DamPiwiYK replicate 2
20 ovaries of 34-day-old Dam-PiwiYK transgenic flies raised at 25C without heat-shock were dissected in ice-cold 1_ PBS.Genomic DNA was isolated by phenol/chloroform extraction. Ethanol-precipitated DNA was resuspended on ice in 500 l 1_ Tris/EDTA (TE) buffer. The sample was treated with 100 g/ml of RNase A (QIAGEN) to digest co-purified RNA molecules at 37C for 30 min. Next, proteinase K was added (100 g/ml) to degrade the RNase A at 37C for 30 min. Following another phenol/chloroform extraction, the pellet was resuspended in 30 l 1_ TE buffer. The isolated DNA was analyzed by agarose gel electrophoresis for purity and stored at 4C to prevent possible fragmentation by freeze-thawing cycles. To cleave methylated GATC sites in the isolated genomic DNA (gDNA), 2.5 g gDNA was digested with DpnI enzyme (10 units, NEB) at 37C for 2 h and the enzyme was heat-inactivated for 20 min at 80C. For the annealing of DamID sequencing adapters, ssDNA oligos (AdRt, AdRb; see Supplemental Figure 1) were incubated at 95C for 5 minutes and slowly cooled down to 25C at -1C/min. 40 pmol of adapters were added to the digested genomic DNA and ligated at 16C for 2 h before the ligase was heat-inactivated at 65C for 10 min. Next, unmethylated GATC sites were digested with DpnII (10 units, NEB) at 37C for 2 h and the enzyme was inactivated at 65C for 20 min. 1% of the thus prepared DamID sample (1 of 50 l) was amplified by Q5 DNA polymerase (NEB; DamID_PCR): 98C, 30 s; (98C, 10 s; 55C, 20 s; 72C, 2 min) _19; 72C, 2 min; 12C, infinite.  The rest was column purified and eluted in ddH2O before removing the DamID adapters by DpnII digestion (10 units, NEB). Following a final round of column purification, 10 ng of the eluted DNA fragments was used in Illumina library preparation (Illumina TruSeq ChIP Sample Preparation Kit). The ligation products were purified and size-selected by agarose gel electrophoresis. Size-selected DNA was then purified and PCR-amplified to enrich for fragments with adapters on both ends. The final purified product was quantitated before cluster generation. Sequencing was carried out using a HiSeq2000 with one sample per lane at 100 bp single-end sequencing.