An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

Mauthe, Mario; Langereis, Martijn; Jung, Jennifer; Zhou, Xingdong; Jones, Alex; Omta, Wienand; Tooze, Sharon A.; Stork, Björn; Paludan, Søren Riis; Ahola, Tero; Egan, Dave; Behrends, Christian; Mokry, Michal; de Haan, Cornelis; van Kuppeveld, Frank; Reggiori, Fulvio

RNA isolation, cDNA synthesis, quantitative real-time PCR, and RNA sequencing Total RNA was isolated from U2OS cells using an RNeasy mini kit (QIA GEN). Isolated RNA was either further processed for RNA sequencing or reverse transcribed into cDNA using a TaqMan reverse transcription reagents (Thermo Fisher Scientific). Real-time PCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) using the iQ SYBR Green Supermix kit (Bio-Rad Laboratories) and specific primers (Table S3) . Alternatively, the Power SYBR Green Cells-to-CT kit (Thermo Fisher Scientific) was used according to manufacturer's protocol to isolate RNA, reverse transcribe the RNA, and synthesize cDNA. Quantitative PCR was performed in a CFX connect Thermocycler (Bio-Rad Laboratories) using specific primers (Table S3 ). RNA quality was controlled for integrity using capillary electrophoresis (Labchip GX; PerkinElmer) before RNA sequencing. Sample preparation was performed from 500 ng total RNA using the QuantSeq 3′ Library Prep kit (Lexogen), and 50-bp single-end samples were sequenced on an Illumina HiSeq2500. Gene expression quantification was performed as follows: The trimmed fastQ files were aligned to the human build b37 reference genome using hisat/0.1.5-β-goolf-1.7.20 (Kim et al., 2015) with default settings (stranded). Before gene quantification, SAMtools/1.2-goolf-1.7.20 (Li et al., 2009 ) was used to sort the aligned reads. The gene level quantification was performed by HTSeq-count HTSeq/0.6.1p1 (Anders et al., 2015) using mode = union. Ensembl version 75 was used as gene annotation database (Genome Analysis Facility of the University Groningen). Differentially expressed genes were identified using the DESeq2 package with standard settings (http ://www .ncbi .nlm .nih .gov /pmc /articles /PMC4302049 /). Genes with log2 fold change >0.2 and p adj <0.1 were considered as differentially expressed. All other genes were considered as unchanged in their expression compared with control cells. The UniProt website (http ://www .uniprot .org /) was used for Gene Ontology and pathway analysis.