GSM3463658: I17-1066-05: SMC3 ChIP HEK293T wt; Homo sapiens; ChIP-Seq
Cells at 70–80% confluence were crosslinked with 1% formaldehyde for 10 minutes and quenched with 125 mM glycine. After washing with PBS, cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA, 1 mM PMSF and Complete Protease Inhibitor (Roche)) and sonicated (Diagenode Bioruptor, Seraing, Belgium) to around 500 bp DNA fragments. Debris were removed by centrifugation and the lysate was diluted 1:4 with IP dilution buffer (20 mM Tris-HCl pH 8.0, 0.15 M NaCl, 2 mM EDTA, 1% TX-100, protease inhibitors) and precleared with Affi-Prep Protein A support beads (Bio-Rad). The respective antibodies were incubated with the lysate overnight at 4ºC, followed by 2 hours incubation at 4ºC with blocked protein A Affiprep beads (Bio-Rad) (blocking solution: 0.1 mg/ml BSA). Beads were washed with washing buffer I (20 mM Tris-HCl pH 8.0, 0.15 M NaCl, 2 mM EDTA, 1% TX-100, 0.1% SDS, 1 mM PMSF), washing buffer II (20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 2 mM EDTA, 1% TX-100, 0.1% SDS, 1 mM PMSF), washing buffer III (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium desoxycholate) and TE-buffer (10 mM Tris-HCl pH 8.0,1 mM EDTA). Beads were eluted twice (25 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% SDS) for 20 minutes at 65ºC. The eluates were treated with proteinase K and RNase for 1 hour at 37ºC and decrosslinked at 65ºC overnight. The samples were further purified by phenol-chloroform extraction and ethanol-precipitated. The pellet was dissolved in TE buffer. For sequencing, the DNA libraries were prepared using the NEXTFlex ChIP-Seq kit (BioO Scientific, Austin, TX, USA). These libraries were sequenced according to the Illumina TruSeq v3 protocol on an Illumina HiSeq2500 sequencer (Illumina, San Diego, CA, USA). Single reads were generated of 50 base pairs in length.