GSM4256783: MZsox19b_K4me3_ChIP; Danio rerio; ChIP-Seq
Fixation of embryos and nuclei extraction. 1000-4000  embryos  were enzymatically dechorionated with pronase E (0.3 mg/ml),homogenized and immediately fixed with 1 % (v/v) Methanol-free Formaldehyde (Thermo scientific , 28906)  for 10 min at room temperature.The fixation was stopped with 0.125 M Glycine. Cells were pelleted by centrifugation and resuspended  in Wardle cell lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.5 % NP-40, 1 ml/1000 embryos), followed by 5 min incubation on ice. Cells were pelleted by 1 min centrifugation, 2700 g at 4 °C. the pellet was washed 2 times with 1 ml ice cold 1x x PBST; a 10ul aliquote was used for scoring the nuclei.  The pellets were frozen atb -80°.   2.5 million nuclei were used to perform one ChIP-Seq experiment.  Chromatin was sonified to 200 bp fragments using Covaris S2 sonicator (DC 20 %, Intensity 5, Cycles of Burst 200, Time = 3*40 cycles with 30 sec each (3*20 min),  1/10 of the sample was taken as an input control. The rest was immunoprecipitated with the anti-H3K27ac or anti-H3K4me3 antibodies. ChIP and Input libraries for each sample were  prepared using NEBNext® Ultra™ DNA Library Prep Kit according to the manufacturer instructions For each sample, ChIP libraries were sequenced to 70 mln reads, Input libraries to 30 mln reads
GEO Accession
GSM4256783