|
| Status |
Public on Mar 05, 2009 |
| Title |
300-800 nt nascent strands, hyb2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
embryonic stem cells, nascent strands
|
| Organism |
Mus musculus |
| Characteristics |
cell type: female pluripotent cells
dna type: nascent strands
|
| Treatment protocol |
No treatment was applied
|
| Growth protocol |
The mouse embryonic stem cell line PGK12.1 was grown as described in Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N (1996; Requirement for Xist in X chromosome inactivation. Nature 379: 131-137). Mouse embryonic fibroblasts (MEFs) were derived from 12.5 dpc CD1 embryos and grown in F12 Nutrient Mixture (Ham) medium supplemented with 10% FCS, 1x105 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L- lutamine, 1x non-essential amino acids, and 50 M beta-mercaptoethanol (Invitrogen). NIH/3T3 cells were cultivated as recommended in the ATCC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA isolation and nascent strands fractionation was performed as described in Gómez M, Antequera F (2008; Overreplication of short DNA regions during S phase in human cells. Genes & Development 22: 375-385)
|
| Label |
Cy3
|
| Label protocol |
The DNA samples hybridised in the arrays were labelled as described on the protocol published by Agilent (Agilent_Technologies, 2005). Briefly, each sample (nascent strands or control) resuspended in 21 μl H2O was mixed with 20 μl of 2.5x Random primer buffer (BioPrime DNA Labeling System, Invitrogen) and incubated at 95º C for 5 minutes. This mix was transferred immediately to ice and the following reagents added: dNTPs (2 mM each of dATP, dGTP and dTTP and 0.5 mM dCTP), 3.75 μl Cy 3-dCTP or Cy 5-dCTP (Amersham), and 20 U Klenow enzyme. The reactions were incubated at 37ºC for 3 hours, an extra 10 U Klenow enzyme added and then incubated for further 2 hours. Corresponding nascent strands and control samples were pooled, diluted in 400 μl of TE pH 8.0 and purified on a MicroCon YM-30 filter column (Millipore) to remove the unincorporated label, following manufacturer’s instructions. The labelled DNA was transferred to a fresh 1.5 ml tube and brought to a final volume of 150 μl with nuclease-free water and stored in the dark at -20ºC until hybridisation.
|
| |
|
| Channel 2 |
| Source name |
embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: female pluripotent cells
dna type: total DNA
|
| Treatment protocol |
No treatment was applied
|
| Growth protocol |
The mouse embryonic stem cell line PGK12.1 was grown as described in Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N (1996; Requirement for Xist in X chromosome inactivation. Nature 379: 131-137). Mouse embryonic fibroblasts (MEFs) were derived from 12.5 dpc CD1 embryos and grown in F12 Nutrient Mixture (Ham) medium supplemented with 10% FCS, 1x105 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L- lutamine, 1x non-essential amino acids, and 50 M beta-mercaptoethanol (Invitrogen). NIH/3T3 cells were cultivated as recommended in the ATCC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA isolation and nascent strands fractionation was performed as described in Gómez M, Antequera F (2008; Overreplication of short DNA regions during S phase in human cells. Genes & Development 22: 375-385)
|
| Label |
Cy5
|
| Label protocol |
The DNA samples hybridised in the arrays were labelled as described on the protocol published by Agilent (Agilent_Technologies, 2005). Briefly, each sample (nascent strands or control) resuspended in 21 μl H2O was mixed with 20 μl of 2.5x Random primer buffer (BioPrime DNA Labeling System, Invitrogen) and incubated at 95º C for 5 minutes. This mix was transferred immediately to ice and the following reagents added: dNTPs (2 mM each of dATP, dGTP and dTTP and 0.5 mM dCTP), 3.75 μl Cy 3-dCTP or Cy 5-dCTP (Amersham), and 20 U Klenow enzyme. The reactions were incubated at 37ºC for 3 hours, an extra 10 U Klenow enzyme added and then incubated for further 2 hours. Corresponding nascent strands and control samples were pooled, diluted in 400 μl of TE pH 8.0 and purified on a MicroCon YM-30 filter column (Millipore) to remove the unincorporated label, following manufacturer’s instructions. The labelled DNA was transferred to a fresh 1.5 ml tube and brought to a final volume of 150 μl with nuclease-free water and stored in the dark at -20ºC until hybridisation.
|
| |
|
| |
| Hybridization protocol |
The labelled DNA was denatured at 95º C for 3 minutes, transferred to ice, mixed with 100 μl H20 and 250 μl 2x Hybridisation buffer (Agilent) and loaded onto a fresh array slide placed into a Agilent SureHyb chamber base. The chamber was sealed and slides hybridised at 65º C for 40 h at 10 rpm. Prior to scanning the slides were washed 5 minutes at room temperature in Wash Buffer 1 and then 5 minutes at room temperature in Wash Buffer 2 (OGT).
|
| Scan protocol |
All labelled samples were sent to Oxford Gene Technology facilities (OGT, Oxford, UK), using the Agilent platform and protocols for the hybridisation and scanning of the arrays.
Data was scanned at 100% and 50% PMT and the 100% PMT data feature extracted using local background and spatial detrending to account for differences in hybridisation intensities across the array. Poor quality features were discarded, as were those with very high signal intensities, following standard Agilent protocols.
|
| Description |
300-800 nt nascent strands, hyb2
|
| Data processing |
Raw datasets from each experiment were loess normalised to remove signal intensity-dependent bias using GeneSpringX software (Agilent).
|
| |
|
| Submission date |
Mar 03, 2009 |
| Last update date |
Mar 04, 2009 |
| Contact name |
Ramon Diaz-Uriarte |
| E-mail(s) |
rdiaz02@gmail.com
|
| Phone |
+34-91-732-8000
|
| Organization name |
Spanish National Cancer Research Centre (CNIO)
|
| Department |
Structural Biology and Biocomputing Programme
|
| Street address |
Melchor Fernandez Almagro 3
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL8246 |
| Series (1) |
| GSE15082 |
Transcription Initiation Activity Sets Replication Origin Efficiency in Mammalian Cells |
|
