HERV-V3 Microarray Protocol: Transcriptome Profiling

The cDNA synthesis and amplification steps were performed from 16 ng of RNA, using the Ovation Pico WTA System V2 kit (Nugen), in accordance with the manufacturer’s instructions. Five micrograms of purified, amplified cDNA was fragmented into 50–200 bp fragments and were 3′-labeled using the Encore Biotin Module kit (Nugen), in accordance with the manufacturer’s instructions. The HERV-V3 microarrays were hybridized at 50 °C for 18 h in an oven, with constant stirring (60 rpm). Washing and staining were performed using the protocol supplied by the manufacturer, using the GeneChip fluidic station 450 (Affymetrix). The arrays were scanned using the GeneChip fluorometric scanner 3000 7G (Affymetrix). Images (DAT files) were converted to CEL files using the GCOS software (Affymetrix) [22 (link)]. The experimental data generated were deposited in the National Center for Biotechnology Information (NCBI) and are available in the GEO DataSets site, under accession number GSE121352.