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| Status |
Public on Aug 07, 2015 |
| Title |
SH-SY5Y nicotine 10min rep2 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
SH-SY5Y cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-SY5Y
material: nucleosomally protected DNA
treatment: Nicotine 10 minutes
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| Treatment protocol |
At the designated time points, cells were fixed and crosslinked for 10 min in 1% PBS-buffered formaldehyde. The reaction was stopped by incubation for 5 min with 125mM glycine.
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| Growth protocol |
We used undifferentiated SH-SY5Y cells (human neuroblastoma cell line; obtained directly from ATCC, Manassas, VA), maintained in culture according to protocols described by ATCC (1:1 F12:EMEM, 10% FBS, 50ug/mL gentamicin). Cells were grown at 37°C until a 150-mm dish was 90% confluent (~2x107cells), at which time the medium was replaced with fresh medium containing nicotine or cocaine (each drug 10µM; Sigma, St. Louis, MO). The cultures were incubated for an additional 10, 60, and 90 min following addition of nicotine; or 5, 20, 40 and 60 min following addition of cocaine.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We used undifferentiated SH-SY5Y cells (human neuroblastoma cell line; obtained directly from ATCC, Manassas, VA), maintained in culture according to protocols described by ATCC (1:1 F12:EMEM, 10% FBS, 50ug/mL gentamicin). Cells were grown at 37°C until a 150-mm dish was 90% confluent (~2x107cells), at which time the medium was replaced with fresh medium containing nicotine or cocaine (each drug 10µM; Sigma, St. Louis, MO). The cultures were incubated for an additional 10, 60, and 90 min following addition of nicotine; or 5, 20, 40 and 60 min following addition of cocaine. Biological replicates were two independently grown cultures treated on nonconsecutive days. At the designated time points, cells were fixed and crosslinked for 10 min in 1% PBS-buffered formaldehyde. The reaction was stopped by incubation for 5 min with 125mM glycine. Nuclei were isolated (0.3M sucrose, 2mM MgOAc2, 1mM CaCl2, 1% Nonidet P-40, 10mM HEPES, pH7.8) by centrifugation at 1000 X g for 5 min at 4°C. Bare, unbound genomic DNA and mononucleosomally-protected DNA were isolated from each sample and purified as described previously (Sexton et al., 2014). A titration of micrococcal nuclease (MNase; 1-1.25U/mL; Worthington Biochemical Corp.) was used to digest bare, unbound DNA and mononucleosomally-protected DNA (Sexton et al., 2014) .
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| Label |
Cy3
|
| Label protocol |
1 µg of mononucleosomally protected and genomic DNA was directly labeled by Klenow (Nimblegen) random priming with Cy3 and Cy5 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
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| Channel 2 |
| Source name |
SH-SY5Y cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-SY5Y
material: bare genomic DNA
treatment: Nicotine 10 minutes
|
| Treatment protocol |
At the designated time points, cells were fixed and crosslinked for 10 min in 1% PBS-buffered formaldehyde. The reaction was stopped by incubation for 5 min with 125mM glycine.
|
| Growth protocol |
We used undifferentiated SH-SY5Y cells (human neuroblastoma cell line; obtained directly from ATCC, Manassas, VA), maintained in culture according to protocols described by ATCC (1:1 F12:EMEM, 10% FBS, 50ug/mL gentamicin). Cells were grown at 37°C until a 150-mm dish was 90% confluent (~2x107cells), at which time the medium was replaced with fresh medium containing nicotine or cocaine (each drug 10µM; Sigma, St. Louis, MO). The cultures were incubated for an additional 10, 60, and 90 min following addition of nicotine; or 5, 20, 40 and 60 min following addition of cocaine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We used undifferentiated SH-SY5Y cells (human neuroblastoma cell line; obtained directly from ATCC, Manassas, VA), maintained in culture according to protocols described by ATCC (1:1 F12:EMEM, 10% FBS, 50ug/mL gentamicin). Cells were grown at 37°C until a 150-mm dish was 90% confluent (~2x107cells), at which time the medium was replaced with fresh medium containing nicotine or cocaine (each drug 10µM; Sigma, St. Louis, MO). The cultures were incubated for an additional 10, 60, and 90 min following addition of nicotine; or 5, 20, 40 and 60 min following addition of cocaine. Biological replicates were two independently grown cultures treated on nonconsecutive days. At the designated time points, cells were fixed and crosslinked for 10 min in 1% PBS-buffered formaldehyde. The reaction was stopped by incubation for 5 min with 125mM glycine. Nuclei were isolated (0.3M sucrose, 2mM MgOAc2, 1mM CaCl2, 1% Nonidet P-40, 10mM HEPES, pH7.8) by centrifugation at 1000 X g for 5 min at 4°C. Bare, unbound genomic DNA and mononucleosomally-protected DNA were isolated from each sample and purified as described previously (Sexton et al., 2014). A titration of micrococcal nuclease (MNase; 1-1.25U/mL; Worthington Biochemical Corp.) was used to digest bare, unbound DNA and mononucleosomally-protected DNA (Sexton et al., 2014) .
|
| Label |
Cy5
|
| Label protocol |
1 µg of mononucleosomally protected and genomic DNA was directly labeled by Klenow (Nimblegen) random priming with Cy3 and Cy5 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
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| Hybridization protocol |
The labeled DNA was precipitated with 1 volume isopropanol, and hybridized according to manufacturer's protocols. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
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| Scan protocol |
Arrays were scanned on a Nimblegen scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
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| Description |
Mnase-chip of 10 minute nicotine-treated SH-SY5Y cells
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| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan CGH arrays.
normalized log2 (mononucleosomally protected DNA/bare genomic DNA) ratio for Nucleosome Distribution experiments.
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| Submission date |
Aug 06, 2015 |
| Last update date |
Aug 07, 2015 |
| Contact name |
Cynthia Vied |
| E-mail(s) |
cynthia.vied@med.fsu.edu
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| Organization name |
Florida State University
|
| Department |
Translational Science Laboratory
|
| Street address |
1115 W. Call St
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| City |
Tallahassee |
| State/province |
Florida |
| ZIP/Postal code |
32306 |
| Country |
USA |
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| Platform ID |
GPL20623 |
| Series (1) |
| GSE71795 |
Nucleosome Repositioning: Nicotine- and Cocaine-induced Changes |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1845845_SH-SY5Y_nicotine_10min_rep2.gff.gz |
1.3 Mb |
(ftp)(http) |
GFF |
| GSM1845845_SH-SY5Y_nicotine_10min_rep2_532.pair.gz |
4.5 Mb |
(ftp)(http) |
PAIR |
| GSM1845845_SH-SY5Y_nicotine_10min_rep2_635.pair.gz |
4.5 Mb |
(ftp)(http) |
PAIR |
| Processed data provided as supplementary file |
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