Ls reloaded scanner

cRNA sample preparation, labeling and hybridization were performed according to the manufacturer’s instructions. Briefly, 0,5 μg of total RNA was used for cDNA synthesis and amplification using the Message™Amp aRNA kit (ThermoFisher Scientific). Then 825 ng of cRNA labeled with Cy3/Cy5 dyes using the Arcturus® TURBO labeling™ Cy™3/Cy™5 Kit (Applied Biosystems, Netherlands) were hybridized to Human 4x44k Oligonucleotide Microarrays (Agilent Technologies, USA) using the HS 400 hybridization station (Tecan, Switzerland). Microarray slides were scanned using a LS Reloaded scanner (Tecan, Switzerland) for microarray image analysis, and the data generated were further analyzed using ImaGene ver. 9.0 (BioDiscovery, USA) and GeneSpring GX ver. 11.0 (Agilent Technologies, USA) software. Loess normalization was performed to adjust microarray data for variation. Gene expression fold change above 1.5 (with p-value < 0.05) was defined as differentially expressed between two conditions. KEGG pathway enrichment analysis was performed using Webgestalt online source [22 (link)]. Network construction analysis using the GeneMANIA plug-in of Cytoscape was performed to predict the most related genes of our gene sets [23 (link)]. All of the microarray data was deposited in a GEO Dataset database, Accession number GSE93228, (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93228).

cRNA sample preparation, labeling and hybridization was performed according to manufacturer’s instructions. Briefly, 1 μg of total RNA was used for cDNA synthesis and amplification using Message™Amp aRNA kit (ThermoFisher Scientific, USA). Then 825 ng of cRNA labeled with Cy3/Cy5 dyes using Arcturus® TURBO labeling™ Cy™3/Cy™5 Kit (ThermoFisher Scientific, USA) were hybridized to Agilent Mouse Whole Genome 4x44k Oligonucleotide Microarrays (Agilent Technologies, USA) using HS 400 hybridization station (Tecan, Switzerland). Three independent replicates of every sample were used. Microarray slides were scanned using LS Reloaded scanner (Tecan, Switzerland). Microarray image analysis and data generated were further analyzed using ImaGene ver. 9.0 (BioDiscovery, USA) and GeneSpring GX v11.5 software (Agilent Technologies, USA). Raw extracted gene expression data were normalized with Loess normalization to adjust microarray data for variation. Genes that showed expression values above fold change 1.5 (with p-value <0.05) were defined as differentially expressed in LLC1 cells between 2D and 3D cell culture conditions. Microarray design and data are available at the GEO database (Accession No. GSE75863 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE75863).

For gel electrophoretic analysis, 3 µl DNA Gel Loading Dye (6 x) (Thermo Fisher Scientific; R0611) were added to the purified (and digested) samples except the RsaI approach. For band visualization, a 2% agarose gel was prepared in 1 × TAE buffer (50 × TAE buffer; PanReac AppliChem; A1691) containing 4 µl peqGreen (VWR; 732-3196) per 100 ml gel. 12–15 µl of the purified and prepared samples were used for electrophoresis at 120 V for 1 h before detection via BioDoc Analyzer (Biometra) using a Canon EOS 1100D. GeneRuler Low Range DNA Ladder (Thermo Fisher Scientific; SM1191) and peqGOLD DNA ladder (VWR; 25-2040) were used for calculation of fragment length.
For the detection of incorporated Cy5-dUTP the DNA gel was scanned with a Tecan LS reloaded scanner using a 3D-printed gel tray.