GSM4173907: ChIP H3K4me1 IP repl1; Drosophila melanogaster; ChIP-Seq
To extract the nuclei the embryos were transferred into Lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1 % TritonX100, 0.5 mM DTT, 0.1 % Sodium Deoxycholate, 10 mM Sodium Butyrate, 0.1% SDS, 0.5% N-Laurosylsarcosine,1X  Protease Inhibitors) and subjected to ultrasound treatment (Covaris E220, 45 sec, peak power 75, duty factor 10, cycles burst 200). The chromatin was sheared using Covaris E220 (900 sec, peak power 140, duty factor 5, cycle burst 200). The quality of the shearing was inspected on a Bioanalyzer 2100 (Agilent).  After sonication and 10 min high-speed centrifugation, fragmented chromatin was recovered in the supernatant. Chromatin was precleared by addition of 50 μl of Protein A-Agarose (PA) suspension (Roche 11134515001) followed by overnight incubation at 4°C. PA was removed by centrifugation, antibodies were added to the supernatant, and samples were incubated for 4 hr at 4°C in a rotating wheel. PA (50 μl) was added, and incubation was continued overnight at 4°C. Antibody-protein complexes were collected by centrifugation at 4,000 rpm for 1 min, and the supernatants were discarded. Samples were washed four times in Lysis buffer (with only 0.05% SDS) and twice in 1 mM EDTA, 10 mM Tris (pH 8) buffer (each wash, 5 min at 4°C). Chromatin was eluted from PA in 150 μl of 10 mM EDTA, 1% SDS, 50 mM Tris (pH 8) at 65°C for 15 min, followed by centrifugation and recovery of the supernatant. The eluate was incubated overnight at 65°C to reverse crosslinks and treated with Proteinase K for 3 hr at 50°C.  The chromatin was purified using ChIP DNA Clean & Concentrator columns (Zymo Research). libraries were prepared using NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturers instructions
GEO
GSM4173907
GEO Accession
GSM4173907