title: GSM2944399: BR.elav.neurons.Dam_DamIDSeq; Drosophila melanogaster; OTHER
library_construction_protocol: Two μl of Proteinase K (20 mg/ml) [Roche, 03115887001] were added to dissected central brains and the sample was incubated at 65°C for 6 hours. Next, 1 μl of RNase A (100 mg/ml) [QIAGEN, 1007885] was added and the sample was incubate at 37°C for 30 minutes. The lysate was transferred to a pre-spun (1 minute at 14,000 rpm just prior to use) phase lock gel light 1.5ml tube [5prime, 2302800] and 100 μl of phenol:chloroform:isoamylalcohol (25:24:1) [Sigma-Aldrich, P2069] were added. The sample was mixed well by shaking and the tube was spun for 5 minutes at 14,000 rpm at room temperature. The upper phase (~90 μl) was transferred into a new tube. To precipitate DNA, 1 µl of glycogen (20 mg/ml) [Fermentas, R0561], 10 µl of 3M NaAc (pH 5.2) and 300 µl of ethanol were added, mixed well by inversion and the solution was kept overnight at -20°C. The tube was spun for 30 minutes at 14,000 rpm at 4°C. The supernatant was removed and the pellet was washed with 500 µl of ice cold 70% ethanol. The tube was spun for 5 minutes at 14,000 rpm at 4°C. The supernatant was carefully removed and the pellet was air dried for 15 minutes and dissolved in 12 µl of double distilled water. DNA concentration was measured using a NanoDrop spectrophotometer. Dam-methylated fragments were amplified as described previously (Vogel, M.J. et al. Detection of in vivo protein-DNA interactions using DamID in mammalian cells. Nat Protoc 2(6), 1467-78, 2007) using 500 ng of DNA as an input. The amplified methylated DNA fragments were purified using QIAquick PCR Purification kit (QIAGEN, 28104) according to the manufacturer's instructions. DNA was eluted in nuclease-free water (Ambion, AM9938). From 200 ng to 3 µg of the purified PCR products were diluted in 100 μl of nuclease-free water and transferred into a snap-cap microTUBE (6 × 16 mm) with AFA fiber (Covaris, 520045). DNA was sheared using the Covaris S2 instrument at the following settings: duty cycle = 10%, intensity = 5, cycles/burst = 200, time = 45 sec (as consequence, power = ~23W and bath temperature = ~4°C). The size of the DNA fragments before shearing was usually a smear in the range of 250–2000 bp with a peak at 600 bp, after shearing this was reduced to become a range of 100–500 bp with a peak around 300 bp. After shearing, the DNA was purified and concentrated using Agencourt magnetic beads (Beckman Coulter, A63881; 160 µl of beads were added to 100 µl of sheared DNA solution). DNA was eluted in 50 µl of nuclease-free water. DNA concentration was measured using an Agilent BioAnalyzer 2100 DNA 7500 chip (Agilent, 5067-1506). One μg of sheared material was used to prepare Illumina sequencing library with TruSeq DNA HT Sample Prep Kit (Illumina, FC-121-2003) according to the manufacturer's instructions. Seven cycles of PCR were performed. This step included sample indexing. The library was quantified on an Agilent BioAnalyzer 2100 DNA 7500 chip that lists the product size distribution and quantifies the concentration in µg/µl and in nM.