title: X90A63 (HG-U133A)

description: Series of 251 tumours


title: X63A62 (HG-U133B)

status: Public on Oct 21 2005

type: RNA

city: Singapore

first: Lance

middle: David

last: Miller

address: 60 Bioplois Street, #02-01 Genome

department: Microarray and Expression Genomics

country: Singapore

institute: Genome Institute of Singapore

description: Series of 251 tumours

hyb_protocol: Standard Affymetrix procedure

scan_protocol: Standard Affymetrix procedure

data_processing: Affymetrix Microarray Suite Version 5.0

label: biotin

molecule: total RNA

organism: Homo sapiens

source_name: Breast Tumor

label_protocol: Approximately 10ug of total RNA was processed to produce biotynylated cRNA targets

extract_protocol: Qiagen RNeasy Mini Kit

tag: Breast Tumor tissue


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


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description: Series of 251 tumours


title: X155B52 (HG-U133B)

description: Series of 251 tumours


title: X170B15 (HG-U133A)

description: Series of 251 tumours


title: X173B43 (HG-U133A)

description: Series of 251 tumours


title: X151B84 (HG-U133B)

description: Series of 251 tumours


title: X16C97 (HG-U133A)

description: Series of 251 tumours


title: X154B42 (HG-U133B)

description: Series of 251 tumours


title: X171B77 (HG-U133B)

description: Series of 251 tumours


title: X171B77 (HG-U133A)

description: Series of 251 tumours


title: X174B41 (HG-U133A)

description: Series of 251 tumours


title: X144B49 (HG-U133B)

description: Series of 251 tumours


title: X150B81 (HG-U133B)

description: Series of 251 tumours


title: X147B19 (HG-U133B)

description: Series of 251 tumours


title: X148B49 (HG-U133B)

description: Series of 251 tumours


title: X152B99 (HG-U133B)

description: Series of 251 tumours


title: X166B79 (HG-U133A)

description: Series of 251 tumours


title: X153B09 (HG-U133B)

description: Series of 251 tumours


title: X14B98 (HG-U133B)

description: Series of 251 tumours


title: X163B27 (HG-U133A)

description: Series of 251 tumours


title: X146B39 (HG-U133B)

description: Series of 251 tumours


title: X145B10 (HG-U133B)

description: Series of 251 tumours


title: X165B72 (HG-U133A)

description: Series of 251 tumours


title: X140B91 (HG-U133B)

description: Series of 251 tumours


title: X168B51 (HG-U133A)

description: Series of 251 tumours


title: X143B81 (HG-U133B)

description: Series of 251 tumours


title: X169B79 (HG-U133A)

description: Series of 251 tumours


title: X13B79 (HG-U133B)

description: Series of 251 tumours


title: X139B03 (HG-U133B)

description: Series of 251 tumours


title: X159B47 (HG-U133A)

description: Series of 251 tumours


title: X162B98 (HG-U133A)

description: Series of 251 tumours


title: X156B01 (HG-U133A)

description: Series of 251 tumours


title: X131B79 (HG-U133B)

description: Series of 251 tumours


title: X161B31 (HG-U133A)

description: Series of 251 tumours


title: X136B04 (HG-U133B)

description: Series of 251 tumours


title: X127B00 (HG-U133B)

description: Series of 251 tumours


title: X137B88 (HG-U133B)

description: Series of 251 tumours


title: X130B92 (HG-U133B)

description: Series of 251 tumours


title: X15C94 (HG-U133A)

description: Series of 251 tumours


title: X150B81 (HG-U133A)

description: Series of 251 tumours


title: X138B34 (HG-U133B)

description: Series of 251 tumours


title: X158B84 (HG-U133A)

description: Series of 251 tumours


title: X154B42 (HG-U133A)

description: Series of 251 tumours


title: X135B40 (HG-U133B)

description: Series of 251 tumours


title: X200B47 (HG-U133A)

description: Series of 251 tumours


title: X152B99 (HG-U133A)

description: Series of 251 tumours


title: X151B84 (HG-U133A)

description: Series of 251 tumours


title: X160B16 (HG-U133A)

description: Series of 251 tumours


title: X134B33 (HG-U133B)

description: Series of 251 tumours


title: X14B98 (HG-U133A)

description: Series of 251 tumours


title: X153B09 (HG-U133A)

description: Series of 251 tumours


title: X124B25 (HG-U133B)

description: Series of 251 tumours


title: X128B48 (HG-U133B)

description: Series of 251 tumours


title: X155B52 (HG-U133A)

