DNA Fragmentation and Labeling for Microarray

Genomic DNA was fragmented by incubating at 37°C for 1 min. in a 20 μl reaction containing 1 × One-Phor-All Plus Buffer (GE Healthcare) and 0.01 units DNase I (GE Healthcare) as previously described (Jackson et al., 2011 (link)), and further modified by Tall et al. (2015 (link)). The fragmented DNA was heat-inactivated at 99°C for 15 min, and was 3′-end labeled by adding 4 μl of 5 × terminal transferase buffer (Promega), 1 μl 1 mM biotin-11-ddATP (PerkinElmer NEL508), and 2 μl (60 units) of terminal transferase enzyme (Promega). The labeling was carried out for 4 h at 37°C followed by heat inactivation at 98°C for 1 min.
Hybridizations were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual for a 49-format array (Affymetrix, 2014 ). Following hybridization, wash and stain procedures were carried out on an Affymetrix FS-450 fluidics station using the mini_prok2v1_450 fluidics script (Affymetrix, 2014 ). Reagents for washing and staining were prepared according to the GeneChip® Expression Analysis Technical Manual (Affymetrix, 2014 ). The following modifications were made to the wash and stain procedure: Streptavidin solution mix (vial 1) was replaced with SAPE solution mix (Life Technologies, Grand Island, NY). Arrays were scanned using Affymetrix GeneChip® Scanner 3000 running on AGCC software.