strain: C57BL/6 gender: female age: 6-8 week old cell line: murine bone marrow transduced with epitope tagged forms of Hoxa9 cell type: Hoxa9-ER cells chip antibody: H3K27trimethyl chip antibody vendor: Millipore chip antibody catalog number: 17-622
Extracted molecule
genomic DNA
Extraction protocol
A total of 150 million cells were crosslinked sequentially with disuccinimidylglutarate (45 min RT) and 1% formaldehyde (15 min RT). Hoxa9 and Meis1 immunoprecipitation was performed with anti-HA antibody (Abcam) pre-conjugated to Protein G magnetic beads (Dynal/Invitrogen). For C/ebpα ChIP, rabbit anti-C/ebpα (Santa Cruz) was compared with pre-immune rabbit IgG. 4-hour incubation (4°C with gentle rotation) was followed by washes using Low Salt, High Salt, LiCl, and Tris-EDTA buffers (Upstate/Millipore). Immunoprecipitates were eluted with 0.1% SDS/0.1M NaHCO3 and DNA-protein crosslinks were reversed overnight at 65° in 0.2M NaCl. DNA was RNAse treated and column purified (Qiaquick, Qiagen). For ChIP-seq, size selection and sequencing were performed at the BC Cancer Agency Genome Sciences Centre (Vancouver, BC) as described previously (Robertson et al. 2007). For ChIP-Chip, DNA was amplified prior to dual hybridization (performed at Nimblegen Systems) of input and immunoprecipitate on a custom tiled mouse genomic array containing putative Hoxa9 and Meis1 target genes (50-mer probes with an average spacing of 35 bp; 15 megabases of total sequence).
Label
Cy5
Label protocol
Labeling was done by Nimblegen, using manufacturer's protocol
strain: C57BL/6 gender: female age: 6-8 week old cell line: murine bone marrow transduced with epitope tagged forms of Hoxa9 cell type: Hoxa9-ER cells sample type: input DNA
Extracted molecule
genomic DNA
Extraction protocol
A total of 150 million cells were crosslinked sequentially with disuccinimidylglutarate (45 min RT) and 1% formaldehyde (15 min RT). Hoxa9 and Meis1 immunoprecipitation was performed with anti-HA antibody (Abcam) pre-conjugated to Protein G magnetic beads (Dynal/Invitrogen). For C/ebpα ChIP, rabbit anti-C/ebpα (Santa Cruz) was compared with pre-immune rabbit IgG. 4-hour incubation (4°C with gentle rotation) was followed by washes using Low Salt, High Salt, LiCl, and Tris-EDTA buffers (Upstate/Millipore). Immunoprecipitates were eluted with 0.1% SDS/0.1M NaHCO3 and DNA-protein crosslinks were reversed overnight at 65° in 0.2M NaCl. DNA was RNAse treated and column purified (Qiaquick, Qiagen). For ChIP-seq, size selection and sequencing were performed at the BC Cancer Agency Genome Sciences Centre (Vancouver, BC) as described previously (Robertson et al. 2007). For ChIP-Chip, DNA was amplified prior to dual hybridization (performed at Nimblegen Systems) of input and immunoprecipitate on a custom tiled mouse genomic array containing putative Hoxa9 and Meis1 target genes (50-mer probes with an average spacing of 35 bp; 15 megabases of total sequence).
Label
Cy3
Label protocol
Labeling was done by Nimblegen, using manufacturer's protocol
Hybridization protocol
Hybridization was performed by Nimblegen, using manufacturer's protocol
Scan protocol
Roche NimbleGen NimbleScan software
Data processing
MA2C (Song et al., 2007) with Median polish and bandwidth 150bp
