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| Status |
Public on Mar 31, 2015 |
| Title |
Rsc8 ChIP |
| Sample type |
SRA |
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| Source name |
Immunoprecipitated chromatin
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: YBC2882
genotype/variation: Rsc8-9xMyc
chip antibody: Anti-c-Myc antibody [9E11]
chip antibody vendor: Abcam
chip antibody cat. #: ab56
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| Growth protocol |
Cells were grown in standard synthetic complete media with 2% glucose at 30C until an optical density at 600 nm of 1.0. Cells were cross-linked by addition of formaldehyde to the media to 1% final concentration for 30 minutes and quenched by the addition of 1M glycine to 0.1M final concentration. Cells were harvested by centrifugation and washed twice with Tris-Buffered Saline. Cells were stored at -80C until preparation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by resuspending the cells in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitors) and bead beating with 0.7 mm zirconium beads for six rounds of 2 minutes, interspersed with cooling on ice. Cells were collected by centrifugation, the supernatant was discarded, and the pellet resuspended in MNase Digestion buffer (50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.1% Triton X-100). Chromatin was digested with MNase at 37C for 15 minutes. The amount of MNase was empirically determined to yield an estimated 60% mono-, 30% di-, and 10% tri-nucleosomes. MNase digestion was stopped by the addition of 0.1X volumes of 10X MNase ChIP Stop Buffer (50 mM Tris pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100, 1% sodium deoxycholate). Cell debris was removed by centrifugation and the soluble chromatin was added to prepared beads. For immunoprecipitation, anti-mouse magnetic Dynabeads (Invitrogen) were prepared by incubating with anti-cMyc (9E11) in phosphate-buffered saline containing 5 mg/ml BSA for several hours with rotation. Normalized amounts of protein was then incubated with the prepared beads in ChIP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). Immunoprecipitations were performed at 4C overnight with gentle rotation. Beads were harvested by magnetic field and washed twice with ChIP buffer, twice with ChIP buffer supplemented to 500 mM NaCl, twice with LiCl wash buffer (10 mM Tris pH 8, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and once with TE. Captured complexes were eluted and cross-links reversed by incubating the beads in buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5% SDS) at 65C overnight. DNA was purified using Qiagen PCR Clean-up columns using the manufacturer's protocol.
ChIP eluate DNA and Input DNA were phosphorylated with T4 kinase (NEB) prior to ligation of adaptors using Illumina Tru-Seq library construction kit according to manufacturers instructions. No other end repair was performed (fill-in, etc). After limited PCR amplification, samples were size selected on agarose gel and fragments corresponding to mononucleosomes were excised and purified.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Rsc8 ChIP
7018X2
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| Data processing |
De-multiplexed Fastq files from the Illumina pipeline were aligned to the Saccharomyces cerevisiae genome release 64 (UCSC SacCer3) using Novoalign version 2.8 (http://www.novocraft.com), allowing for one random repeat, base recalibration, and a minimum alignment quality score of 13. Alignments were converted to sorted, indexed Bam files using samtools.
Proper paired-end alignments were filtered for an insert size of 99-195 bp using the BioToolBox (http://code.google.com/p/biotoolbox/) extra program split_bam_by_isize, using the --at option. Midpoint and fragment coverage normalized maps were generated using the BioToolBox program bam2wig using the --pos option of mid and span, respectively, combined with the options --pe and --rpm. Wig files were converted to bigWig files using UCSC utilities.
ChIP enrichment was calculated using MACS2 (http://pypi.python.org/pypi/MACS2) with the callpeak mode options '--keep-dup all --shift-control -g 12.1e6 --slocal 0 --llocal 0'. Tracks representing QValue FDR scores and Fold Enrichment were generated with the MACS2 bdgcmp mode. High level gene analysis was performed using BioToolBox programs get_datasets and get_relative_data.
Genome_build: SacCer3
Supplementary_files_format_and_content: Log2 Fold Enrichment of ChIP over Input as generated by MACS2
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| Submission date |
Feb 04, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Timothy J Parnell |
| E-mail(s) |
timothy.parnell@hci.utah.edu
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| Organization name |
Huntsman Cancer Institute
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| Street address |
2000 Circle of Hope
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| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
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| Platform ID |
GPL13272 |
| Series (2) |
| GSE65592 |
RSC and ISW1 Chromatin Remodelers Display Functional and Chromatin-based Promoter Antagonism [ChIP-seq] |
| GSE65594 |
The Chromatin Remodelers RSC and ISW1 Display Functional and Chromatin-based Promoter Antagonism |
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| Relations |
| BioSample |
SAMN03328988 |
| SRA |
SRX864899 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1600614_Rsc8_Myc_log2FE.bw |
34.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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