybridization at 65°C to the SurePrint G3 Mouse GE 8 × 60K Microarrays (Agilent Technologies). All procedures were carried out according to the manufacturer's instructions. The hybridized microarrays were scanned with the Agilent DNA Microarray Scanner (G2505C). Signal intensity was quantified from the scanned image by using Feature Extraction software version 11.0.1.1 (Agilent Technologies). These experiments were performed by Imaxio (Clermont-Ferrand, France). The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus [79 (link)] and are accessible through GEO Series accession number GSE73758 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73758).