title: GSM3671366: Input1_st7_embryo; Xenopus laevis; ChIP-Seq
library_construction_protocol: Preparation sperm and egg extract treated sperm chromatin for ChIP-seq: Chromatin fractionation and chromatin immunoprecipitation (ChIP) were performed as described before (Hisano et al., 2013 and Teperek et al., 2016) with slight modifications. Xenopus sperm were purified from their testes and permeabilised before ChIP. Sperm or egg extract treated sperm chromatin was resuspended in 50 l of Buffer 1 (0.3 M Sucrose, 15 mM Tris pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT) and added 50 l of Buffer 1 with detergent (Buffer 1 including 0.5% NP40 and 1% NaDOC). Samples were incubated for 10 min on ice. 100 l of MNase buffer (0.3 M Sucrose, 85 mM Tris, 3 mM MgCl2, 2　mM CaCl2, 2.5U of micrococcal nuclease: Roche 10107921001) was added in to each tube (0.5 million of cells per tube). Tubes were incubated at 37C for 30 min in pre-warmed water bath. Reaction was stopped by adding 2 l of 0.5 M EDTA pH8.0 in the same order as started. Tubes were vortexed and placed on ice for at least 5 min. Supernatant and pellet were separated by centrifugation at 13,000 rpm for 10 min at room temperature. Supernatant was transferred to new tube and stored on ice. For ICeChIP, semi-synthetic nucleosomes were spiked in as described (Grzybowski et al., 2015) before MNase digestion. Preparation Chromatin for embryo ChIP-seq: ChIP was performed as described previously (Gentsch and Smith, 2014) with the following modifications. Blastula (stage 7) embryos were generated by in vitro fertilization. For each ChIP experiment, 50 embryos were fixed in 2 mL of 1% Formaldehyde in 1x MMR for 25 min at room temperature, followed by 4 washes with 1 ml of 1xMMR and equilibration in 500 μl HEG solution (50 mM HEPES-KOH pH 7.5, 1 mM EDTA, 20% Glycerol) at 4°C, then excess buffer was removed and samples were frozen at −80°C. To extract chromatin, the samples were transferred to 2 ml tube or 15 ml Falcon tube and homogenized in 200-250 μl of sonication buffer (20 mM Tris-HCl pH 8.0, 70 mM KCl, 1 mM EDTA pH 8.0, 10% Glycerol, 5 mM DTT, 0.125% NP40, 1x complete protease inhibitors), by pipetting up and down in a 1 ml pipette tip. 250 μl of diagenode beads were transferred to diagenode falcon tube (Diagenode, C1020031). 750 μl of embryo lysates were added and diagnode falcons were transferred to the sonicator bath (diagnode bioruptor). Sonication is carried out in 30 cycles (with 30 s on/off cycles). Sonicated embryo extract was transferred into eppendorf tubes. Chromatin was collected by centrifugation for 5 min at top speed in tabletop centrifuge at 4°C. Chromatin extract was transferred to new tube and stored on ice. ChIP-seq library preparation was performed using the TruSeq DNA kit (Illumina, FC-121-2001) according to the manufacturer's protocol.
attributes.tag: GEO Accession
attributes.value: GSM3671366