title: GSM3638164: Stage 10.5 vegetal H3K4me1 ChIP-seq replicate 2; Xenopus tropicalis; ChIP-Seq
library_construction_protocol: Vegetal masses were dissected from 100 fixed embryos and were then 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 3 ug anti-H3K4me1 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: GSM3638164