title: GSM4141457: HEK293_SP1_Input.rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: 5 million cells were used for all ChIP reactions. Crosslinked cells were resuspend in ice-cold lysis buffer (50 mM HEPES, pH=7.9, 5 mM MgCl2, 0.2% Triton X-100, 20% glycerol, 300 mM NaCl) and incubated on ice for 10 min before centrifuged at 500 g for 5 min. The pellets were resuspended in 0.1% SDS lysis buffer (50 mM HEPES, pH=7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl) and incubate on ice for 10 min and then sheared by using Bioruptor Pico at 30s-on/30s-off setting for 20 cycles. 5% of sheared chromatin was saved as input and the rest was subjected to pre-clear and then mixed with 30 µL protein A/G bead coated with 5-10 µg antibodies. Immunoprecipitation was performed overnight and the beads were washed twice with 0.1% SDS lysis buffer, high salt buffer (50 mM HEPES, pH=7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 0.1% SDS, 350 mM NaCl), LiCl wash buffer (10 mM Tris-HCl, pH=8.0, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 250 mM LiCl) and once with TE buffer (10 mM Tris-HCl, pH=8.0, 1 mM EDTA, 0.2% Triton X-100). Enriched chromatin was eluted from the beads by incubating with elution buffer (50 mM NaHCO3, 10 mM EDTA, 1% SDS) at room temperature for 1 h. The eluent and the input were subjected to reverse crosslink and proteinase K digestion, before DNA were purified from the mixture by using DNA Clean & Concentrator-5 kit (Zymo). Recovered DNA was used for library construction by Kapa HyperPlus kit (Kapa, KK8515).
attributes.tag: GEO Accession
attributes.value: GSM4141457