product of ChIP with an anti-HEB antibody from U937 cell line (human cell line established from a diffuse histiocytic lymphoma and displaying monocytic characteristics) transfected with HA-tagged AML1/ETO under the control of the mouse metallothionine promoter treated for 8h with 100µM ZnSO4 to induce transgene expression.
Treatment protocol
Cell lines were treated for 8h with 100uM ZnSO4 prior to RNA extraction.
Growth protocol
U937 cell lines were maintained in RPMI-1640 supplemented with 10% FCS and 2mM glutamine at 37°C in a humidified atmosphere containing 5% CO2.
Extracted molecule
genomic DNA
Extraction protocol
Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, harvested and washed twice with 1x PBS. Pellet was resuspended in ChIP lysis buffer (150mM NaCl, 1% Triton-X 100, 0,1% SDS, 500microM DTT, 10mM Tris-HCl, 5mM EDTA) and sonicated to obtain an average chromatin length of 500 bp. 5x106 cells were used for each IP and incubated at 4°C overnight with immunopurified anti-HA antibody from clone 12CA5, or commercially available antibodies against ETO (Santa Cruz sc-9737), AML1/RUNX1 (Abcam ab23980), HEB (Santa Cruz sc-357), or trimethylated H3K4 (Abcam ab8580),previously coated on Dynabeads Protein A magnetic beads (Invitrogen, USA). Beads were then washed twice with each of the following buffers: Mixed Micelle Buffer (150mM NaCl, 1% Triton-X 100, 0,2% SDS, 20mM Tris-HCl, 5mM EDTA, 65% sucrose), LiCl/detergent wash (250mM LiCl, 0.5% Na deoxycholate, 0,5% NP-40, 10mM Tris-HCl, 1mM EDTA), Buffer 500 (500mM NaCl, % Triton-X 100, 0.1% Na deoxycholate, 25mM HEPES, 10mM Tris-HCl, 1mM EDTA) an a final wash was performed with 1x TE. Finally, beads were resuspended in 1x TE containing 1% SDS and incubated at 65°C for 10 minutes to elute immunocomplexes. Elution was repeated twice, and the samples were further incubated overnight at 65°C to reverse cross-linking, along with the untreated input (2,5% of the starting material). After treatment with 0,5 mg/ml proteinase K for 3 hours, DNA was purified with Wizard SV Gel and PCR Clean-up system (Promega, USA) according to manufacturer’s protocol and eluted in nuclease-free water. ChIP products and input DNA were sent to NimbleGen Services (Madison, Wisconsin) for labeling, hybridization, scanning and preliminary data processing.
Label
Cy5
Label protocol
Experimental samples are labeled with Cy5, total DNA samples used as reference are labeled with Cy3 using 9mer primers that have Cy3 and Cy5 dyes attached and Klenow added.
Channel 2
Source name
U937 myelomoncytic cells, total input DNA, transfected with HA-tagged AML1/ETO
total DNA from U937 cell line (human cell line established from a diffuse histiocytic lymphoma and displaying monocytic characteristics) transfected with HA-tagged AML1/ETO under the control of the mouse metallothionine promoter treated for 8h with 100µM ZnSO4 to induce transgene expression.
Treatment protocol
Cell lines were treated for 8h with 100uM ZnSO4 prior to RNA extraction.
Growth protocol
U937 cell lines were maintained in RPMI-1640 supplemented with 10% FCS and 2mM glutamine at 37°C in a humidified atmosphere containing 5% CO2.
Extracted molecule
genomic DNA
Extraction protocol
Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, harvested and washed twice with 1x PBS. Pellet was resuspended in ChIP lysis buffer (150mM NaCl, 1% Triton-X 100, 0,1% SDS, 500microM DTT, 10mM Tris-HCl, 5mM EDTA) and sonicated to obtain an average chromatin length of 500 bp. 5x106 cells were used for each IP and incubated at 4°C overnight with immunopurified anti-HA antibody from clone 12CA5, or commercially available antibodies against ETO (Santa Cruz sc-9737), AML1/RUNX1 (Abcam ab23980), HEB (Santa Cruz sc-357), or trimethylated H3K4 (Abcam ab8580),previously coated on Dynabeads Protein A magnetic beads (Invitrogen, USA). Beads were then washed twice with each of the following buffers: Mixed Micelle Buffer (150mM NaCl, 1% Triton-X 100, 0,2% SDS, 20mM Tris-HCl, 5mM EDTA, 65% sucrose), LiCl/detergent wash (250mM LiCl, 0.5% Na deoxycholate, 0,5% NP-40, 10mM Tris-HCl, 1mM EDTA), Buffer 500 (500mM NaCl, % Triton-X 100, 0.1% Na deoxycholate, 25mM HEPES, 10mM Tris-HCl, 1mM EDTA) an a final wash was performed with 1x TE. Finally, beads were resuspended in 1x TE containing 1% SDS and incubated at 65°C for 10 minutes to elute immunocomplexes. Elution was repeated twice, and the samples were further incubated overnight at 65°C to reverse cross-linking, along with the untreated input (2,5% of the starting material). After treatment with 0,5 mg/ml proteinase K for 3 hours, DNA was purified with Wizard SV Gel and PCR Clean-up system (Promega, USA) according to manufacturer’s protocol and eluted in nuclease-free water. ChIP products and input DNA were sent to NimbleGen Services (Madison, Wisconsin) for labeling, hybridization, scanning and preliminary data processing.
Label
Cy3
Label protocol
Experimental samples are labeled with Cy5, total DNA samples used as reference are labeled with Cy3 using 9mer primers that have Cy3 and Cy5 dyes attached and Klenow added.
Hybridization protocol
Hybridization is performed for 16 - 20 hours at 42°C.
Scan protocol
NimbleGen two-color arrays are scanned with a GenePix 4000B Scanner, to obtain images from for the 532nm and 635nm wavelengths.
Description
U937 cells expressing AML1/ETO-HA - ChIP with anti-HEB antibody
Data processing
Data is pre-processed by NimbleGen Services using the NimbleGen NimbleScan software. Pair reports contain the raw data, listing the probe intensities of each array. Scaled Log2-Ratio Data is calculated for each sample from the experimental and reference conditions.
