Genechip command console v4

Total RNA of 1 × 10⁶ MPC or MSC, each cultured either in growth medium or 6 days in differentiation medium, was isolated by the RNEasy Kit (QIAGEN, Hilden, Germany) according to the manufacturers’ instructions. Sample preparation for microarray hybridization was carried out as described in the NuGEN Ovation PicoSL WTA System V2 and NUGEN Encore Biotin Module manuals (NuGEN Technologies, Inc., San Carlos, CA, USA). Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. The Affymetrix GeneChip Command Console v4.1.3 software controlled fluidics and scan functions. The Affymetrix Service Provider and Core Facility, “KFB - Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany) performed the sample processing.

Sample preparation for microarray hybridization was carried out as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual (Affymetrix, Inc., Santa Clara, CA, USA).
In brief, 200 ng of total RNA was used to generate double-stranded cDNA. Subsequently, synthesized cRNA (15 µg) was purified and reverse transcribed into sense-strand (ss) cDNA, into which unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1), followed by terminal labeling with biotin. Then, 5.5 µg fragmented and labeled ss cDNA were hybridized to Affymetrix Clariom D human arrays for 16 h at 45 °C in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by Affymetrix GeneChip Command Console v4.1.3 software.
Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB—Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de, accessed on 20 November 2017).