Human gene 1.1 st array plate

RNA isolate from three independent biological replicate experiments was used for microarray analysis. Total RNA yield was measured using photometry (DeNovix, USA). RNA quality was determined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). RNA was only used when the RNA integrity number (RIN) exceeded 8.0. One hundred nanogram of RNA was converted to cDNA and labelled (Ambion WT expression kit, Life Technologies, Bleiswijk, The Netherlands). Samples were hybridized to an Affymetrix Human Gene 1.1 ST array plate according to the standard Affymetrix instructions (Affymetrix, Santa Clara, CA, USA). The robust multi-array average (RMA) pre-processing algorithm in the Bioconductor library AffyPLM was used to obtain normalized expression estimates [47 (link)]. Probe sets were defined and assigned as described by Dai et al. [48 (link)]. Differences in gene expression between static and SWMS conditions per cell type were analyzed using the Intensity Based Moderated T statistics (IBMT) [49 (link)], using p values <0.05 as threshold. The Venn diagram was created using Venny 2.1 [50 ]. Microarray data has been submitted to the Gene Expression Omnibus (GEO) at the NCBI (GSE173729).

Purified RNA was labeled with the Affymetrix WT PLUS reagent kit (Affymetrix, Santa Clara, CA) and hybridized to an Affymetrix Human Gene 1.1 ST array plate (Affymetrix). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform according to manufacturer's instructions. Arrays were analyzed using the R package Oligo (Carvalho and Irizarry 2010) following standard procedures for quality checks and calculation of normalized expression values.

The analyses were conducted in the analytic center using the vendors’ recommended methods. The Ovation Pico WTA System v2 (NuGEN Technologies, San Carlos CA, USA) was used with 50 ng of input total RNA, providing linear expansion of all transcripts without interference from ribosomal or globin RNAs. The resulting cDNA was labeled with biotin (Encore Biotin Module; NuGEN) and hybridized to Human Gene 1.1 ST Array Plates (Affymetrix, Santa Clara CA, USA) followed by scanning on an Affymetrix GeneTitan instrument and inspection of quality control metrics and probeset summarization with Expression Console software. Microarray data sets were analyzed using Partek Genomics Suite (Partek, St. Louis MO, USA) with normalization by the RMA algorithm (Irizarry et al., 2003 (link)).