title: GSM3891457: Dam-HP1 DamID-seq, rep 2; Drosophila melanogaster; OTHER
library_construction_protocol: 160-200 ovaries from 0-4 days old females were manually dissected using dissection needles in PBS at 4°C. 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 16 cycles of PCR (1 min at 94°C, 1 min at 65°C, 2 min at 68°C) for Dam-only and Dam-HP1 DNA samples. Adaptors 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 and Dam-HP1 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.