title: GSM2428255: Endoderm_H3K4me3_ChIP_rep1 (Group7); Xenopus laevis; ChIP-Seq
library_construction_protocol: ChIP and ChIP-seq preparation: Chromatin Immunoprecipitation (ChIP) was performed as described. Neurula (stage 18) embryos were generated by in vitro fertilization. For each ChIP experiment, 50 embryos were dissected in 1xMBS to obtain the endoderm tissue. Samples were fixed in 2 ml of 1% Formaldehyde in 0.1x MMR for 25 min at room temperature, followed by 4 washes with 1 mL 0.1xMMR and equilibration in 500 μl HEG solution (50mM 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 homogenized in 2 ml buffer E1 (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% Glycerol, 0.5% Igepal CA-630, 0.25% Triton X-100, 1mM DTT, complete protease inhibitors (Roche)). Chromatin was collected by centrifugation for 2 min at 3500 rmp, 4°C and then washed two times with 2 ml E1, three times with 2ml buffer E2 (10mM Tris pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, complete protease inhibitors (Roche)) and three times with 500 μl Buffer E3 (10mM Tris pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, complete protease inhibitors (Roche)). Chromatin was fragmented by sonication for 20 cycles (30s on and 30s off) using a Bioruptor (Diagenode). The samples were centrifuged at 15min, 4°C at full speed, the supernatant was collected and Triton X-100 was added to 1%. 25 μl of the solution were put aside to serve as Input for later analysis. Before ChIP, primary anti-H3K4me3 (Abcam ab8580, 0.5 μg per 50 embryos) antibodies were bound to PBS washed magnetic beads conjugated with secondary antibody (Invitrogen 11204D, 25 μl per 50 embryos) in 500 μl 1xPBS 0.1% BSA overnight at 4°C on a rotating wheel. Beads were washed 3 times with 1xPBS 0.1% BSA, added to the fragmented chromatin solution and incubated overnight at 4°C on a rotating wheel. Beads were then washed 6 times with RIPA buffer (50mM Hepes-KOH pH 7.5, 500mM LiCl, 1mM EDTA, 1% Igepal CA-630, 0.7% Na-deoxycholate, complete protease inhibitors (Roche)) and twice with TEN buffer (10mM Tris pH 8.0, 1mM EDTA, 150mM NaCl, complete protease inhibitors (Roche)) for each 10 min. For crosslink reversal, the beads were resuspended in 150 μl Stop buffer (40mM Tris pH 8.0, 10mM EDTA, 1% SDS) and 125 μl Stop buffer was added to the input fraction. The samples were supplemented with Proteinase K (0.3 μg/μl), NaCl (250 mM) and incubated at 65°C overnight. RNase A (DNase free) was added to a final concentration of 200 μg/μl and DNA was Phenol/Chloroform extracted. 150 μg/μl Glycogen was added and DNA was recovered by Ethanol precipitation. The pellet was resuspended in 30 μl H2O, and half was subjected for ChIP-seq library preparation with the TruSeq DNA kit (Illumina, FC-121-2001). Two independent biological replicates were generated for each H3K4me3 ChIP experiment. Library construction: 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: GSM2428255