HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37oC in 5% CO2 to a density of 0.75 x 10*6 cells/ml (medium and supplemental components were purchased from Invitrogen). Cells were then induced with 1 uM all-trans-retinoic acid (ATRA, Sigma) for the period of time specified. At the appropriate time, suspension cells are fixed with 1% formaldehyde (J.T. Baker) at room temperature for 10 minutes. Subsequently, 1/20 volume of 2.5 M glycine (American Bioanalytical) was added and incubation proceeded for 5 minutes. Crosslinked cells were extensively washed resulting in nuclear pellet formation. A nuclear pellet, equivalent to 2 x 10*8 cells, was treated with 20U of MNase (USB) for 10 min at 37C to generate fragmented DNA of 500-1000 bp average size, followed by sonication. Cellular debris was separated from solubilized chromatin by high-speed centrifugation. Isolated chromatin, equivalent to 2.5 x 10*7 cells, was pre-cleared for 30 min with Protein A/G sepharose beads (Amersham Biosciences) at 4C. This pre-cleared chromatin was separated from the beads and immunoreacted with a monoclonal antibody against RNA Polymerase II, RNAP (Covance Research Products) overnight at 4C. Protein A/G resin were subsequently added and allowed to bind for 3 hours at 4C. Recovered, immunoprecipitated chromatin was extensively washed, crosslinks reversed, and purified immunoprecipitated (IP) DNA recovered using a QIAquick PCR purification kit (Qiagen). A mock IP was preformed in parallel to isolate total DNA referred to as INPUT. Two rounds of linear amplification were separately performed on 30% of IP and INPUT DNA using Sequenase (USB, per manufacturer's protocol) and a random primer (GTTTCCCAGTCACGATCNNNNNNNNN). DNA was recovered using a QIAquick PCR purification kit (Qiagen). PCR was performed using Invitrogen products, per manufacturer's protocol, on 75% of linearly amplified DNA using a primer (GTTTCCCAGTCACGATC) for 30 cycles. DNA was purified via a QIAquick PCR purification kit (Qiagen), and 90% of PCR amplified DNA was subjected to an additional 25 rounds of PCR, with subsequent DNA recovered as stated above. Amplified DNA was verified on 1.5% agarose gel to be an average size of 300 bp. PCR amplified DNA, 5-10 ug, was subjected to further fragmentation to 50-100 bp by DNAse I (1U/ul; Epicentre) treatment in 1X One-Phor-All buffer (Pharmacia). Following DNAse I inactivation at 99C for 10 min, the size ditsribution of fragmented DNA was verified on a 1% agarose gel. The PCR amplified, fragmented DNA from DNAse I treatment was end-labeled with 70nM of bio-ddATP (Perkin Elmer) using 6-10U of terminal transferase (TdT, Roche) per 1ug of fragmented DNA in 1X TdT buffer (Roche) and 5mM CoCl2 (Roche) for 2 hours at 37C. The labeled DNA material was subsequently hybridized to the chips (previously prehybridized for 1 hour in 1X MES-triton at 45C) for 18 hours at 45C in a 3 M TMAC/1X MES-based solution. Keywords = Homo sapiens, RNAP, HL-60 Cells, 32 hours Retinoic Acid
'signal' an estimate of ln(target_source1/target_source2) using a Wilcoxon Rank Sum Scan Statistic where the median of all pairwise differences of ln(max(PM-MM,1)) for target_source1 and target_source2 is calculated for all probe pairs within a sliding window of 1001bp of chromosome genomic position using quantile normalizing replicate arrays whose median array intensity was scaled to 25.
ABS_CALL
the call in an absolute analysis that indicates if at ID_REF target_source1 is significantly more enirched than target_source2 with P indicating target_source1 is present over the control, target_source2, and A indicating absent. This call was produced by applying a cutoff of 50 in the -10log10(P-VALUE) and calling all probes P within 500bp of probes which pass the threshold.
DETECTION -10log10(P-VALUE)
'detection log transformed p-values' p-value that indicates the significance of the enrichment of target_source1 over target_source2 using the Wilcoxon Rank Sum Scan Statisic.
