established cell line; grown under standard conditions
Extracted molecule
genomic DNA
Extraction protocol
ChIP analysis was performed as described by Si et al. (J Virol. 2006 Sep;80(18):9017-30) and recommended by the array manufacturer (Agilent Mammalian ChIP-on-chip protocol V10.0, May 2008), with some modifications. For chromatin immunoprecipitation, protein from 5x10^6 to 2x10^7 cells was cross-linked to DNA with 1 % formaldehyde in PBS for 10 min (adherent cells) or 20 min (suspension cells) at room temperature. The reaction was quenched by adding 1/20th volume 2.5 M glycine, cells were washed with PBS, scraped off the dish and collected. Cells were washed twice with ice cold PBS. All following steps were performed at 4°C. Lysis and wash buffers contained 1x protease inhibitor cocktail (Roche) and 1 mM Pefabloc® SC-Protease Inhibitor (Roth). Nuclei were isolated by incubation of cross-linked cells with 1 ml buffer I (50 mM Hepes-KOH, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10 min on ice and pelleted by centrifugation (1,350 x g 5 min). The nuclei were subsequently washed with 1 ml buffer II (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), pelleted again and resuspended in 1 ml buffer III (1% SDS, 10 mM EDTA, 50 mM Tris-HCL). Chromatin was fragmented by sonication using a BioruptorTM (Diagenode) to an average length if 100-500 bp. After addition of 100 µl 10% Triton X-100, cell debris was pelleted by centrifugation (20,000 x g, 4°C) and supernatants were collected. For each individual IP, chromatin from 1x106 cells in a maximum volume of 200µl was diluted with dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16,7 mM Tris-HCl, 167 mM NaCl) to a final volume of 2 ml. To reduce non-specific background, chromatin was pre-incubated with 60 µl salmon-sperm DNA protein-A agarose beads (Upstate). Antibodies (2 to 10 µg, depending on the antibody) specific for the histone modifications H3K9/K14-Ac (Upstate: #06-599), H3K4-me3 (Upstate: #04-745), H3K9-me3 (Upstate: #17-625) or H3K27-me3 (Upstate: #07-449) were added and incubated for 16 hrs at 4°C on a rotating wheel. 60 µl agarose beads were added to precipitate the chromatin-immunocomplexes for 1 hr at 4°C. Beads were washed once with low-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl), once with high-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), once with LiCl-wash buffer (0.25 M LiCl, 1 % Nonidet P-40, 1 % Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl) and twice with TE buffer. Chromatin was eluted from the beads in 210 µl elution-buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS) for 30 min at 65°C. 8 µl of a 5 M NaCl solution were added to 200 µl of the supernatant, and chromatin was de-crosslinked overnight at 65°C. Samples were diluted by adding 200 µl TE and RNA was degraded with 8 µl RNAseA (10 mg/ml) for 2 hrs at 37°C. For degradation of protein, 7 µl of CaCl2 solution (300 mM CaCl2 in 10 mM Tris-HCl) and 4 µl ProteinaseK (20 mg/ml Peqlab) were added and samples were incubated for 1h at 55°C. DNA was purified two times by phenol-chloroform extraction followed by a single chloroform extraction. DNA (400 µl in total) was precipitated with 1055 µl ethanol (100 %), 24 µl NaCl (5 M) and 3 µl glycogen (10 mg/ml) at -80°C for at least 30 min. After centrifugation (20.000 x g, 4°C, 15 min), pellets were washed once with ethanol (70 %) centrifuged again and dried in a vacuum centrifuge. DNA was resuspended in 70 µl of 10 mM Tris-HCl. For preparation of input controls, 1/4th of the amount of chromatin that was used in the immunopreciptations was employed. Input samples were diluted in dilution buffer to a final volume of 200 µl and were treated identical to IP samples, starting with the decrosslinking step. Both samples were subsequenctly subjected to whole genome amplification and labeling using a linker mediated PCR protocol (Agilent Mammalian ChIP-on-chip protocol V10.0, May 2008), followed by microarray hybridization.
