sample type: isolated, non amplified, nucleolar DNA cell line: IMR90
Treatment protocol
Nucleolus-associated DNA was isolated as discribed below
Growth protocol
5% CO2, 37°C, 10% FCS, DMEM, 1g/L Glucose
Extracted molecule
genomic DNA
Extraction protocol
1x10^8 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. After washing cells 2 times with ice-cold PBS they were scraped off and pooled in 100 mL 1xPBS and centrifuged (750 g, 5 min, 4°C). Supernatant was discarded or used for DNA precipitation and cells were resuspended in 1 mL HMg buffer (10 mM Hepes pH 7.4, 0.88 M sucrose, 12 mM MgCl2, 1 mM TCEP, PI). Cells were sonicated on ice for 3x10 sec with fine tip (Branson sonifier, 65% output, 70% duty cycle). After centrifugation (15000 g, 30 sec) the supernatant was set aside for DNA precipitation or discarded. The pellet was resuspended in 1 mL LMg buffer (10 mM Hepes pH 7.4, 0.88 M sucrose, 1 mM MgCl2, 1 mM TCEP, PI), sonicated on ice for 2x10 sec (as before) and centrifuged (15000 g, 2 min). Supernatant was set aside for DNA precipitation and the pellet was resuspended in 200 µL 20 mM Tris pH 8.1. RNA was digested by adding 3 µL of RNAse A (10 mg/mL) and incubation for 1 h at 37°C. Proteins were deigested by adding 5 µL Proteinase K and incubation for 8 h at 37°C and the cross-link was reversed for 8 h at 65°C. Finally DNA was precipitated.
Label
Cy3
Label protocol
2µg of DNA was submitted to Nimblegen and processed there. Nucleolus-associated DNA was labelled with Cy3 and the control DNA with Cy5.
Non-nucleolar DNA was isolated from supernatants of the nucleolus-isolation with ethanol precipitation
Extracted molecule
genomic DNA
Extraction protocol
1x10^8 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. After washing cells 2 times with ice-cold PBS they were scraped off and pooled in 100 mL 1xPBS and centrifuged (750 g, 5 min, 4°C). Supernatant was discarded or used for DNA precipitation and cells were resuspended in 1 mL HMg buffer (10 mM Hepes pH 7.4, 0.88 M sucrose, 12 mM MgCl2, 1 mM TCEP, PI). Cells were sonicated on ice for 3x10 sec with fine tip (Branson sonifier, 65% output, 70% duty cycle). After centrifugation (15000 g, 30 sec) the supernatant was set aside for DNA precipitation or discarded. The pellet was resuspended in 1 mL LMg buffer (10 mM Hepes pH 7.4, 0.88 M sucrose, 1 mM MgCl2, 1 mM TCEP, PI), sonicated on ice for 2x10 sec (as before) and centrifuged (15000 g, 2 min). Supernatant was set aside for DNA precipitation and the pellet was resuspended in 200 µL 20 mM Tris pH 8.1. RNA was digested by adding 3 µL of RNAse A (10 mg/mL) and incubation for 1 h at 37°C. Proteins were deigested by adding 5 µL Proteinase K and incubation for 8 h at 37°C and the cross-link was reversed for 8 h at 65°C. Finally DNA was precipitated.
Label
Cy5
Label protocol
2µg of DNA was submitted to Nimblegen and processed there. Nucleolus-associated DNA was labelled with Cy3 and the control DNA with Cy5.
Hybridization protocol
Hybridization was performed at Nimblegen due to their protocols.
Scan protocol
Scanning was perfomred at Nimblegen due to their protocols.
Description
Nucleolus-associated DNA from proliferating IMR90 cells was labelled with Cy3 and hybridized against non-nucleolar DNA labelled with Cy5 on NimbleGen Human CGH 4.2M Whole-Genome Tiling Array v1.0 arrays.
Data processing
Data were preprocessed at Nimblegen, due to their protocols.
