title: Sample 10632, Slide 32010_E

description:
Sample collected and immediately mixed and stored with RNAlater.
All procedures were followed according to manufacturer's protocols

tag: Sample 10632, Slide 32010_E, Male, Captive


title: Genome Input G0 phase Replicate 2

status: Public on Apr 17 2007

type: genomic

city: Beijing

first: Xiaopeng

last: Zhu

address: 15 Datun Street

country: China

institute: Institute of Biophysics, Chinese Academy of Sciences

description: Input chromatin from G0-arrested T98G cells, replicate 2 out of 3

hyb_protocol: Affymetrix fluidics protocol EukGE-WS2v4_450

scan_protocol: Scanning was carried out on an Affymetrix scanner GCS3000.  The scan protocol was contained in a .cif file for chip type Hs_PromPR_v02.

data_processing:
Affymetrix GCOS processed image files to generate .CEL files.
MAT (Model-based Analysis for Tiling Arrays) analysis algorithm used to detect regions enriched by chromatin immunoprecipitation (ChIP) on Affymetrix Human Promoter Array 1.0R (ChIP-chip).

label: biotin

molecule: genomic DNA

organism: Homo sapiens

source_name: Input chromatin from T98G cells

label_protocol:
The chromatin immunprecipitated DNA (ChIP) or 2 ng of whole genomic DNA (Input) were amplified by ligation-mediated PCR, digested with Dnase I to give fragments of ~50 bp.
The DNA fragments were end-labelled with biotin in a TdT-catalyzed reaction.

growth_protocol: Human T98G cells grown till confluency in DMEM supplemeted with 10 % fetal calf serum

extract_protocol: Chromatin was prepared from formalin-fixated cells exactly as described in Rayman, Takahashi et al. (2002) Genes Dev 16, 933-947

treatment_protocol: Cells were incubated in serum-free medium for 3 days to induce G0 arrest

tag: human glioblastoma cell line T98G CRL-1690


title: p130 G0 phase Replicate 2

description: Chromatin from G0-arrested T98G cells immunoprecipitated with anti-p130 antibody, replicate 2 out of 3

source_name: p130 ChIP from G0 arrested T98G cells


title: E2F4 G0 phase Replicate 1

description: Chromatin from G0-arrested T98G cells immunoprecipitated with anti-E2F4 antibody, replicate 1 out of 3

source_name: E2F4 ChIP from G0 arrested T98G cells


title: LIN9 G0 phase Replicate 1

description: Chromatin from G0-arrested T98G cells immunoprecipitated with anti-LIN9 antibody, replicate 1 out of 3

source_name: LIN9 ChIP from G0 arrested T98G cells


title: p130 G0 phase Replicate 1

description: Chromatin from G0-arrested T98G cells immunoprecipitated with anti-p130 antibody, replicate 1 out of 3


title: p130 G0 phase Replicate 3

description: Chromatin from G0-arrested T98G cells immunoprecipitated with anti-p130 antibody, replicate 3 out of 3


title: Genome Input G0 phase Replicate 3

description: Input chromatin from G0-arrested T98G cells, replicate 3 out of 3


title: E2F4 G0 phase Replicate 3

description: Chromatin from G0-arrested T98G cells immunoprecipitated with anti-E2F4 antibody, replicate 3 out of 3


title: Sample 111080, Slide 32010_C

status: Public on Jun 13 2007

type: RNA

city: Raleigh

first: Erin

last: Kennerly

state: NC

country: USA

institute: NCSU

description:
All samples collected in wild and immediately mixed with RNAlater to perserve RNA integrity
All procedures were followed according to manufacturer's protocols

hyb_protocol: Arrays were hybridized overnight following Illumina's Ref-8 hybridization protocol

scan_protocol: Arrays were scanned using Illumina's Beadstation Scanner on the Default settings

data_processing: Raw data extracted using Illumina Beadstudio version 1.5.0.34. Normalized VALUEs represent median standardized log base 2 signal intensities.

label: Biotin

molecule: total RNA

organism: Canis rufus

source_name: Whole blood

label_protocol: Total RNA was biotin labeled using Ambion's Illumina RNA Amplification Kit

extract_protocol: Whole Blood stored in RNALater was extracted using Ambion's RiboPure Blood RNA extraction KitWhole Blood stored in RNALater was extracted using Ambion's RiboPure Blood RNA extraction Kit

tag: Sample 111080, Slide 32010_C, Male, Free Range


title: Genome Input G0 phase Replicate 1

description: Input chromatin from G0-arrested T98G cells, replicate 1 out of 3

data_processing:
Affymetrix GCOS processed image files to generate .CEL files.
MAT (Model-based Analysis for Tiling Arrays) analysis algorithm used to detect regions enriched by chromatin immunoprecipitation (ChIP) on Affymetrix tiling array (ChIP-chip).


