GSM2263598: stage_8_Input_DNA; Xenopus tropicalis; ChIP-Seq
The fixed embryos were homogenized in RIPA buffer and incubated on ice for 10 minutes. Samples were then microfuged at 14,000rpm for 15 minutes at 4°C. Pellets were resuspended in RIPA buffer and sonicated on ice to an average size of 200-500bp. The resulting samples were microfuged at 14,000 rpm for 20 minutes at 4°C to remove insoluble cellular debris. The sheared chromatin was “pre-cleared” by incubating with Protein A-coated Dynabeads (Invitrogen) for 2 hour at 4°C with rotation. Antibodies were pre-bound to blocked Protein A Dynabeads by incubating at 4°C for 30 min. A sample of sheared chromatin was frozen for use as input control. Pre-cleared chromatin was added to antibody-bound Dynabeads, and incubated overnight at 4°C on an end-over-end rotator. The next day, the beads were washed, and DNA was eluted off the beads with TE buffer containing 1% SDS, and reverse-crosslinked at 65°C overnight. Sonicated input control was diluted 3-fold with elution buffer and also incubated at 65°C. All samples were treated with RNAse A, Proteinase K, phenol/chloroform extracted, and ethanol precipitated overnight. DNA pellets were resuspended in Qiagen EB solution. 10-30ng of DNA was used for library construction using the Bioo Scientific NEXTflex™ ChIP-seq Kit (Bioo Scientific #5143-01). Stage 8 Tle library was constructed using the TruSeq ChIP Sample Prep kit (Illumina).