GSM8112094: 126_ZT2_IN; Drosophila melanogaster; ChIP-Seq
Fly heads were homogenizedin homogenization buffer (10mM HEPES-KOH at pH 7.5, 10mM KCl, 1.5mM MgCl2, 0.8M sucrose, 0.5mM EDTA, 1mM DTT, 1× protease inhibitor, 1× phosphatase inhibitor cocktail) at 4°C. Homogenates were loaded on equal volumes of sucrose cushion buffer (with 1.0M sucrose and 10% glycerol in the homogenization buffer) and centrifuged in a HB-6 rotor (Sorvall) at 11,000rpm for 10min at 4°C.Nuclear pellets were suspended in 1ml 1X PBS with 1% formaldehyde and fixed for 15 min at room temperature and quenched by 0.125 M glycine. Chromatin was extensively washed by 1X PBS and sheared using a biorupter (Diagenode) in 500 ul sonication buffer (20mM Tris-HCl at pH 7.5, 150mM NaCl, 10% glycerol, 1% SDS). For each IP, 25 ul of sheared chromatin was reserved as input sample. The remaining chromatin sample were diluted by 10 times of IP buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 10% glycerol, 1% NP40 and protease inhibitor tablet) and incubated with 30 ul anti-V5 agarose beads (Sigma) overnight at 4°C.Beads were washed and eluted and decrosslinked as described previously (Yu et al. 2006). The DNA was purified using Minelute columns (Qiagen). 30μl of DNA from each IP or 50ng of Input DNA were used to generate ChIP-seq libraries accroding to illumina ChIP-seq protocol. After adaptor ligation, DNA samples were separated by 2% agarose TAE gel and gel slices corresponding to 250-450bps were recovered and purified using QIAGEN gel purification kit. ChIP-seq libraries were sequenced on a Nextseq 550 (Illumina) and the resulting datasets were mapped to the Drosophila genome (dm6) using Bowtie2 and analyzed using MACS2