description: Series of 251 tumours


title: X122B81 (HG-U133B)

description: Series of 251 tumours


title: X120B73 (HG-U133B)

description: Series of 251 tumours


title: X11B47 (HG-U133B)

description: Series of 251 tumours


title: X148B49 (HG-U133A)

description: Series of 251 tumours


title: X145B10 (HG-U133A)

description: Series of 251 tumours


title: X112B55 (HG-U133B)

description: Series of 251 tumours


title: X114B68 (HG-U133B)

description: Series of 251 tumours


title: X113B11 (HG-U133B)

description: Series of 251 tumours


title: X111B51 (HG-U133B)

description: Series of 251 tumours


title: X142B05 (HG-U133A)

description: Series of 251 tumours


title: X13B79 (HG-U133A)

description: Series of 251 tumours


title: X147B19 (HG-U133A)

description: Series of 251 tumours


title: X10B88 (HG-U133B)

description: Series of 251 tumours


title: X143B81 (HG-U133A)

description: Series of 251 tumours


title: X140B91 (HG-U133A)

description: Series of 251 tumours


title: X139B03 (HG-U133A)

description: Series of 251 tumours


title: X110B34 (HG-U133B)

description: Series of 251 tumours


title: X105B13 (HG-U133B)

description: Series of 251 tumours


title: X172B19 (HG-U133A)

description: Series of 251 tumours


title: X134B33 (HG-U133A)

description: Series of 251 tumours


title: X104B91 (HG-U133B)

description: Series of 251 tumours


title: X136B04 (HG-U133A)

description: Series of 251 tumours


title: X103B41 (HG-U133B)

description: Series of 251 tumours


title: X106B55 (HG-U133B)

description: Series of 251 tumours


title: X102B06 (HG-U133B)

description: Series of 251 tumours


title: X137B88 (HG-U133A)

description: Series of 251 tumours


title: X138B34 (HG-U133A)

description: Series of 251 tumours


title: X101B88 (HG-U133B)

description: Series of 251 tumours


title: X135B40 (HG-U133A)

description: Series of 251 tumours


title: X128B48 (HG-U133A)

description: Series of 251 tumours


title: X163B27 (HG-U133B)

description: Series of 251 tumours


title: X100B08 (HG-U133B)

description: Series of 251 tumours


title: X122B81 (HG-U133A)

description: Series of 251 tumours


title: X127B00 (HG-U133A)

description: Series of 251 tumours


title: X142B05 (HG-U133B)

description: Series of 251 tumours


title: X124B25 (HG-U133A)

description: Series of 251 tumours


title: X112B55 (HG-U133A)

description: Series of 251 tumours


title: X11B47 (HG-U133A)

description: Series of 251 tumours


title: X114B68 (HG-U133A)

description: Series of 251 tumours


title: X130B92 (HG-U133A)

description: Series of 251 tumours


title: X120B73 (HG-U133A)

description: Series of 251 tumours


title: X131B79 (HG-U133A)

description: Series of 251 tumours


title: X111B51 (HG-U133A)

description: Series of 251 tumours


title: wild-type pNM12#2 +FeSO4 + arabinose

status: Public on Aug 31 2005

city: Sherbrooke

first: Eric

last: Masse

state: Quebec

address: 3001 12e Avenue

department: Biochemistry

country: Canada

institute: Universite de Sherbrooke

description: Hybridization, washing and staining of microarrays was performed as recommended in the Affymetrix technical manual.  Briefly, the cDNA fragments were hybridized for 16 hours at 45oC to an Affymetrix GeneChip E. coli antisense microarray.  Arrays were washed at 25oC with non-stringent wash buffer, followed by a wash at 50oC with stringent buffer.  Then, the arrays were stained with phycoerythrin-conjugated streptavidin (Molecular Probes).  The arrays were read at 570 nm with a GeneChip Scanner 3000 and the data were analyzed with the Affymetrix Microarray Suite 5.0 software.  Sample variations were standardized by scaling the average of all genes to constant target intensity of 500 for all arrays used.  The statistical significance of the data was analyzed by using rank-based (non-parametric) methods to generate p-values for each gene.  The data were passed through a first screen to discard absent genes, which have p-values > 0.065.  Then, from the present (p-value < 0.05) or moderate (p-value between 0.05 and 0.065) genes, only those with a signal ratio (pNM12/pBAD-ryhB) higher than 2 fold, positive or negative, were kept for further studies.  Operons were included in the Tables only if at least one gene within the operon had a ratio of over 2.  Usually, when the detection level was low (signal less than 200), the genes were discarded, unless otherwise indicated in the text (e.g. bfr).  Array experiments were done in duplicate and the reproducibility of the experiments is shown on the scatter plot of the values for all present genes (supplemental data: S2).  Genes repressed by RyhB in all three experiments (wild-type, fur, wild-type with added FeSO4) have been placed in Table 1.  Table 2 includes genes that are repressed by RyhB only in a fur+ background (unless indicated otherwise in the text).  Table 3 includes genes that were increased in expression upon RyhB expression.