title: Sample 11136, Slide 32010_B

description:
All samples collected in the wild and immediately perserved with RNA later.
All procedures were followed according to manufacturer's protocols

source_name: Whole Blood

tag: Sample 11136, Slide 32010_B, Male, Free Range


title: Sample 10406, Slide 32010_A

description: All procedures were followed according to manufacturer's protocols

tag: Sample 10406, Slide 32010_A, Male, Captive


title: Sample 10537, Slide 32011_G

description:
Whole blood was collected in EDTA tubes and immediately mixed and stored in RNAlater.
All protocols were followed according to manufacturer's instructions

data_processing: Raw values were extracted from Array image using Illumina's BeadStudio Software version 1.5.0.34. Normalized VALUEs represent median standardized log base 2 signal intensities.

tag: Sample 10537, Slide 32011_G, Male, Captive


title: HeLa_RNA_rep 1

status: Public on Oct 11 2006

city: PROVIDENCE

first: Alexander

last: Brodsky

state: RI

address: 593 Eddy St.

department: MCB

institute: Rhode Island Hospital/Brown University

description: RNA samples were isolated from HeLa S3 cells and purified with trizol (Invitrogen) and RNeasy (Qiagen).double-stranded cDNA was generated using Invitrogen Superscript cDNA synthesis kit. cDNA (1-1.5 ug) was hybridized to the tiled array. Three biological replicates were performed for each RNA array.

data_processing: To take into account probe-to-probe variability we used a generalization of the Wilcoxon signed-rank test for blocked data to generate p-values. All input and IP normalized, sign(PM-MM)max(1,|PMMM|) intensities (where PM are perfect match and MM are mismatched probes) interrogating the same chromosomal location were assigned to the same block.  Probes were mapped to the genomic coordinates to ensure that no probe mapped to more than one location in any 1,000-bp window and that no two probes map to the same genomic location.

source_name: HeLa_RNA_rep 1

tag: HeLa_RNA_rep 1


title: Input_rep1

description: DNA was isolated from chromatin immunoprecipitation.  Briefly, HeLa S3 cells were first crosslinked with dimethyl adipimidate (DMA) (Pierce) for 10 min, washed with PBS and then crosslinked with formaldehyde for 10 min.  Briefly, 8WG16 (Covance) and 4H8 (AbCam) antibodies wereincubated with a 50:50 mix of Dynal protein A/G beads for more than 16 h at 4°C in PBS with 5 mg/ml BSA. After washing in PBS, beads with bound antibody were incubated with chromatin from approximately 2 × 107 cells for more than 16 h at 4°C. Beads were washed eight times with RIPA buffer (50mM HEPES pH 7.6, 1 mM EDTA, 0.7% DOC, 1% IGEPAL, 0.5M LiCl) before DNA was eluted at 65°C in TE/1% SDS.  Crosslinks were reversed by incubating at 65°C for more than 12 h followed by proteinase K treatment, phenol extraction and RNase treatment. Isolated DNA was then amplified isothermally using random nonamer primers and Klenow polymerase (Invitrogen) for more than 4 h, yielding approximately 2 ?g of DNA per IP. DNA was prepared and hybridized on Affymetrix ENCODE oligonucleotide tiled arrays using the fragmentation, hybridization, staining and scanning procedure described by Kennedy et al. [24]. Affymetrix ENCODE microarrays have interrogating 25mer oligonucleotide probes tiled every 20 bp on average. A sample of chromatin was set aside before IP and used to represent the input DNA.

source_name: HeLa S3 Input_rep1

tag: HeLa S3 Input_rep1


title: Input_rep5

source_name: HeLa S3 Input_rep5

tag: HeLa S3 Input_rep5


title: HeLa_RNA_rep 3

source_name: HeLa_RNA_rep 3

tag: HeLa_RNA_rep 3


title: HeLa_RNA_rep 2

source_name: HeLa_RNA_rep 2

tag: HeLa_RNA_rep 2


title: Input_rep2

source_name: HeLa S3 Input_rep2

tag: HeLa S3 Input_rep2


title: Input_rep4

source_name: HeLa S3 Input_rep4

tag: HeLa S3 Input_rep4


title: Input_rep3

source_name: HeLa S3 Input_rep3

tag: HeLa S3 Input_rep3


title: Input_rep6

source_name: HeLa S3 Input_rep6

tag: HeLa S3 Input_rep6


title: PolIIa_rep2

source_name: HeLa S3 PolIIa IP

tag: HeLa S3 PolIIa IP


title: PolII_rep1

source_name: HeLa S3 PolII IP

tag: HeLa S3 PolII IP


title: PolII_rep2


title: PolII_rep3


title: PolIIa_rep3


title: Slide 2

status: Public on Feb 01 2008

city: Cambridge

first: Elizabeth

last: Herbolsheimer

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute

description: Array #2

hyb_protocol: Labeled DNA was combined (5 - 6 ug each of immunoenriched and whole cell extract DNA) and hybridized to arrays in Agilent hybridization chambers for 40 hours at 40C.

scan_protocol: Slides were scanned using an Agilent DNA microarray scanner BA. PMT settings were set manually to normalize bulk signal in the Cy3 and Cy5 channel.

