title: GSM3317448: Stage 8 Vegt ChIP-seq replicate 2; Xenopus tropicalis; ChIP-Seq
library_construction_protocol: The fixed embryos were homogenized in RIPA buffer and incubated on ice for 10 minutes. Samples were then centrifuged at 14,000rpm for 10 minutes at 4°C. Pellets were resuspended in RIPA buffer and sonicated on ice to an average size of 100-500bp. The resulting samples were centrifuged at 14,000 rpm for 30 minutes at 4°C to remove insoluble cellular debris. The sheared chromatin was pre-cleared by incubating with 20 mL Protein G-coated Dynabeads (Invitrogen) for 2 hours at 4°C with rotation. Meanwhile, another 20 mL Protein G Dynabeads were incubated in a tube rotator in 1 mg/mL torula RNA and 0.5% BSA in 1x PBS for 30 minutes at 4°C to block non-specific binding. This second 20 mL Protein G Dynabeads were then pre-bound with 2 mg anti-vegt antibody at 4°C for > 1 hour. The pre-cleared chromatin was added to antibody-bound Dynabeads, and incubated overnight at 4°C on a tube 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. ChIP-seq libraries were generated using Nextflex ChIP-seq kit (Bioo Scientific), analyzed using an Agilent Bioanalyzer 2100, quantified using KAPA qPCR and sequenced using Illumina instruments.
attributes.tag: GEO Accession
attributes.value: GSM3317448