data_processing: Microarray suite 5.0

label: Biotinylated

organism: Escherichia coli

source_name: wild-type pNM12#2 +FeSO4 + arabinose

tag: wild-type pNM12#2 +FeSO4 + arabinose

tag: experiment #306


title: X113B11 (HG-U133A)

description: Series of 251 tumours


title: X104B91 (HG-U133A)

description: Series of 251 tumours


title: wild-type pBAD-ryhB#2 +FeSO4 + arabinose


title: X102B06 (HG-U133A)

description: Series of 251 tumours


title: wild-type pBAD-ryhB#1 +FeSO4 + arabinose

tag: experiment #303


title: X100B08 (HG-U133A)

description: Series of 251 tumours


title: fur mutant pBAD-ryhB#1 + arabinose

source_name: fur mutant pBAD-ryhB#1 + arabinose

tag: fur mutant pBAD-ryhB#1 + arabinose

tag: experiment #203


title: wild-type pNM12#1 +arabinose

source_name: wild-type pNM12#1 +arabinose

tag: EM1456 wild-type pNM12#1 +arabinose

tag: experiment #115


title: fur mutant pBAD-ryhB#2 + arabinose

source_name: fur mutant pBAD-ryhB#2 + arabinose

tag: fur mutant pBAD-ryhB#2 + arabinose

tag: experiment #204


title: wild-type pNM12#2 + arabinose

source_name: wild-type pNM12#2 + arabinose

tag: wild-type pNM12#2 + arabinose

tag: experiment #116


title: X10B88 (HG-U133A)

description: Series of 251 tumours


title: wild-type pNM12#1 +FeSO4 + arabinose

source_name: wild-type pNM12#1 +FeSO4 + arabinose

tag: wild-type pNM12#1 +FeSO4 + arabinose

tag: experiment #305


title: wild-type pBAD-ryhB #2 + arabinose

source_name: wild-type pBAD-ryhB #2 + arabinose

tag: wild-type pBAD-ryhB #2 + arabinose


title: fur mutant pNM12#1 + arabinose

source_name: fur mutant pNM12#1 + arabinose

tag: fur mutant pNM12#1 + arabinose

tag: experiment #205


title: wild-type pBAD-ryhB#1 + arabinose

source_name: wild-type pBAD-ryhB#1 + arabinose

tag: Strain EM1453 wild-type pBAD-ryhB#1 + arabinose


title: fur mutant pNM12#2 + arabinose

source_name: fur mutant pNM12#2 + arabinose

tag: fur mutant pNM12#2 + arabinose

tag: experiment #206


title: X106B55 (HG-U133A)

description: Series of 251 tumours

tag: Breast Tumor tissue


title: X105B13 (HG-U133A)

description: Series of 251 tumours


title: Plasma membrane from Arabidopsis collected from two-phase partitioning (Sample 2)

status: Public on Jun 21 2005

type: protein

city: Madison

first: Michael

middle: R

last: Sussman

state: WI

address: 425 Henry Mall

department: Biotechnology Center

country: USA

institute: University of Wisconsin

description: 18O Isotopic labeling experiments conducted in order to identify proteins with PM localization.

data_processing: Following digestion, peptides were separated via strong cation exchange and analyzed via RP LC MS.  The raw data from each LC-MS analysis was dumped to text files using DataBridge 4.0 (Waters) and all further processing was done using Mathematica (Wolfram Research) and software programs written in C++ using Visual Studio 6.0 (Microsoft) and perl.  EICs for the zero, one, and two incorporation events were extracted, 4.5 minutes wide centered on the sequencing event for all relavent peptides smoothed using a Savitzky-Golay filter and fitted to a Gaussian peak.  Data values within two standard deviations of the peak center from the unsmoothed chromatograms were then used to calculate linear regressions between the monoisotopic peak and single incorporation peak as well as the monoisotopic peak and double incorporation peak in a method similar to that reported by Washburn and colleagues.  Using this method, the slope of these regressions represented values for the single and double incorporation peaks that were normalized to the monoisotopic peak where the monoisotopic peak had a value of one.  Peptides were only quantified if the r-squared value from both regressions was 0.8 or greater.

