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Sample GSM52988 Query DataSets for GSM52988
Status Public on Jun 17, 2005
Title SIRT1 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBIv34)
Sample type genomic
 
Channel 1
Source name SIRT1 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours
Organism Homo sapiens
Extracted molecule genomic DNA
 
Channel 2
Source name Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 2 hours
Organism Homo sapiens
Extracted molecule genomic DNA
 
 
Description HL60 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 SIRT1 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, SIRT1, HL60 Cells, 2 hours Retinoic Acid
 
Submission date Jun 07, 2005
Last update date Dec 13, 2005
Contact name Thomas R Gingeras
E-mail(s) tom_gingeras@affymetrix.com
Organization name Affymetrix, Inc.
Street address 3380 Central Expressway
City Santa Clara
State/province CA
ZIP/Postal code 95051
Country USA
 
Platform ID GPL1789
Series (1)
GSE2798 ENCODE: SIRT1 ChIP-chip of retinoic acid-stimulated HL60 cells at 4 timepoints (NCBIv34)

Data table header descriptions
ID_REF chromosome_genomic position
VALUE '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 (each biolocial replicate individually) and target_source2 (biological replicates 1, 2 and 3) is calculated for all probe pairs within a sliding window of 1001bp of ID_REF using quantile normalizing replicate arrays whose median array intensity was scaled to 22. A composit value is found by taking the median of all individual biological replicate processed data.
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. Positive regions were selected using the following approach: Putative seed binding sites of size 1001bp on average are generated per biological replicate dataset based on a p-value, maxgap nad minrun threshold. The thresholds are: -10log10(P-VALUE) = 20 and a maxgap=500bp and minrun=0bp. This process clusters neighboring set of probes -neighboring in genomic space -into intervals or seed sites. A median pValue is generated per each seed site. These seed sites are then ranked, individually in each of the replicates, based on their median p-values. For a seed site completely absent in a biological replicate a surrogate rank corresponding to the worst rank in the data set is assigned to it. At this point three distributions are generated by combining ranked data per seed site across all replicates. (1) The distribution of average ranks?(AvgRank) per site across all replicates is computed. (2)The distribution of the sum of absolute rank differences (SAD) is computed per site by considering all partwise combination of replicates. Since SAD is a metric of consistency or dispersion, it has a high correlation with the standard deviation. The rationale behind this metric is to rank sites based on minimization of pair-wise rank differences. Average rank is a measure of centrality. The SAD versus average of pair-wise ranks as described above ranks sites based on consistency and significance simultaneously. Ideal sites are the ones with the highest consistency and significance - that is have low ranks on both scores. Sites with very low or stringent average p-value, alternatively defined as highly significant sites have lower ranks. Similarly sites with maximal consistency have lowest ranks. (3) A combined p-Value,(CombpValue) based on a chi square distribution is computed per site across replicates. This metric measures the overall confidence of a site across all available replicates and hence is a surrogate for error. A metric r =sqrt((SAD)^2 + (AvgRank)^2+(CombpValue)^2) is computed per site. All seed sites passing a threshold r metric(r_thresh), is selected as sites. Inc omputing r_thresh values corresponding to the 25th percentile of the SAD, AvgRank and CombPvalue are used. Following this site selection any sites with low complexity repeats are removed by using a uniqueness filter on the pValue. The start and stop coordinates for a given site can vary across replicates. The final startand stop of a site is defined by the most conservative approach, where the interval as defined by the intersect of basepairs across all replicates is considered.
DETECTION -10log10(P-VALUE) 'detection log transformed p-values' median p-value (from the individual biological replicate data) that indicates the significance of the enrichment of target_source1 over target_source2 using the Wilcoxon Rank Sum Scan Statisic.

Data table
ID_REF VALUE ABS_CALL DETECTION -10log10(P-VALUE)
chr1_148374651 0 A 1.04765
chr1_148374672 0 A 1.738
chr1_148374692 0 A 1.738
chr1_148374719 0 A 1.738
chr1_148374740 0 A 1.738
chr1_148374767 0 A 1.738
chr1_148374787 0 A 1.738
chr1_148374809 0 A 1.738
chr1_148374824 0 A 1.738
chr1_148374846 0 A 1.738
chr1_148374865 0 A 1.738
chr1_148374886 0 A 1.738
chr1_148374906 0 A 1.738
chr1_148374927 0 A 1.738
chr1_148374949 0 A 1.738
chr1_148374968 0 A 1.738
chr1_148374991 0 A 1.738
chr1_148375007 0 A 1.738
chr1_148375029 0 A 1.738
chr1_148375050 0 A 1.738

Total number of rows: 737680

Table truncated, full table size 20365 Kbytes.




Supplementary file Size Download File type/resource
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B1_T1.cel.gz 9.6 Mb (ftp)(http) CEL
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B1_T2.cel.gz 9.7 Mb (ftp)(http) CEL
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B2_T1.cel.gz 9.5 Mb (ftp)(http) CEL
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B2_T2.cel.gz 9.4 Mb (ftp)(http) CEL
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B3_T1.cel.gz 9.6 Mb (ftp)(http) CEL
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B3_T2.cel.gz 9.6 Mb (ftp)(http) CEL
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B5_T1.cel.gz 9.9 Mb (ftp)(http) CEL
GSM52988_EC_AS_HL60_DN_RA_SIRT1_02hr_C01_B5_T2.cel.gz 9.5 Mb (ftp)(http) CEL

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