GSM5569840: ChIPseq_GFPUBR7_GFPIP; Homo sapiens; ChIP-Seq
2x15cm 90% confluent plates of GFPUBR7 293T cells were used for each ChIP-seq experiment. The cells were fixed with 1% paraformaldehyde in DMEM for 15 m at 37C, then quenced with  0.125M glycine (final concentration) for 5m 37C. The cells were washed with ice-cold PBS containing Protease inhibitors (cOmplete, Roche). Cells were sonicated with a Branson sonicator at  40% amplitude, 0.7s on 1.3s off, for 10 m. Samples were then spun at max speed for 10m at 40C. The supernatant was collected and 50uL retained as input sample. The lysate was pre-cleared with beads.  4ug anti-GFP antibody (Supplementary Table 3) was added and the samples were incubated overnight with rotation at 4C. The immune complex was collected by incubating with Protein-A Sepharose beads on a rotator at 4°C. The beads were washed 5x with washing buffer (50 mM HEPES pH 8.0, 1% NP-40, 0.7% DOC, 0.5 M LiCl, freshly added protease inhibitors) then 1x with TE buffer. Immunoprecipitated complexes were eluted twice in serial by resuspending beads with 100 μl elution buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS) and incubating for 30 m at 65°C. The supernatant was collected and treated with proteinase K for 2h at 55°C, then purified DNA by phenol-chloroform extraction. ChIPseq libraries were prepared with the KAPA HTP Library Preparation Kit (Illumina KK8234) using NEXTFLEX Adapters (Illumina 514104), according to manufacturer protocols, from 10ng of starting DNA. Libraries were size selected using SPRI beads (Beckman Coulter A63987). Libraries were assessed on an Agilent Bioanalyzer, and sequenced on a NextSeq500 (Illumina) to generate 50bp single end reads.