Microarray analysis was performed as previously described (Pai et al., 2014 (link); Rallis et al., 2013 (link)). Experiments were conducted in duplicate with a dye swap. RNAs from two independent biological replicates have been utilized for cDNA production. Fig. 6 C shows average expression ratios from the two repeats. Original data have been deposited in ArrayExpress under accession number E-MTAB-6795. In brief, Alexa Fluor 555- or 647-labelled cDNA was produced from the RNA, using a Superscript direct cDNA labelling system (Invitrogen) and Alexa Fluor 555 and 647 dUTP mix. cDNAs were then purified using an Invitrogen PureLink PCR Purification system and hybridized to the array using a Gene Expression Hybridization kit (Agilent). The arrays are Agilent custom-designed containing 60-mer oligonucleotides synthesized in situ containing 15,000 probes. Following hybridization for at least 17 h, the arrays were washed using a Gene Expression Wash Buffer kit (Agilent) and scanned in an Agilent Array Scanner. Signals were extracted using GenePix software.
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