Label
Cy5
Label protocol
500 ng of amplified ChIP-input controls and 500 ng of amplified immunoprecipitated ChIP material were labeled with Cy3 and Cy5 using Agilent Genomic DNA Labeling Kit PLUS according to Agilents recommendations. For normalization purposes, 0.1 ng of Adenovirus Type 5 DNA were added to each samples prior to the labeling procedure. After labeling, samples were purified using Microcon YM-30 filter columns (Milipore).
Channel 2
Source name
Input DNA from SLKP cells (long-term KSHV infected SLK cells)
established cell line; grown under standard conditions
Extracted molecule
genomic DNA
Extraction protocol
ChIP analysis was performed as described by Si et al. (J Virol. 2006 Sep;80(18):9017-30) and recommended by the array manufacturer (Agilent Mammalian ChIP-on-chip protocol V10.0, May 2008), with some modifications. For chromatin immunoprecipitation, protein from 5x10^6 to 2x10^7 cells was cross-linked to DNA with 1 % formaldehyde in PBS for 10 min (adherent cells) or 20 min (suspension cells) at room temperature. The reaction was quenched by adding 1/20th volume 2.5 M glycine, cells were washed with PBS, scraped off the dish and collected. Cells were washed twice with ice cold PBS. All following steps were performed at 4°C. Lysis and wash buffers contained 1x protease inhibitor cocktail (Roche) and 1 mM Pefabloc® SC-Protease Inhibitor (Roth). Nuclei were isolated by incubation of cross-linked cells with 1 ml buffer I (50 mM Hepes-KOH, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10 min on ice and pelleted by centrifugation (1,350 x g 5 min). The nuclei were subsequently washed with 1 ml buffer II (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), pelleted again and resuspended in 1 ml buffer III (1% SDS, 10 mM EDTA, 50 mM Tris-HCL). Chromatin was fragmented by sonication using a BioruptorTM (Diagenode) to an average length if 100-500 bp. After addition of 100 µl 10% Triton X-100, cell debris was pelleted by centrifugation (20,000 x g, 4°C) and supernatants were collected. For each individual IP, chromatin from 1x106 cells in a maximum volume of 200µl was diluted with dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16,7 mM Tris-HCl, 167 mM NaCl) to a final volume of 2 ml. To reduce non-specific background, chromatin was pre-incubated with 60 µl salmon-sperm DNA protein-A agarose beads (Upstate). Antibodies (2 to 10 µg, depending on the antibody) specific for the histone modifications H3K9/K14-Ac (Upstate: #06-599), H3K4-me3 (Upstate: #04-745), H3K9-me3 (Upstate: #17-625) or H3K27-me3 (Upstate: #07-449) were added and incubated for 16 hrs at 4°C on a rotating wheel. 60 µl agarose beads were added to precipitate the chromatin-immunocomplexes for 1 hr at 4°C. Beads were washed once with low-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl), once with high-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), once with LiCl-wash buffer (0.25 M LiCl, 1 % Nonidet P-40, 1 % Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl) and twice with TE buffer. Chromatin was eluted from the beads in 210 µl elution-buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS) for 30 min at 65°C. 8 µl of a 5 M NaCl solution were added to 200 µl of the supernatant, and chromatin was de-crosslinked overnight at 65°C. Samples were diluted by adding 200 µl TE and RNA was degraded with 8 µl RNAseA (10 mg/ml) for 2 hrs at 37°C. For degradation of protein, 7 µl of CaCl2 solution (300 mM CaCl2 in 10 mM Tris-HCl) and 4 µl ProteinaseK (20 mg/ml Peqlab) were added and samples were incubated for 1h at 55°C. DNA was purified two times by phenol-chloroform extraction followed by a single chloroform extraction. DNA (400 µl in total) was precipitated with 1055 µl ethanol (100 %), 24 µl NaCl (5 M) and 3 µl glycogen (10 mg/ml) at -80°C for at least 30 min. After centrifugation (20.000 x g, 4°C, 15 min), pellets were washed once with ethanol (70 %) centrifuged again and dried in a vacuum centrifuge. DNA was resuspended in 70 µl of 10 mM Tris-HCl. For preparation of input controls, 1/4th of the amount of chromatin that was used in the immunopreciptations was employed. Input samples were diluted in dilution buffer to a final volume of 200 µl and were treated identical to IP samples, starting with the decrosslinking step. Both samples were subsequenctly subjected to whole genome amplification and labeling using a linker mediated PCR protocol (Agilent Mammalian ChIP-on-chip protocol V10.0, May 2008), followed by microarray hybridization.
