RNA-seq of pancreatic CAF-derived cell line 0082T treated with Autotaxin inhibitors (IOA-289 and PF-8380) against DMSO treated controls
RNA-seq of pancreatic CAF-derived cell line 0082T treated with Autotaxin inhibitors (IOA-289 and PF-8380) against DMSO treated controls
After 24 hours of treatment, 0082T cells were lysed in RLT plus lysis Buffer (Qiagen) 0082T cells were maintained in high glucose (4.5 g/L) Dulbecco's modified eagle's medium (DMEM) with L-glutamine and pyruvate supplemented with 10% FBS in humidified atmosphere with 5% CO2 at 37 degrees The day after seeding, cells were washed twice with serum free DMEM and treated with either 0.1% DMSO, 12 µM of IOA-289, or 12 µM of PF8380 in serum free DMEM supplemented with 1 mM Glucose, 0.5 mM Glutamax, and 0.5% fatty acid free BSA for 24 hours. RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with RNA Kit on Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA).