gender: male cell line: IMR91L sample type: DNA fragment ~ 4 kb
Treatment protocol
Apoptosis was induced by staurosporine (Cell Signaling Technology, Inc., Danvers, USA), which was dissolved in dimethyl sulfoxide (DMSO, Sigma, Saint Louis, USA) to a stock concentration of 1 mmol/L. About 2 x 10^6 cells were exposed to either 1 µmol/L staurosporine/0.1%DMSO or 0.1% DMSO alone as a control for 4 hours at 37°C.
Growth protocol
Both cell lines were cultured in Eagle´s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM UltraGlutamine 1, 1 mM sodium pyruvate and 100units/ml penicillin/streptomycin. The fibroblasts were maintained at 37°C with a humidified atmosphere of 5% CO2 and ambient oxygen.
Extracted molecule
genomic DNA
Extraction protocol
ChIP was done according to the Transcription Factor ChIP kit protocol (Diagenode, Liège, Belgium). In brief, lysed cells were sonicated for 60 minutes using the Bioruptor UCD-200 device (Diagenode, Liège, Belgium), followed by overnight incubation of 1x106 cells with 5 µg of antibodies against unmodified histone H3 (ab1791; Abcam, Cambridge, UK) and histone H4 lysine 8 acetylation (pAb-103-050; Diagenode, Liège, Belgium), respectively. The subsequent chromatin reverse crosslinking, elution and purification of ChIP and input DNA were done employing the IPure Kit (Diagenode, Liège, Belgium). Analysis of apoptotic DNA: after treatment cells were washed once in PBS, detached by five minutes treatment with trypsin-EDTA and the cell pellets were stored at -80°. For DNA isolation cells were treated with lysis buffer (0.40 M Tris-HCl pH 8.0, 0.06 M Na-EDTA, 0.15 M NaCl, 1% SDS) and RNA was digested for 1 hour at 37°C using 15 µg/mL RNase A. 1 M sodium perchlorate and one volume chloroform were added to deproteinize cell lysates. After phase separation and collection of the aqueous phase DNA was precipitated. 3 µg of genomic DNA were loaded onto a 1% low melt agarose gel, electrophoresed for 90 minutes at 120 V and stained with ethidium bromide for 30 minutes. DNA bands responding to non-fragmented (> 48 kb) and fragmented DNA (approximately 4 kb) were cut with a scalpel and collected. For the extraction of DNA from the gel pieces β-Agarase I (New England Biolabs, Ipswich, USA) was used according to the manufacturer´s protocol.
gender: male cell line: IMR91L sample type: DNA fragment > 48 kb
Treatment protocol
Apoptosis was induced by staurosporine (Cell Signaling Technology, Inc., Danvers, USA), which was dissolved in dimethyl sulfoxide (DMSO, Sigma, Saint Louis, USA) to a stock concentration of 1 mmol/L. About 2 x 10^6 cells were exposed to either 1 µmol/L staurosporine/0.1%DMSO or 0.1% DMSO alone as a control for 4 hours at 37°C.
Growth protocol
Both cell lines were cultured in Eagle´s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM UltraGlutamine 1, 1 mM sodium pyruvate and 100units/ml penicillin/streptomycin. The fibroblasts were maintained at 37°C with a humidified atmosphere of 5% CO2 and ambient oxygen.
Extracted molecule
genomic DNA
Extraction protocol
ChIP was done according to the Transcription Factor ChIP kit protocol (Diagenode, Liège, Belgium). In brief, lysed cells were sonicated for 60 minutes using the Bioruptor UCD-200 device (Diagenode, Liège, Belgium), followed by overnight incubation of 1x106 cells with 5 µg of antibodies against unmodified histone H3 (ab1791; Abcam, Cambridge, UK) and histone H4 lysine 8 acetylation (pAb-103-050; Diagenode, Liège, Belgium), respectively. The subsequent chromatin reverse crosslinking, elution and purification of ChIP and input DNA were done employing the IPure Kit (Diagenode, Liège, Belgium). Analysis of apoptotic DNA: after treatment cells were washed once in PBS, detached by five minutes treatment with trypsin-EDTA and the cell pellets were stored at -80°. For DNA isolation cells were treated with lysis buffer (0.40 M Tris-HCl pH 8.0, 0.06 M Na-EDTA, 0.15 M NaCl, 1% SDS) and RNA was digested for 1 hour at 37°C using 15 µg/mL RNase A. 1 M sodium perchlorate and one volume chloroform were added to deproteinize cell lysates. After phase separation and collection of the aqueous phase DNA was precipitated. 3 µg of genomic DNA were loaded onto a 1% low melt agarose gel, electrophoresed for 90 minutes at 120 V and stained with ethidium bromide for 30 minutes. DNA bands responding to non-fragmented (> 48 kb) and fragmented DNA (approximately 4 kb) were cut with a scalpel and collected. For the extraction of DNA from the gel pieces β-Agarase I (New England Biolabs, Ipswich, USA) was used according to the manufacturer´s protocol.
scanned on an Agilent DNA Microarray Scanner G2565BA, Scan resolution: 2µm, Scan region: 61 x 21.6 mm, Green PMT: 100%, Red PMT: 100%
Description
Regional preferences in apoptotic DNA degradation were determined by co-hybridizing high molecular (> 48 kb) and degraded apoptotic DNA (~4 kb), which we have extracted from 1% low melting agarose and amplified by means of the GenomePlex Whole Genome Amplification Kit.
