Quantitative Analysis of Rsp Satellite DNA

Genomic DNA from 5 males was extracted using the NucleoSpin Tissue XS kit (Macherey-Nagel, ref #740901). To amplify the Rsp satDNA region, we used two sets of primers: 5’-CCAGGCGAACAGAAGATACC-3’ and 5’-TTTTGACCGCTTAAAATGACA-3’; and 5’-AAGTTATGTCATTTTAAGCGGTCA-3’ and 5’-AACTTAGGCAATTTACTGTTTTTGC-3’. As a control, we amplified a fragment into nup62 gene (primers 5’-GGCACCTACTGCTGGTATCG-3’ and 5’-AATCCAAAGGCTGGTGGAG-3’). Quantitative PCR analysis was performed with 5ng of template gDNA in a 25μl reaction using the TB Green Premix Ex Taq II (Takara, ref #RR820L) and the CFX Connect (Biorad CFX Connect) system. For each set of primers, standard and melting curve analyses were performed to check for, respectively, PCR efficiency and specificity. qPCR analysis was done using technical duplicates on three biological replicates. The nup62 gene was used as internal control with a known copy number (two) so that genomic DNA levels were normalized for each sample to the levels of nup62. Based on [28 (link)], we considered that cn¹bw¹/SD-Mad flies carry 1000 repeats. The copy number in the Rsp satDNA is relative to cn¹bw¹/SD-Mad and was calculated using the comparative quantification ΔΔCT method [66 (link)].