molecule: protein

organism: Arabidopsis thaliana

source_name: Plasma Membrane

extract_protocol: Arabidopsis thaliana ecotype Columbia was grown at 22°C in 24 hour light in liquid culture consisting of:  0.5% (w/v) MES (pH 5.7), 2.15% (w/v) Murashige and Skoog salts (Sigma), and 1% (w/v) sucrose.  At two weeks of age whole seedlings were harvested.

treatment_protocol: Sample was digested using porcine trypsin in 18O enriched water.

tag: PM enriched from two-phase partitioning

source_name: Microsomes (PM-depleted)

treatment_protocol: Sample was digested using porcine trypsin in 16O (natural abundance) enriched water.

tag: Microsomes enriched from two-phase partitioning


title: Plasma membrane from Arabidopsis collected from two-phase partitioning (Sample 1)

growth_protocol: Arabidopsis thaliana ecotype Columbia was grown at 22°C in 24 hour light in liquid culture


title: TAK1_KO_IgM

status: Public on Aug 11 2005

city: Suita

first: Shizuo

last: Akira

state: Osaka

address: 3-1, Yamada-oka

department: Host Defense

country: Japan

institute: Research Institute for Microbial Diseases Osaka University

description: CD19-Cre; Tak1 flox/-; anti IgM 4 h

data_processing: MAS5.0, scaled to 500

label: PE

organism: Mus musculus

source_name: B cells

tag: Strain

value: C57BL/6 x 129Ola

tag: Gender

value: male

tag: Age

value: 8 weeks

tag: Cell

value: Splenic B cells (CD43 negative)


title: TAK1_WT_unstim

description: CD19-Cre; Tak1 flox/+ unstim.


title: TAK1_KO_unstim

description: CD19-Cre; Tak1 flox/- unstim.


title: TAK1_WT_IgM

description: CD19-Cre; Tak1 flox/+; anti IgM 4 h


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 0 hours (ENCODE) (NCBIv34)

status: Public on Jun 17 2005

city: Santa Clara

first: Thomas

last: Gingeras

state: CA

address: 3380 Central Expressway

institute: Affymetrix, Inc.

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C 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.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and 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 fragmented cDNA was then 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, PolyA+ RNA, HL-60 Cells, 0 hours Retinoic Acid

molecule: polyA RNA


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 2 hours (ENCODE) (NCBIv34)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C 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.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and 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 fragmented cDNA was then 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, PolyA+ RNA, HL-60 Cells, 2 hours Retinoic Acid


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 8 hours (ENCODE) (NCBIv34)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C 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.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and 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 fragmented cDNA was then 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, PolyA+ RNA, HL-60 Cells, 8 hours Retinoic Acid


title: PolyA+ RNA from cytosol of GM06990 Cells (NCBIv34)

description:
GM06990 suspension cells were grown in Rosswel Park Memorial Institute (RPMI) 1640 medium; supplemented with 15% FBS (heat-inactivated), and 1% Penicillin-Streptomycin, at 37C in 5% CO2 (medium and serum were puraches from HyClone; supplemental components were purchased from Invitrogen).
Cytosolic RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and 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 fragmented cDNA was then 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.
Sample author: Tom,,Gingeras
Keywords = Homo sapiens, Cytosolic PolyA+ RNA, GM06990 Cells

molecule: cytoplasmic RNA

tag: Coriell cell line repository identifier


title: PolyA+ RNA from cytosol of HeLa Cells (NCBIv34)

description:
HeLa adherent cells were grown in Dulbecco's Modified Eagle's Medium (DMEM); supplemented with 10% FBS, and 1% Penicillin-Streptomycin, at 37C in 5% CO2 to confluency (medium and serum were puraches from HyClone; supplemental components were purchased from Invitrogen).
Cytosolic RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and 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 fragmented cDNA was then 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.
Sample author: Tom,,Gingeras
Keywords = Homo sapiens, Cytosolic PolyA+ RNA, HeLa Cells

source_name: PolyA+ RNA from cytosol of HeLa Cells


title: RARecA ChIP from Retinoic Acid Stimulated HL60 Cells, 32 hours (NCBIv34)

type: genomic

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 RARecA 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, RARecA, HL60 Cells, 32 hours Retinoic Acid

molecule: genomic DNA

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 32 hours


title: P300 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBIv34)

