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| Status |
Public on Jul 02, 2013 |
| Title |
BG3_NHS |
| Sample type |
SRA |
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| Source name |
tissue culture cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: BG3
treatment: no heat shock
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| Treatment protocol |
Cells were heat shocked by replacing the media with media that had been prewarmed to 37 degrees C. The dish of cells was held at 37 degrees C in a water bath for 15 min. At the end of the heat shock, an appropriate volumn of ice cold media was added to adjust the temperature to 24 degrees C.
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| Growth protocol |
Cells were grown in Schneider's media with 10% FBS at 24 degrees C
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde for 10 min and quenched with 125 mM glycine. Cells were dislodged from the culture dish and washed once in PBS followed by incubation in 10 mM KMnO4 in PBS for 1 min at room temperature. The permanganate reaction was stopped by adding an equal volume of PBS containing 0.8 M beta-mercaptoethanol and 40 mM EDTA. After washing once with PBS, cells were lysed and chromatin was sheared as previously described by Petesch, S.J. and J.T. Lis (2008) Cell 134, p 74-84. Chromatin from at least 20 million cells was used in one permanganate-ChIP-seq experiment, and RNA polymerase II was immunoprecipitated with antibody against Rpb3.
Polishing ChIP’ed DNA, first adaptor (P2) ligation and nick translation were done on protein A sepharose resin as described by Rhee and Pugh (2011), Cell 147, p. 1408. DNA with P2 adaptors on both ends was eluted from the resin with buffer containing 1% SDS and 0.1M NaHCO3. After reversing the crosslinks, the sample was digested with proteinase K, and extracted with phenol/chloroform. DNA was precipitated with ethanol and suspended in 90 ul of TE buffer (10 mM Tris pH 7.5, 1 mM EDTA). 10 ul of piperidine was added and the sample was incubated at 90 °C for 30 minutes. Piperidine was then removed by isobutanol and ether extraction and the DNA was ethanol precipitated as previously described by Gilmour and Fan (2009) Methods, 48, p. 368. The following steps of library preparation were done in solution as previously described Rhee and Pugh (2011). Briefly, the DNA was denatured at 95 °C and a primer hybridized to the P2 adaptor was extended with phi29 polymerase. After ligating the second adaptor (P1) to the piperidine cleaved ends, the DNA was purified with AMPure beads to remove free adaptors. After nick translation and PCR amplification with both P1 and P2 primers, the DNA was separated on a 2% agarose gel and fragments between 150-250 bp were selected for parallel sequencing. Emulsion PCR and SOLiD Sequencing were done at the Penn State Nucleic Acid facility on a SOLiD 2.0 sequencer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 2.0 |
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| Description |
Permanganate ChIP-seq, nonheat shocked BG3 cells
Library Strategy: Permanganate ChIP-seq
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| Data processing |
2 processed data files were generated: one identifying breaks occcuring at all nucleotides and the other identifying breaks at thymines.
Supplementary_files_format_and_content: all_reads file: Provides chromosome, location, and number of reads for all 4 nucleotides on the forward or reverse strand
Supplementary_files_format_and_content: T_reads file: Provides chromosome, location, and number of reads for T's on the forward or reverse strand
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| Submission date |
Jul 01, 2013 |
| Last update date |
Jul 02, 2013 |
| Contact name |
David Scott Gilmour |
| E-mail(s) |
[email protected]
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| Phone |
814-863-8905
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| Organization name |
Penn State University
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| Department |
Biochemistry and Molecular Biology
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| Lab |
Gilmour
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| Street address |
465 North Frear
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| City |
University Park |
| State/province |
Pennsylvania |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL9521 |
| Series (1) |
| GSE46620 |
Permanganate ChIP-seq analysis of Pol II in nonheat shocked and heat shocked Drosophila S2R+ cells and nonheat shocked BG3 cells |
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| Relations |
| BioSample |
SAMN02222833 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1179207_BG3_NHS_T_reads.txt.gz |
7.1 Mb |
(ftp)(http) |
TXT |
| GSM1179207_BG3_NHS_all_reads.txt.gz |
8.7 Mb |
(ftp)(http) |
TXT |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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