data_processing: We used thirteen different algorithms to make predictions of enriched regions from the array measurements. While most of the algorithms function only on a single platform, we used two algorithms (MA2C21, Splitter22) for multiple platforms. To standardize the results across algorithms, we required that each prediction consist of a rank-ordered list of predicted spike-in regions, with each region represented by a single chromosome coordinate and a quantitative value that corresponded to a predicted enrichment level. We considered a region to be predicted correctly if the single predicted coordinate was within the spike-in region. Because the total number of spike-ins was unknown to the predictors, each predictor was also asked to estimate a cutoff score, above which the selected predictions were considered significant. We then used the spike-in key to assess the performance of each microarray platform, amplification method, and analysis algorithm .

source_name: Spike-in Controls

label_protocol: Amplified DNA was labeled and purified using Invitrogen Bioprime random primer labeling kits; immunoenriched DNA was labeled with Cy5 fluorophore.

extract_protocol: I. Preblock and binding of antibody to magnetic beads 1. wash 100 µL Dynal magnetic beads (per reaction) in 1 ml fresh BSA/PBS 2. collect the beads by spinning at 3000 RPM for 3 minutes. 3. wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet. 4. Add 6-10 µg of Ab +250µL of PBS/BSA. 5. incubate 4hr to O.N. on a rotating platform at 4∞C. 6. wash beads 3 times in 1.5 Ml PBS/BSA. 7. Resuspend in 100µL PBS/BSA. II. Chromatin Immunoprecipitation 1. Add 100 µL Ab prebound Dynal magnetic beads from step I to cell lysate from prior protocol. 2. Rock 4°C O/N. III. Washing, eluting, and reverse cross-linking 1. Transfer to centrifuge tubes, continue working in the cold room until step 6. 2. Use the magnetic stand to precipitate the beads. 3. Wash 4-8 times with 1 mL wash buffer 4. Wash once with 1 ml TE-plus-50 mM NaCl 5. Spin 3k for 2-3 min and aspirate any residual TE buffer. 6. Add 100 µl of elution buffer. 7. Elute at 65∞C for 10-15 min with brief vortexing every 2 min. 8. Spin down beads 14k for 1 min. 9. Remove all 100 µl of supernatant. 10. Reverse x-link O/N 65∞C. 11. Thaw input, add 3 vol of elution buffer and reverse x-link O/N 65∞C. IV. RNase, Proteinase K 1. Add 1 vol of TE to IP and input fraction. 2. Add RNase A so final is 0.2µg/µL (~5 µL/250 µL rxn). Incubate 37∞C 1-2hr. 3. Add proteinase K so final is 0.2µg/µL (~2.5 µL/250 µL rxn). Incubate 55∞C 2hr. 4. Extract once w/ 1 vol of phenol. 5. Extract once w/ 1 vol of phenol:chl:IA (made by mixing 1 vol of phenol w/ 1 vol of Chloroform:isoamyl alcohol). 6. extract once w/ 1 vol of chl:IA. 4-6. Or instead extract 1x w/ 1 vol phenol:chl:IA using phaselock tubes 7. add 30 µg (1.5 µL) of glycogen. 8. Add 5M NaCl so final is 0.2M (10 µL/250 µL rxn). 9. Add 2 vol of EtOH and incubate -80∞C 30 min. 10. Spin and wash with 500 µL 75% EtOH. 11. Dry and resuspend pellets in 60 µL 10mM Tris HCl pH 8. Save 5 µL of IP sample for checkpoints to the right. Normalize the input/wce fraction to 100 ng/µL using the Nanodrop. Wash buffer (RIPA buffer) 100ml: final concentration 5ml of 1M Hepes (pH 7.6) 50 mM 200µL of 0.5M EDTA 1 mM 7 ml of 10% DOC (Na deoxycholate) 0.7% 10 ml of 10% NP-40 (IPGEL) 1% 10 ml of 5M LiCl or 2.12g powder 0.5 M Elution buffer: 50mM Tris pH8 10mM EDTA 1% SDS

tag: To create our simulated ChIP spike-in mixture, we first randomly selected 100 cloned genomic DNA sequences (average length 497 bp) corresponding to predicted promoters in the human genome.

source_name: Whole Cell Extract

label_protocol: Amplified DNA was labeled and purified using Invitrogen Bioprime random primer labeling kits; whole cell extract DNA was labeled with Cy3 fluorophore.

tag: Genomic DNA


title: PolIIa_rep1


title: Slide 1

description: Array #1

source_name: Control Spike in data

tag: To create our simulated ChIP spike-in mixture, we first randomly selected 100 cloned genomic DNA sequences (average length 497 bp) corresponding to predicted promoters in the human  genome

tag: whole cell extract DNA


title: Slide 3

description: Array #3

source_name: Spike-in Control Testing


title: Nalm29 t(9;22) #2

status: Public on Dec 21 2005

city: Stanford

last: Stanford Microarray Database

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

description:
Simple annotation: acute lymphoblastic leukemia, cell line
Image: http://smd.stanford.edu/MicroArray/gifs/2001-01/12635.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: Common Reference F

tag: Common Reference F


title: Slide 4

description: Array #4

source_name: Spike-in Control