After fixation and permeabilization, cells were washed in TS buffer (100 µM tris-HCl, 150 mM NaCl, pH 7.0). Endogenous peroxidase activity was quenched with 0.5% H2O2 in TS for 10 minutes at RT. After another TS wash, samples were blocked in 5% donkey serum in TS for 20 minutes. mAb 5E10 (a gift from R. van Driel, University of Amsterdam, The Netherlands) was used to detect PML protein. A secondary antibody conjugated with horseradish peroxidase was then added to the samples. Biotinylated tyramide (Invitrogen) was diluted in 0.1M boric acid and 0.003% H2O2 and then incubated on the samples for 2 minutes. Washing the cells three times in TS buffer stopped the reaction. After this, the samples were either processed for immunofluorescence microscopy or for affinity purification of the PML NB-associated chromatin.
Growth protocol
Jurkat cells were cultured in RPMI (Invitrogen) supplemented with 10% FBS (Wisent), 10 µg/ml penicillin and streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen).
Extracted molecule
genomic DNA
Extraction protocol
For affinity purification, the cells were scrapped off the slides into an eppendorf tube containing PBS. The samples were pelleted at 2900g at 4 °C for 25 minutes. The pellet was resuspended in 5M urea, 2M NaCl, and 10 nM EDTA. This suspension was then sonicated in a Misonix 3000 sonicator equipped with a Cup Horn in an iced water bath with the following program (5 sec pulse/15 sec pause repeated 15 times at power level 4). The program was repeated 4 more times with changes of the iced water bath between repetitions. The sample was then centrifuged at 13000 rpm for 15 minutes at 4 °C. The supernatant was then dialyzed overnight in PBS. Streptavidin agarose beads (Invitrogen) were washed three times in PBS and the supernatant was incubated with the beads for 3-4 hours at 4 °C. The beads were washed with the following buffers in this order: PBS, TSE150 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl), TSE 500 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 500 mM NaCl), and TE (10 mM tris pH 8.0, 1 mM EDTA). The beads were collected in TE buffer and the formaldehyde cross-links were reversed by shaking the samples at 65 °C overnight. The slurry was then treated with RNase A (2 µg/ml) (Fermentas) for 30 minutes at 37 °C and then with proteinase K (100 µg/ml) (Fermentas) for 6 hours at 37 °C.
Label
Cy3
Label protocol
The DNA was phenol extracted and ethanol precipitated using glycogen as carrier. The DNA was amplified using the Whole Genome Amplification (WGA) kit (Sigma).
After fixation and permeabilization, cells were washed in TS buffer (100 µM tris-HCl, 150 mM NaCl, pH 7.0). Endogenous peroxidase activity was quenched with 0.5% H2O2 in TS for 10 minutes at RT. After another TS wash, samples were blocked in 5% donkey serum in TS for 20 minutes. mAb 5E10 (a gift from R. van Driel, University of Amsterdam, The Netherlands) was used to detect PML protein. A secondary antibody conjugated with horseradish peroxidase was then added to the samples. Biotinylated tyramide (Invitrogen) was diluted in 0.1M boric acid and 0.003% H2O2 and then incubated on the samples for 2 minutes. Washing the cells three times in TS buffer stopped the reaction. After this, the samples were either processed for immunofluorescence microscopy or for affinity purification of the PML NB-associated chromatin.
Growth protocol
Jurkat cells were cultured in RPMI (Invitrogen) supplemented with 10% FBS (Wisent), 10 µg/ml penicillin and streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen).
Extracted molecule
genomic DNA
Extraction protocol
For affinity purification, the cells were scrapped off the slides into an eppendorf tube containing PBS. The samples were pelleted at 2900g at 4 °C for 25 minutes. The pellet was resuspended in 5M urea, 2M NaCl, and 10 nM EDTA. This suspension was then sonicated in a Misonix 3000 sonicator equipped with a Cup Horn in an iced water bath with the following program (5 sec pulse/15 sec pause repeated 15 times at power level 4). The program was repeated 4 more times with changes of the iced water bath between repetitions. The sample was then centrifuged at 13000 rpm for 15 minutes at 4 °C. The supernatant was then dialyzed overnight in PBS. Streptavidin agarose beads (Invitrogen) were washed three times in PBS and the supernatant was incubated with the beads for 3-4 hours at 4 °C. The beads were washed with the following buffers in this order: PBS, TSE150 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl), TSE 500 (20 mM tris pH 8.0, 1% triton X-100, 0.1% SDS, 2 mM EDTA, 500 mM NaCl), and TE (10 mM tris pH 8.0, 1 mM EDTA). The beads were collected in TE buffer and the formaldehyde cross-links were reversed by shaking the samples at 65 °C overnight. The slurry was then treated with RNase A (2 µg/ml) (Fermentas) for 30 minutes at 37 °C and then with proteinase K (100 µg/ml) (Fermentas) for 6 hours at 37 °C.
Label
Cy5
Label protocol
The DNA was phenol extracted and ethanol precipitated using glycogen as carrier. The DNA was amplified using the Whole Genome Amplification (WGA) kit (Sigma).
Hybridization protocol
A 5 µg Cy5-labeled and 5 µg of Cy3-labeled DNAs were combined in a total volume of 158 µl. The 158 µl were mixed with 50 µl of 1 mg/ml of Human Cot-1 DNA, 52 µl of 10x Agilent Blocking Agent, 260 µl of 2x Agilent Hybridization Buffer. Samples were heated at 95°C for 3 min, and then incubated at 37°C for 30 min. 490 µl of the sample were applied to the 1x244 promoter array assembled in a chamber and incubated in a rotisserie hybridization oven at 65°C and 20 r.p.m. for 40 h. After hybridization the microarray slide was washed with Oligo aCGH/ChIP-on-chip Wash Buffer for 5 min at room temperature, followed by a wash for 5 min at 31°C.
Scan protocol
Agilent Feature Extraction software v9.5 was used.
Data processing
Data were pre-processed using the variance-stabilizing normalization algorithm with default parameter settings, as implemented in the vsn package (v3.8.0) of the BioConductor open-source library
