title: GSM4064086: Vok.NLE.input; Nomascus leucogenys; ChIP-Seq
library_construction_protocol: 10x106 fixed cells were used per ChIP assay. After washing the cells twice with cold 1X PBS, we lysed the fixed cells in Lysis buffer (0.1% SDS, 0.5% Triton X-100, 20mM Tris-HCl pH=8.0, 150mM NaCl, 1x Proteinase inhibitor (Roche)) and used the Bioruptor Pico sonicator (Diagenode) to shear lysates. Each ChIP assay was incubate with 6ul of the PU.1 antibody (MA5-15064, ThermoFisher Scientific) overnight. Next, we added 20ul Pierce ChIP-grade Protein A/G Magnetic Beads (Pierce) and let samples rotate for 2hrs at 4°C. We then washed the beads consecutively (rotating at 4°C) with TBST buffer (3 times for 15min), Lysis buffer (once for 1hr), 1xTE pH=8 (once for 1hr, then once for 10min). Following washes, we eluted chromatin in 250ul fresh Elution buffer (1% SDS, 0.1M NaHCO3). We incubated the ChIP and input samples overnight at 65°C in presence of NaCl to reverse crosslinks and then digested them consecutively with 8ug RNAse A (30 minutes at 37°C) and 80ug Proteinase K (2 hour at 55°C). Lastly, we purified the samples using traditional phenol:chloroform extraction and ethanol precipitation. We quantified all samples using the Qubit dsDNA High Sensitivity kit (Thermo Fisher Scientific). libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs)
identifiers.namespace: GEO
identifiers.id: GSM4064086