|
| Status |
Public on Dec 08, 2010 |
| Title |
Early_S-phase_Hela_cells_exp1_hyb1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Early S-phase Hela cellular DNA
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
tissue type: cervix, epithelial, adenocarcinoma
cell line: Hela
genomic dna: Replication Initiating genomic DNA
|
| Treatment protocol |
The earlytrap data set was generated from HeLa cells that were synchronized by treating with 2 mM thymidine for 14 hr to collect the population in S-phase or at the G1/S boundary, and then were released into drug-free medium for 9 hr to allow the culture to clear S-phase. Mimosine (200 uM) was added for 13 hr to collect the population at or near the G1/S border, followed by a wash with serum free media and transfer into drug-free medium. Eighty min later, cells were harvested and replication intermediates prepared.
|
| Growth protocol |
HeLa cells (earlytrap and bubbletrap data sets) were obtained from the American Type Culture Collection and were maintained as monolayer cultures in 5% CO2 on Minimal Essential Medium supplemented with 10% Fetal Clone II (Hyclone), 1% Glutamax and 0.1% Gentamicin (both from Invitrogen) . GM06990 cells (gmtrap data set) were obtained from Coriell Institute for Medical Research and were maintained as suspension cultures in 5%CO2 in flasks in RPMI 1640 media supplemented with 15% Fetal Clone II, 1% Glutamax and 0.1% Gentamicin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
GM06990 cells were harvested when the culture reached a cell density of ~5X10(5) cells per ml (~10(10) cells per library) by centrifugation at 500Xg for 8m. Hela log-phase or synchronized cell populations (~5X10(9) cells for each library) were scraped from plates, and replication intermediates (RIs; from both cell types) were partially purified by preparing nuclear matrix/DNA halo structures exactly as described in (Mesner et al.(2009b), Methods Mol. Biol. 521, 121-137) and digesting the DNA with EcoRI. The matrix-affixed DNA was isolated and RIs further purified by adsorption to BND-cellulose and subsequent elution with caffeine. Bubble-containing EcoRI fragments were isolated exactly as described in (Mesner and Hamlin(2009), Methods Mol. Biol. 521, 315-328 and Mesner et al (2006), Mol. Cell 21, 719-726). Briefly, RI preparations were mixed with low-melting agarose, the molten mixture was drawn up into a 1ml syringe, without the needle, and allowed to congeal. The resulting cylindrical plug was extruded into an electrophoresis tank after excising the syringe tip. After exhaustive electrophoresis to get rid of linear, single-forked, and X-shaped fragments, the trapped bubble-containing fragments were isolated by agarase digestion and purified twice by ethanol precipitation. Twenty percent of the sample was used for 2D-analysis and the remainder cloned into the EcoRI site of pGem7 (Promega). After transformation into E.coli strain DH10B, library clones were obtained after less than one cell division in liquid medium to avoid the propagation of sibs, and were spread onto LB-agarose plates and allowed ~10h until colonies appeared as pin pricks to the unaided eye. They were then scraped, pooled, aliquoted, and frozen at –80oC. Approximately 10(6) individual clones were obtained for each of the 4 libraries in which >95% contained vector inserts.
|
| Label |
biotin
|
| Label protocol |
Bubble trap and genomic DNA from HeLa cells were fragmented to 50-100bp size using DNaseI. The fragmented DNA was labeled either with biotin-dATP using random priming (rp) or biotin-ddATP (tt) using terminal tranferase.
