GSM3504399: HIF1_GFP_8510; Homo sapiens; ChIP-Seq
K562 cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature at a concentration of 15x106 cells/ml. The fixed cells were sonicated for 4 minutes (30 sec on; 30 sec off) using the Diagnode Bioruptor and centrifuged at maximum speed for 10 minutes. Chromatin of about 5x105 cells was incubated overnight in dilution buffer (167mM NaCl, 16,7 mM Tris (pH 8), 1,2mM EDTA, 1% Triton X-100) with 1 µg antibody at 4C. ProtA/G beads were blocked and incubated with the chromatin-Ab for one hour at 4C. Beads were washed with three different wash buffers and chromatin was eluted from the beads. DNA-proteins were de-crosslinked (200mM NaCl and 4 µl proteinase K (10mg/ml)), by incubation for four hours at 65C and samples were purified using the Qiaquick MinElute PCR purification kit according manufacturer's protocol. Sequencing samples were prepared according to the manufacturer's protocol (Illumina). End repair was performed using the precipitated DNA using Klenow and T4 PNK. A 3' protruding A base was generated using Taq polymerase and adapters were ligated. The DNA was loaded on gel and a band corresponding to ~300 bp (ChIP fragment + adapters) was excised. The DNA was isolated, amplified by PCR and used for cluster generation on the NextSeq500 genome analyzer. The 50 bp tags were mapped to the human genome HG19 using BWA (Li and Durbin, 2009). For processing and manipulation of SAM/BAM files SAMtools was used (Li et al., 2009). For each base pair in the genome the number of overlapping sequence reads was determined and averaged over a 10 bp window and visualized in the UCSC genome browser (Kent et al., 2002).
GEO Accession
GSM3504399