GSM6774688: Dam-Elys DamID-seq, rep 2; Drosophila melanogaster; OTHER
Approximately two hundred 16-18-h embryos from these crosses were collected, washed with H2O, dechorionated with 2.5% hypochlorite for 3 min and again washed three times with H2O. Isolation of genomic DNA was performed by DNeasy Blood and Tissue kit (Qiagen), following the protocol for cultured cells. DNA was concentrated by centrifugation in 0.5 ml Amicon-Ultra columns (Millipore) for 10 min at 18000 × g and its concentration was measured using a NanoDrop spectrophotometer. For the selective amplification of methylated GATC-GATC fragments, genomic DNA was first digested by DpnI, followed by ligation of adaptors, DpnII digestion and PCR-amplification as described previously (Vogel et al. 2007) with a minor modification: ~0.5 µg instead of 2.5 µg of genomic DNA was used as an input.We applied 18 cycles of PCR (1 min at 94°C, 1 min at 65°C, 2 min at 68°C) for Dam-only and Dam-Elys and 16 cycles for Dam-Lam DNA samples. Adapters were removed by DpnII digestion for 2 h at 37°C. The amplified methylated DNA fragments were purified using QIAquick PCR Purification kit (Qiagen), concentrated by centrifugation in 0.5 ml Amicon-Ultra columns (Millipore) for 10 min at 18000 × g, and quantified on a NanoDrop spectrophotometer.  0.5 µg of Dam-only, Dam-Elys and Dam-Lam DNA samples (each in two biological replicates) were subjected to next generation sequencing (NGS) on Illumina HiSeq 2500 resulting in ~30 million 100 nt single-end reads per sample.