Gene chip mta 1

For transcriptome profiling, we used the GeneChip® Mouse Transcriptome Array (MTA) 1.0 (released in May, 2015; currently termed Clariom™ D assay, Affymetrix Inc., Santa Clara, USA) with more than 66,100 coding and non-coding transcripts. We used two separate sets of microarray experiments (denoted as 1st and 2nd array set). The 1st array set was based on 8 testicular samples hybridized to individual gene chips. For validation, a 2nd array set was utilized, comprising 8 biologically independent replicates pooled in equivalent amounts and hybridized to a single ‘pool-microarray’ for each mouse line. RNA labeling and hybridization were conducted at the Core Facility for Microarray Analysis, University of Rostock. Briefly, 200 ng of quality controlled total RNA was used for cDNA preparation and labeling with the GeneChip® WT PLUS Reagent Kit (Affymetrix, Santa Clara, USA). The fragmented (~100 bp) and biotinylated cDNA was hybridized for 16 h at 45 °C to Affymetrix Gene Chip® MTA 1.0, followed by washing and staining using the Affymetrix Fluidics Station 450 according to standard instructions. The chips were subsequently scanned at 0.7-μm resolution (GeneChip Scanner 3000 7G, Affymetrix). Furthermore, all hybridizations were assessed for quality requirements, and raw data were submitted to the Gene Expression Omnibus (GEO) database according to MIAME guideline (GSE86063).