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. 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.

本数据集针对敲低ARAF（shARAF）的A549细胞，分别以空载体对照（+EV）、野生型ARAF（+ARAF）或二聚体缺陷型ARAF激酶（+R362H）进行回复补全，旨在鉴定受ARAF及其激酶活性差异调控的分子。研究发现，ARAF通过抑制ERBB3/AKT信号通路成员的基因表达，调控参与细胞增殖与恶性生长的相关基因。同时，借助topGO对差异表达（DE）基因进行功能富集分析，结果显示ARAF在调控细胞黏附、细胞运动、上皮细胞迁移等诸多生物学过程相关基因中发挥关键作用。
本研究设置两组生物学重复：对敲低ARAF（shARAF）的A549细胞，分别以空载体对照（+EV）、野生型ARAF（+ARAF）及其激酶失活突变体（+R362H）进行回复补全后，将细胞接种于6孔细胞培养板，培养48小时后进行RNA提取。采用预冷PBS洗涤细胞后，一部分用EDTA消化细胞，另一组直接在培养板中使用罗氏（Roche）High Pure RNA Isolation试剂盒（货号Cat#11828665001）按照厂商说明书完成细胞裂解与RNA提取。总RNA浓度通过Qubit 2.0荧光计（Invitrogen）定量，RNA质量与完整性则采用Agilent Bioanalyzer 2100搭配RNA6000 Nano芯片进行检测。仅选取RNA完整性指数（RNA Integrity Number, RIN）>8的样本开展后续RNA文库构建。
取500 ng总RNA，使用NEBnext Poly(A) mRNA Magnetic Isolation Module与NEBNext Ultra RNA Library Prep Kit for Illumina（NEB）按照说明书构建带条形码的cDNA文库。文库浓度通过Qubit 2.0配合Invitrogen Qubit HS检测试剂盒评估，文库片段大小则采用Agilent Bioanalyzer 2100搭配HS DNA检测芯片测定。
将带条形码的RNA-seq文库以8pM浓度使用HiSeq Rapid SR Cluster Kit v2完成上机簇生成，随后在Illumina HiSeq2500平台上采用HiSeq Rapid SBS Kit v2进行51 bp单端测序。