Transcriptome profiling of MYO5B mutants

Stably transfected SK-N-AS cells were seeded onto T75 flasks (1x10⁶ cells), then cultured and harvested at 24, 48 and 72 hours. The procedure was repeated twice (at passage 23 and passage 30). Cells were pelleted and RNA was extracted using Maxwell 16 LEV SimplyRNA kit (Promega) according to manufacturer’s protocol. RNA quality assessment was performed by measuring absorbance (A260/280; 1.9–2.1) on Nanodrop (Denovix DS-11 spectrophotometer) and RNA integrity number (RIN) values (>9.0) with High Sensitivity RNA Screen tape on 2200 Tape station (Agilent Technologies). Expression analysis was performed with Clariom S arrays for five constructs (empty vector, MYO5B^WT, p.L587P, p.G1611S, and p.R1641C) at three time points of proliferation (24h, 48h, 72h) in duplicates by Eurofins Genomics (www.eurofinsgenomics.eu). Expression data on the 40 top-ranked differentially expressed genes from all three MYO5B mutants, i.e. genes present as differential expressed top-candidates in at least two out of three time points in at least two mutants, or at least in one time point in all three mutants, are presented in S5 Table. Next, Gene Ontology analysis by GOrilla (http://cbl-gorilla.cs.technion.ac.il [38 (link)]) and gene set enrichment analysis (GSEA, [39 (link),40 (link)]) was performed on the gene lists. For further details on microarray and enrichment analyses see S1 File.