CATCH-IT was performed as previously described (PMID: 20508129) with some modifications. Briefly, tissue culture medium was replaced with DMEM media without methionine (Invitrogen Cat# 21013-024) supplemented with 1X glutamine, 0.2 mM L-cystine, 10% FBS, and 1% penicillin-streptomycin, and cells were grown at 37C for starvation fro 30 minutes, followed by 30 minutes incubation at 37C with 4 mM azidohomoalanine (Anaspec Cat# 63669) added to the media. Cells were then harvested, washed with 1X PBS, and resuspended in ice-cold TM2 buffer (10 mM Tris, pH=7.5, 2mM MgCl2). Cells were lysed using 0.2% of NP-40 and nuclei were isolated, followed by digestion of micrococcal nuclease for 10 minutes to produce mostly mononucleosomes. Biotin coupling, chrmatin extraction, streptavidin pulldown, urea wash, and DNA isolation were then performed as previously described.
CATCH-IT was performed as previously described (PMID: 20508129) with some modifications. Briefly, tissue culture medium was replaced with DMEM media without methionine (Invitrogen Cat# 21013-024) supplemented with 1X glutamine, 0.2 mM L-cystine, 10% FBS, and 1% penicillin-streptomycin, and cells were grown at 37C for starvation fro 30 minutes, followed by 30 minutes incubation at 37C with 4 mM azidohomoalanine (Anaspec Cat# 63669) added to the media. Cells were then harvested, washed with 1X PBS, and resuspended in ice-cold TM2 buffer (10 mM Tris, pH=7.5, 2mM MgCl2). Cells were lysed using 0.2% of NP-40 and nuclei were isolated, followed by digestion of micrococcal nuclease for 10 minutes to produce mostly mononucleosomes. Biotin coupling, chrmatin extraction, streptavidin pulldown, urea wash, and DNA isolation were then performed as previously described.
Label
Cy3
Label protocol
Standard NimbleGen protocol
Hybridization protocol
Standard NimbleGen protocol
Scan protocol
Standard NimbleGen protocol
Description
CATCH-IT profiling of Atm -/- SCC cells after 4 hours of doxorubicin treatment.
Data processing
The dye-bias correction of Peng, et al (PMID PMC1913544) was applied to bi-weight mean adjusted log base 2 ratios of Cy5/Cy3 computedby NimbleGen. The supplementary gff and wig files contain the bi-weight mean adjusted log base 2 ratios in MM9 coordinates.
