title: Human STAT1 ChIP-chip on Hela S3 Biological Sample 3 (Maskless Photolithographic 50mer ENCODE Array)

status: Public on May 27 2005

city: New Haven

first: Joel

last: Rozowsky

state: CT

address: 266 Whitney Ave, Bass 437

department: Molecular Biophysics & Biochemistry

institute: Yale University

description:
HeLa S3 cells were grown in suspension in Minimum Essential Medium modified for Suspension (S-MEM), supplemented with 10% FBS, 1% Antibiotic-Antimycotic and 2 mM L-glutamine (all medium and supplements from Invitrogen), at 37C in 5% CO2, to a density of 6 x10^5 cells/ml. Per IP, 2x10^8 cells were either induced with 5 ng/ml of human recombinant interferon gamma (R&D Systems) for 30 min or left untreated and then fixed with 1% formaldehyde at room temperature for 10 min. Fixation was terminated with 125 mM glycine (2 M stock made in 1x PBS). Cells were washed twice in cold 1x DulbeccoÕs PBS (Invitrogen), and then swelled on ice in 10 ml hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, 0.1 mM sodium orthovanadate, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 10 ml of 1x RIPA buffer per 2 x 10^8 cells (1x RIPA buffer: 10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, 0.1 mM sodium orthovanadate, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (power setting 2, 100% duty cycle, three 30-second pulses) to an average of 500 bp as verified on a 1.5% agarose gel. Clarified lysates were agitated overnight at 4C with 15 ug of anti-STAT1 alpha p91 (C-24) rabbit polyclonal antibody (Santa Cruz sc-345) per 1x10^8 cells. Following this incubation Protein A agarose (Upstate) was added and allowed to bind for 60 min at 4C. Immunoprecipitates were washed three times in 1x RIPA, once in 1x PBS, and then eluted in 1xTE/1%SDS. Crosslinks were reversed overnight at 65C. ChIP DNA was purified by incubation with 200 ug/ml Proteinase K for 2 h at 45C, phenol:chloroform:isoamyl alcohol extraction, and precipitation with 0.1 volumes of 3M sodium acetate, and 2 volumes of 100% ethanol.
For Maskless arrays, 8 ug of ChIP DNA (as measured by Nanodrop spectrophotomer) were random primed with Klenow (enzyme and primers from BioPrime DNA Labeling System, Invitrogen) and aminoallyl-dUTP (Sigma) was incorporated. Subsequently Alexa Fluor dyes (Invitrogen; Alexa 647, ChIP DNA isolated from interferon gamma-stimulated cells and Alexa 555, ChIP DNA isolated from unstimulated cells) were coupled to the aminoallyl-dUTP.  Coupling reactions were terminated with hydroxylamine. Alexa 555 and Alexa 647 coupled ChIP DNA samples were combined and recovered using a CyScribe GFX Purification Kit (Amersham) according to the manufacturerÕs protocol. Labeled ChIP DNA was precipitated with 30 ug of COT1 DNA (Invitrogen) per array and pellets were resuspended in 45 ul of 1x hybridization buffer (40% formamide, 5x SSC, 0.1% SDS, and 0.2x TE). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature NSWB (6x SSPE, 0.01% Tween-20, 1 mM DTT), 0.2x SSC, and 0.05x SSC. Arrays were scanned on an Axon 4200A scanner.
Keywords = Transcription Factor Binding, STAT1, ChIP-chip, Human, Genome Tiling Arrays

source_name: STAT1 ChIP from Interferon gamma Stimulated HeLa S3 Cells, 30 min

source_name: STAT1 ChIP from Unstimulated HeLa S3 Cells


title: Human STAT1 ChIP-chip on Hela S3 Biological Sample 2 (Maskless Photolithographic 50mer ENCODE Array)

type: genomic

country: USA

molecule: genomic DNA

organism: Homo sapiens


title: M211 E- TN 2nd rep

status: Public on Jan 28 2005

type: RNA

city: Columbia

first: Sachin

middle: S

last: Bhusari

state: MO

address: 920 East Campus Drive

department: Animal Sciences

institute: Univ of Missouri

description:
M211 mouse belongs to mice group fed endophyte-free fescue diet (E-). This is second technical replicate of this sample.
Keywords = Endophyte
Keywords = fescue
Keywords = Thermonetrality (TN).

