Fusion Pten-NOLC1 Promotes C-Met and EGFR Signals in Human Cancers (Chip-Seq part 5)
Prostate Chip-Seq data
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Chromatin immunoprecipitation (ChIP). The Pten-NOLC1 knockout clones, dKO1, dKO2, mKO1, mKO2 and their parental cells, Du145 and MCF7, were used for ChIP analysis. MagnaChIP A/G kit (Millipore, USA) was used and the manufacturer’s protocol was followed. Briefly, 2-3 x 105cells at 80% confluence in culture were incubated in cold 4% formaldehyde at room temperature for 10 minutes to cross-link proteins and DNA, followed by quenching with glycine for 5 minutes at room temperature, washing 3 times with ice cold 1xPBS, and spinning down the cell pellets. The cells were then lysed with lysis buffer containing protease inhibitors on ice for 15 minutes and centrifuged at 800g in 4°C for 5 minutes. The pellet was further lysed with nuclear lysis buffer. The nuclear lysates (protein/DNA) were then sheared to the DNA fragment with size of 100 – 800 bps by sonication. A Focused ultrasonicator M220 (COVARIS) was used with setting at power 7.5, 200 burst/cycle and factor 10%. The fragmented and cross-linked chromatin was immunoprecipitated with antibodies against the C-terminus of NOLC1 or the N-terminus of Pten. The A/G magnetic beads were used to collect the immune complex including DNA fragments. The DNA were then eluded and purified for library preparation (Illumina, CA). Similar assays were also performed on lysates from PC3-PNOL-Flag, RWPE1-PNOL-Flag and their control PC3-Flag and RWPE1-Flag; and PC3 by using antibodies specific for FLAG tag. ChIP sequencing: The manufacturer recommended procedure was followed (Illumina): Quantity and size of fragment DNAs were analyzed in a Bio-analyzer (Agilent); the DNA was first blunt-ended with the end repairing reagents, followed by 3’ end adenylation and ligation with indexing adaptor; the modified DNA was purified with Ampure XP magnetic beads, and resolved in 2% agarose with SYBR Gold gel. The DNA sizes of 250 to 300 bps were excised, purified with MinElute Gel Extraction Kit, and enriched with 17 heat cycles of PCR with TruSeqTM reagents (Illumina). Each library was quantified again in Agilent Bioanalyzer and normalized to 2 nM for each sample for loading. The process of sequencing in Illumina Highseq 2500 followed the manufacturer’s standard protocol. Two lanes of 70 G sequencing capacity were used for sequencing 15 ChIP samples.