title: GSM3330863: Smc1 ChIP-seq Smc1 RNAi replicate 2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Chromatin was prepared and ChIP-seq was conducted as described in Dorsett and Misulovin. 2017. Methods Mol Biol 1515:125-139, DOI: 10.1007/978-1-4939-6545-8_8. For early S phase DNA synthesis, cells were removed from the plate after treatment by scraping, fixed with 1% formaldehyde in 20 mL volume per 100 million cells for 12 min at room temperature and fixation was stopped by adjusting to 0.25 M glycine. Cells were washed twice with phosphate buffered saline (PBS). Cells were permeablized in PBS containing 0.25% Triton X-100 (20 mL per 100 million cells) for 30 min at room termperature, and washed twice with PBS containing 0.5% bovine serum albumin. A click reaction (1 mL per 100 million cells) was conducted in PBS containing 100 micromolar Azide-PEG3-biotin conjugate, 10 mM sodium ascorbate and 2 mM copper sulftate for 60 min in the dark at room temperature. The cells were washed 3 times with PBS and chromatin was prepared as described for the ChIP-seq except the cells were suspended in 1 mL sonication buffer per 100 million cells, the sonication buffer contained 0.4% sodium dodecyl sulfate, and sonication was conducted for 5 cycles. The sonicated suspension was adjusted to 0.5% lauryl sarcosine and clarifed by centrifugation at 15 g for 10 min. Crosslinks were reversed by diluting 1 mL chromatin with 1 mL TE, adjusting to 800 micrograms per mL RNAse A, 500 micrograms per mL Proteinase K and 0.5% sodium dodecyl sulfate, and incubating at 65 degrees overnight. The solution was extracted with phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.5) and the DNA purified using Qiagen QIAquick PCR purification columns, eluting twice with 55 microliters of provided elution buffer. EdU-containing DNA was isolated from the purified DNA using magnetic streptavidin beads (Dynabeads® M-280 Streptavidin, ThermoFisher). Twenty microliters of beads were washed twice before use with 1 mL of WB (5 mM Tris.HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.1% Tween 20). 100 microliters of DNA was diluted with 100 microliters of 2X WB and incubated with the streptavidin beads for 30 min at room temperature. Beads were collected with a magnet, and washed 3 times with 1 mL WB for 5 min. DNA was eluted from the beads by suspending in 400 microliters of 0.3X TE containing 1% SDS and 0.5 micrograms per mL Proteinase K and incubating at 65 degrees for 20 min. The bead suspension was extracted with 400 microliters of phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.5) and DNA was purified from the supernatant using a column (Qiagen MinElute PCR Purification). Ion Torrent IonXpress barcoded sequencing libraries were constructed using the Ion Xpress Fragment Library Kit (ThermoFisher) according to the manufacturer's directions except that size selection was performed using Beckman Agencourt magnetic beads.