scan_protocol: with Axon genepix scanner.

data_processing: log2(ch1/ch2) normalized using plant control genes.

molecule: total RNA

organism: Mus musculus

source_name: M211 fed E- diet at TN

label_protocol: indirect amino-allyl labeling.

extract_protocol: RNAquious-Midi, Ambion inc, Austin, Tx

treatment_protocol: Fed E- control diet at thermoneutrality.

tag: ICR strain, Male, 27d old, liver.

source_name: universal mouse reference RNA

tag: RNA extracted from 11 different mouse cell lines.


title: Human STAT1 ChIP-chip S3 on Hela Biological Sample 4 (Nimblegen ENCODE Array)

description:
HeLa S3 cells were grown in suspension in Minimum Essential Medium modified for Suspension (S-MEM), supplemented with 10% FBS, 1% Antibiotic-Antimycotic and 2 mM L-glutamine (all medium and supplements from Invitrogen), at 37C in 5% CO2, to a density of 6 x10^5 cells/ml. Per IP, 2x10^8 cells were either induced with 5 ng/ml of human recombinant interferon gamma (R&D Systems) for 30 min or left untreated and then fixed with 1% formaldehyde at room temperature for 10 min. Fixation was terminated with 125 mM glycine (2 M stock made in 1x PBS). Cells were washed twice in cold 1x DulbeccoÕs PBS (Invitrogen), and then swelled on ice in 10 ml hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, 0.1 mM sodium orthovanadate, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 10 ml of 1x RIPA buffer per 2 x 10^8 cells (1x RIPA buffer: 10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, 0.1 mM sodium orthovanadate, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (power setting 2, 100% duty cycle, three 30-second pulses) to an average of 500 bp as verified on a 1.5% agarose gel. Clarified lysates were agitated overnight at 4C with 15 ug of anti-STAT1 alpha p91 (C-24) rabbit polyclonal antibody (Santa Cruz sc-345) per 1x10^8 cells. Following this incubation Protein A agarose (Upstate) was added and allowed to bind for 60 min at 4C. Immunoprecipitates were washed three times in 1x RIPA, once in 1x PBS, and then eluted in 1xTE/1%SDS. Crosslinks were reversed overnight at 65C. ChIP DNA was purified by incubation with 200 ug/ml Proteinase K for 2 h at 45C, phenol:chloroform:isoamyl alcohol extraction, and precipitation with 0.1 volumes of 3M sodium acetate, and 2 volumes of 100% ethanol.
For NimbleGen arrays, 16 ug (as measured by Nanodrop spectrophotomer) were directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers (ChIP DNA isolated from interferon gamma-stimulated cells) or Cy3 nonamers (ChIP DNA isolated from unstimulated cells). The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and then hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, and 0.1 ug/ul herring sperm DNA. Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Arrays were scanned on an Axon 4000B scanner.
Keywords = Transcription Factor Binding, STAT1, ChIP-chip, Human, Genome Tiling Arrays


title: Human STAT1 ChIP-chip S3 on Hela Biological Sample 2 (Maskless Photolithographic 36mer ENCODE Array)


title: Human STAT1 ChIP-chip S3 on Hela Biological Sample 3 (Nimblegen ENCODE Array)


title: Human STAT1 ChIP-chip on Hela S3 Biological Sample 1 (Maskless Photolithographic 50mer ENCODE Array)


title: Human STAT1 ChIP-chip S3 on Hela Biological Sample 1 (Nimblegen ENCODE Array)


title: YoungOld Calvaria2

status: Public on Jul 09 2005

city: Stanford

first: Oliver

last: Aalami

state: CA

address: 257 Campus Dr.

institute: Stanford University

description:
Simple annotation: Development, Aging
Image: http://smd.stanford.edu/MicroArray/gifs/2002-11/35174.gif; format: GIF

data_processing: VALUE is Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)

source_name: adult calvaria

tag: adult calvaria

source_name: young calvaria

tag: young calvaria


title: Human STAT1 ChIP-chip S3 on Hela Biological Sample 1 (Maskless Photolithographic 36mer ENCODE Array)


title: Human STAT1 ChIP-chip S3 on Hela Biological Sample 5 (Nimblegen ENCODE Array)


title: Human STAT1 ChIP-chip S3 on Hela Biological Sample 2 (Nimblegen ENCODE Array)