|
| |
|
| Channel 2 |
| Source name |
control DNA
|
| Organism |
Homo sapiens |
| Characteristics |
control: control DNA
|
| Growth protocol |
HeLa cells (earlytrap and bubbletrap data sets) were obtained from the American Type Culture Collection and were maintained as monolayer cultures in 5% CO2 on Minimal Essential Medium supplemented with 10% Fetal Clone II (Hyclone), 1% Glutamax and 0.1% Gentamicin (both from Invitrogen) . GM06990 cells (gmtrap data set) were obtained from Coriell Institute for Medical Research and were maintained as suspension cultures in 5%CO2 in flasks in RPMI 1640 media supplemented with 15% Fetal Clone II, 1% Glutamax and 0.1% Gentamicin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
GM06990 cells were harvested when the culture reached a cell density of ~5X10(5) cells per ml (~10(10) cells per library) by centrifugation at 500Xg for 8m. Hela log-phase or synchronized cell populations (~5X10(9) cells for each library) were scraped from plates, and replication intermediates (RIs; from both cell types) were partially purified by preparing nuclear matrix/DNA halo structures exactly as described in (Mesner et al.(2009b), Methods Mol. Biol. 521, 121-137) and digesting the DNA with EcoRI. The matrix-affixed DNA was isolated and RIs further purified by adsorption to BND-cellulose and subsequent elution with caffeine. Bubble-containing EcoRI fragments were isolated exactly as described in (Mesner and Hamlin(2009), Methods Mol. Biol. 521, 315-328 and Mesner et al (2006), Mol. Cell 21, 719-726). Briefly, RI preparations were mixed with low-melting agarose, the molten mixture was drawn up into a 1ml syringe, without the needle, and allowed to congeal. The resulting cylindrical plug was extruded into an electrophoresis tank after excising the syringe tip. After exhaustive electrophoresis to get rid of linear, single-forked, and X-shaped fragments, the trapped bubble-containing fragments were isolated by agarase digestion and purified twice by ethanol precipitation. Twenty percent of the sample was used for 2D-analysis and the remainder cloned into the EcoRI site of pGem7 (Promega). After transformation into E.coli strain DH10B, library clones were obtained after less than one cell division in liquid medium to avoid the propagation of sibs, and were spread onto LB-agarose plates and allowed ~10h until colonies appeared as pin pricks to the unaided eye. They were then scraped, pooled, aliquoted, and frozen at –80oC. Approximately 10(6) individual clones were obtained for each of the 4 libraries in which >95% contained vector inserts.
|
| Label |
biotin
|
| Label protocol |
Bubble trap and genomic DNA from HeLa cells were fragmented to 50-100bp size using DNaseI. The fragmented DNA was labeled either with biotin-dATP using random priming (rp) or biotin-ddATP (tt) using terminal tranferase.
|
| |
|
| |
| Hybridization protocol |
The fragmented and labeled DNA was hybridized to ENCODE01-Forward tiling arrays (P/N 900543) All the array hybridizations were done at 45°C for 16 hours at 60rpm using an Affymetrix hybridization oven
|
| Scan protocol |
Each microarray was scanned and analyzed for signal intensities using the ChIP® Scanner 3000 and GeneChIP Operating Software (GCOS) from Affymetrix.
|
| Description |
Hela Early S-phase Replication Origins
|
| Data processing |
Data was quantile normalized and analyzed using the Affymetrix Tiling Array Software (TAS) or Tilecore. Log2(Bubble Trap/Ctrl DNA) fold changes were generated in separate BAR files using a two-sample Wilcoxon test over a sliding window with a bandwidth of 500bp (Note: window size = 2*Bandwidth + 1).
results file descriptions: *.bar were generated by the TAS software. *.bar files for the Bubble Trap assay in HeLa and GM06990 cells contain log2(Bubble Trap/Ctrl DNA) fold changes for each probe. The data is the center probe within a sliding window (500bp bandwidth) and the corresponding Hodges-Lehmann estimator calculated using all the probes within the sliding window. *.sgr are text file versions of the binary *.bar files.
|
| |
|
| Submission date |
Mar 29, 2010 |
| Last update date |
Dec 08, 2010 |
| Contact name |
Stefan Bekiranov |
| E-mail(s) |
sb3de@virginia.ed
|
| Organization name |
University of Virginia
|
| Department |
Biochemistry & Molecular Genetics
|
| Lab |
Bekiranov
|
| Street address |
1340 Jefferson Park Avenue
|
| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22908 |
| Country |
USA |
| |
|
| Platform ID |
GPL3725 |
| Series (1) |
| GSE21110 |
Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription |
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