GSM1138202: duf:CD2- rep. 2; Drosophila melanogaster; OTHER
Crude genomic DNA was prepared by suspending cells in Gloor & Engel's extraction buffer (25 mM NaCl, 10 mM Tris pH 7-8, 1 mM EDTA, 200 micrograms/mL proteinase K) and incubating 30 minutes at 37 degrees, then 10 minutes at 95 degrees to inactivate proteinase K, then stored at -20 degrees. Stored extracts were pooled by sample type, split five ways, and used as template for nested PCR of specific transgenic inserts. PCR products were gel-purified and pooled by sample type. 5 micrograms of each PCR pool was fragmented by Covaris sonication and prepared for Illumina sequencing using standard protocols (end repair with the EpiCentre Biotechnologies End-IT kit, A-tailing with New England Biolabs Klenow exo-, and adapter ligation with New England Biolabs Quick T4 DNA ligase), with the Illumina Multiplexing Sample Preparation Oligonucleotide kit, and 10 cycles of enrichment PCR after adapter ligation.