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 P300 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, P300, HL60 Cells, 2 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 2 hours


title: PolyA+ RNA from Retinoic Acid Stimulated HL-60 Cells, 32 hours (ENCODE) (NCBIv34)

description:
HL-60 suspension cells were grown in Iscove's Modified Dulbecco's Medium (IMDM), GlutMAX; supplemented with 20% FBS, and 1% Penicillin-Streptomycin, at 37C 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.
Total RNA was extracted with an RNAeasy kit (Qiagen) using supplied reagents and following manufacturer?s protocol.  PolyA+ enriched fraction was obtained using Oligotex kit (Qiagen) following the manufacturer?s protocol.  PolyA+ fraction was treated with RNAse-free DNAse I (Roche), 1U/ug of RNA, for 20 min at 37C.
First-strand cDNA synthesis was achieved using SuperScript II reverse transcriptase (Invitrogen) in the reaction volume of 105 ul for 15 of starting RNA material or scaled accordingly using the following protocol. RNA was mixed with random hexamers (83.3 ng/ g of mRNA; Invitrogen) and the bacterial control transcripts (see below) and subjected to the following cycling conditions in PE GeneAmp9600 PCR System: 70?C- 10 min and 20min ramp to 15C after which the 5x Superscript II First Strand buffer (Life Technologies), DTT and four dNTPs were added to the following final concentrations of 1x, 10mM and 0.5mM, respectively, followed by a 30 min incubation at 15C. At this point, Superscript II RTase was added (200Units/ g of mRNA; Life Technologies) followed by a 20 min ramp to 42C and 60 min incubation at 42C. At this point the SuperScript II was inactivated by 15 min incubation at 75C.
The first strand cDNA synthesis reaction was used for the second-strand cDNA synthesis in the presence of 50U of E. coli DNA Ligase, 200U of E. coli DNA polymerase I, and 10U of E. coli RNAse H supplemented with 0.2mM dNTPs in the final volume of 750 ul at 16oC (all reagents were from Invitrogen) based on 15ug of starting RNA or scaled appropriately.  Upon treatment with RNAse H (Epicentre) and RNAse A/T1 (Ambion), double-stranded cDNA was extracted using QIAquick PCR purification kit (Qiagen) and 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 fragmented cDNA was then 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, PolyA+ RNA, HL-60 Cells, 32 hours Retinoic Acid


title: SIRT1 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBIv34)

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


title: P300 ChIP from Retinoic Acid Stimulated HL60 Cells, 32 hours (NCBIv34)

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 P300 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, P300, HL60 Cells, 32 hours Retinoic Acid


title: SIRT1 ChIP from Retinoic Acid Stimulated HL60 Cells, 0 hours (NCBIv34)

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, 0 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 0 hours


title: RARecA ChIP from Retinoic Acid Stimulated HL60 Cells, 8 hours (NCBIv34)

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 RARecA 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, RARecA, HL60 Cells, 8 hours Retinoic Acid

source_name: Whole Cell Extract (WCE) DNA from Retinoic Acid Stimulated HL60 Cells, 8 hours


title: TFIIB-R ChIP from Retinoic Acid Stimulated HL60 Cells, 32 hours (NCBIv34)

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 TFIIB-R 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, TFIIB-R, HL60 Cells, 32 hours Retinoic Acid


title: RARecA ChIP from Retinoic Acid Stimulated HL60 Cells, 0 hours (NCBIv34)

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 RARecA 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, RARecA, HL60 Cells, 0 hours Retinoic Acid


title: Pol2 ChIP from Retinoic Acid Stimulated HL60 Cells, 8 hours (NCBIv34)

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 Pol2 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, Pol2, HL60 Cells, 8 hours Retinoic Acid


title: PU1 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBIv34)

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 PU1 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, PU1, HL60 Cells, 2 hours Retinoic Acid


title: Pol2 ChIP from Retinoic Acid Stimulated HL60 Cells, 0 hours (NCBIv34)

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 Pol2 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, Pol2, HL60 Cells, 0 hours Retinoic Acid


title: PU1 ChIP from Retinoic Acid Stimulated HL60 Cells, 0 hours (NCBIv34)

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 PU1 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, PU1, HL60 Cells, 0 hours Retinoic Acid


title: PU1 ChIP from Retinoic Acid Stimulated HL60 Cells, 8 hours (NCBIv34)

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 PU1 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, PU1, HL60 Cells, 8 hours Retinoic Acid


title: Pol2 ChIP from Retinoic Acid Stimulated HL60 Cells, 2 hours (NCBIv34)

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 Pol2 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, Pol2, HL60 Cells, 2 hours Retinoic Acid