GSM5087951: SUZ12_ERT2-Cre_Pcgf1Mutant_DMSO_rep1; Mus musculus; ChIP-Seq
10^6-10^7 cells were cross-linking by 1% formaldehyde for 10 min at room temperature. Cross-linked cells were washed once with PBS, added swelling buffer (0.1% NP-40, 1 mM DTT in PBS) and chilled on ice for 10 minutes. After centrifuge, pellets were suspended with RIPA buffer (10 mM Tris-HCl pH8.0, 1mM EDTA, 140 mM NaCl, 1%Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate), and then were sonicated by using a BioRuptor sonicator (Diagenode). After sonication, DNA concentration is measured with Qubit 1x dsDNA HS Assay Kit (Thermo, cat. Q33230). On the other hand, protein A/G beads were prepared as washed twice with beads blocking buffer and suspended with beads blocking buffer with approximately 5 µg of antibody and incubated at 4ºC for more than 2 hours. Then, antibody attached beads were washed twice with beads blocking buffer and the chromatin samples were added to those antibody-attached-beads. After overnight incubation at 4ºC, chromatin samples bound beads were washed with RIPA for 6 times, RIPA high salt buffer(10 mM Tris-HCl pH8.0, 1mM EDTA, 500 mM NaCl, 1%Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate) twice, LiCl buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate) twice and TE twice. The chromatins were eluted with elution buffer (10 mM Tris-HCl pH8.0, 5 mM EDTA, 300 mM NaCl, 0.1 % SDS) overnight at 65ºC and treated with RNase for 30 min for 37°C and further incubation with Proteinase K for 1 hour at 37ºC. Then DNA samples were purified and used for further ChIP-qPCR or ChIP-seq. ChIP-seq libraries were prepared by using NEBNext Ultra II FS DNA prep kit (NEB E7805) following manufacturer's instruction.