title: GSM5343690: TEX10-HEK293FT_RING1DKO_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells for ChIP were cultured in 15 cm plates. Cell were first washed with cold PBS, crosslinked at room temperature with 10 mM DMP (ThermoFisher Scientific) for 30 min, and then 1% formaldehyde (ThermoFisher Scientific) for 15 min. Crosslinking reactions were quenched by addition of 125 mM glycine for 5 min. Crosslinked cells were separated by 3 min treatment of 0.05% trypsin (Gibco), and then washed with cold PBS 3 times. In every wash, cells were centrifuged for 3 min at 1000xg at 4℃.Cell were then resuspended in sonication buffer (pH 7.9, 50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, and 0.5% SDS) and sonicated to shear chromatin into ~300 bp fragments using a Branson sonicator. Sonicated samples were diluted 5-fold with ChIP dilution buffer (pH 7.9, 50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate) to obtain a final concentration of 0.1% SDS. Diluted samples were centrifuged at13,000 rpm for 10 min. The supernatant was pre-cleared with protein A/G or Dynabeads M-280 Streptavidin beads (ThermoFisher) and immunoprecipitated for 3-12 h using 3 μg antibodies and 40 μl protein A/G or Dynabeads M-280 Streptavidin beads. The beads were washed twicewith high salt wash buffer A (pH 7.9, 50 mM Hepes, 500 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, and 0.1% SDS), and once with wash buffer B (pH 7.9, 50 mM Hepes, 250 mM LiCl, 1 mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, 0.5% NP-40). The bound chromatin fragments were eluted with elution buffer (pH 8.0, 50 Mm Tris, 10 mM EDTA, 1% SDS) for 5 min at 65℃. Eluted DNA-proteins complexes were treated with RNase A and crosslinks were reversed overnight at 65℃. Proteinase K was then added to digest proteins for 1 h at 55℃. DNA was further purified using PCR Purification Kit (QIAGEN) and analyzed by PCR on a QuantStudio 7 Flex Real Time PCR System (Applied Biosystem). PCR parameters were 95℃for 2 min and 40 cycles of 95℃ for 15s, 60℃ for 15s, and 72℃ for 15 s, followed by 72℃ for 1 min. All the ChIP-qPCR data presented were at least three biological replicates. Primer sequences are in the table. Error bar represent standard deviation (three biological replicates). For ChIP-seq, sequencing library was constructed using TruSeq DNA sample Prep Kits (Illumina) and adapter dimers were removed by agarose gels electrophoresis. Sized selected and purified DNA libraries were sequenced on an Illumina Hiseq 2500 machine (Bauer core facility at Harvard University) to obtain 50 bp single-end reads.
attributes.tag: GEO Accession
attributes.value: GSM5343690