Euchromatic FISH Probe Preparation

Euchromatic FISH probes were made as previously described (Buster 2013 (link); Smith 2013 (link)) from BAC clones (CHORI BACPAC Resources) as follows: X1, BACR30C13 and BACR18F10; X2, BACR20K01 and BACR35A18; 2L (1), BACR30M19 and BACR29P12; and 2L (2), BACR14I17 and BACR15P08. BAC clones were mapped and picked using the UCSC genome browser (genome.ucsd.edu). Clones were cultured, DNA was purified using the Plasmid Midi Kit (Qiagen), and purified DNA was amplified using the Whole Genome Amplification kit (Sigma). Amplified DNA (µg) was digested using a restriction enzyme cocktail consisting of AluI, Rsa, MseI, MspI, HaeIII, and BfuCl (New England BioLabs) overnight at 37° and then ethanol-precipitated. DNA was denatured at 100° for 1 min and 3′-end-labeled with aminoallyl dUTP and terminal deoxynucleotidyl transferase (Roche) for 2 hr at 37°. Five mM EDTA was added to terminate the reaction and, after ethanol precipitation, DNA was resuspended in 10 uL ddH₂O and conjugated to fluorophores using ARES Alexa Fluor DNA labeling kits (Invitrogen) according to the manufacturer’s instructions. Probes were then cleaned using Qiagen PCR clean-up kit (Qiagen), ethanol-precipitated, and resuspended in 10 µL EB buffer (Qiagen).