Transcriptome analysis of human lung andenocarcinoma cell line A549, in which ARAF was depleted and reconstituted with an empty vector control, ARAF wildtype, or a dimer deficient and kinase inactive mutant.. Transcriptome analysis of human lung andenocarcinoma cell line A549, in which ARAF was depleted and reconstituted with an empty vector control, ARAF wildtype, or a dimer deficient and kinase inactive mutant.

RNA sequencing analysis of A549 cells that were depleted for ARAF (shARAF) and reconstituted with either empty vector control (+EV) or ARAF wildtype (+ARAF) or dimer deficient ARAF kinase (+R362H) respectively to identify factors that are differentially regulated by ARAF and its kinase activity. In particular, we discovered that ARAF regulates genes that are involved in proliferation and malignant growth by surpressing gene expression of members of the ERBB3/ AKT signaling pathway. At the same time, a functional interpretation of the differentially expressed (DE) genes with topGO indicated an important role of ARAF in regulation of genes involved in, among others, cell adhesion, cell motility, epithelial cell migration. Overall design: Two biological replicates of A549 cells that were depleted for ARAF (shARAF), reconstituted with either empty vector control (+EV) or ARAF wildtype (+ARAF) and its kinase deficient mutant (+R362H) were seeded in a 6-well cell culture dish and left for growing (48h) before RNA isolation. After ice cold PBS-wash, cells were either detached with EDTA or directly lysed on plate using High Pure RNA Isolation kit (Cat#11828665001, Roche) according to maufacturer´s instructions. Total RNA was quantified by a Qubit 2.0 fluorometer (Invitrogen) and RNA quality and integrity was determined using a RNA6000 Nano chip on Bioanalyzer 2100 (Agilent). Samples with RNA integrity number (RIN) > 8 were further subjected for RNA library preparation. Barcoded cDNA libraries were prepared from 500 ng of total RNA using the NEBnext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) according to the provided instruction. Library quantity was assessed on a Qubit 2.0 using Invitrogen’s Qubit HS assay kit and library size was determined using Agilent’s Bioanalyzer 2100 and a HS DNA assay chip. Barcoded RNA-Seq libraries were onboard clustered using HiSeq Rapid SR Cluster Kit v2 using 8pM and 51 bps were sequenced on an Illumina HiSeq2500 using a HiSeq Rapid SBS kit v2.