Label
Cy3
Label protocol
500 ng of amplified ChIP-input controls and 500 ng of amplified immunoprecipitated ChIP material were labeled with Cy3 and Cy5 using Agilent Genomic DNA Labeling Kit PLUS according to Agilents recommendations. For normalization purposes, 0.1 ng of Adenovirus Type 5 DNA were added to each samples prior to the labeling procedure. After labeling, samples were purified using Microcon YM-30 filter columns (Milipore).
Hybridization protocol
Labelled input and ChIP samples were blocked using Agilent blocking solution and human cot-1 DNA (Invitrogen), and hybridized using the Agilent Oligo aCGH/ChIP-on-chip Hybridization Kit at 65°C for 24 hrs in a rotating oven. Arrays were washed once with Oligo aCGH/ChIP-on-chip Wash Buffer 1 (Agilent 5188-5221) at RT for 5 min and in Oligo aCGH/ChIP-on-chip Wash Buffer 2 (Agilent 5188-5222) at 37°C for 1 min.
Scan protocol
Microarrays were scanned using a GenePix Personal 4100A scanner (Axon Instruments)
Description
Analysis of H3K9me3 patterns in SLKP cells (long-term KSHV infected SLK cells)
Data processing
Primary array analysis and data normalization was carried out using GenePix®Pro 6.0 software (Axon Instruments). All ChIP datasets were normalized using the adenovirus type 5 DNA that was added as a spike-in prior to labeling, hence correcting for errors during labelling, hybridization or scanning of the samples (probes Ad5f/r_K_475 to _590 were excluded due to a sequence deletion). To eliminate false positive spots, we hybridized DNA from KSHV-negative SLK cells and identified all probes which exhibited high levels of background hybridization (i.e., fluorescence levels that exceeded the mean value plus 1x the standard deviation of all KSHV-specific spots on the negative control array). These probes (which mapped almost exclusively to repeat regions) were permanently flagged in all datasets and not used for further analysis. While our arrays carry probes specific for the M and P types of the KSHV genome, the KSHV genomes from the BCBL1 and AP3 lines have not been fully sequenced and thus may deviate from the reference genomes at a few locations. To control for such sequence differences, we flagged all spots which exhibited fluorescence levels which did not exceed a background fluorescence treshold in the input channel, which was set to the mean fluorescence plus twice the standard deviation of all negative control features (i.e. empty array features as well as spots containing irrelevant sequences, corresponding to all Agilent probes in the datasets which are labeled with NC2_ and (-)3xSLv1). Note that, if sequence diversification leads to only a reduction of hybridization efficiency (e.g. due to single nucleotide polymorphism, which will not abolish hybridization), this will not falsify our results as the hybridization efficiency will be reduced in input as well as the immunoprecipitated sample; the ratio will thus be unaffected. In addition to above quality controls, in each dataset we flagged all probes which exhibited more than 30 % variance between duplicate spots. The 30% treshold corresponds to the mean variance plus twice the standard deviation exhibited by all KSHV-specific probes in all ChIP experiments, thus removing all probes which show a significantly increased variance between individual spot repeats. After normalization, an enrichment score was calculated for each of the probes, represented by the ratio of fluorescence signal intensities in the immunoprecipitated samples relative to the input control.
