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| Status |
Public on Oct 01, 2014 |
| Title |
ER4_Mitotic_Dnase rep1 |
| Sample type |
SRA |
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| Source name |
G1E-ER4+E2
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| Organism |
Mus musculus |
| Characteristics |
strain background: 129
cell line: G1E-ER4+E2
cell type: inducible GATA-1 rescue subline of G1E cells
cell markers: Ter119+CD71+
treated with: 100 nM estradiol for 22h and nocodazole (200 ng/ml) for 7h prior to harvest
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| Treatment protocol |
G1E-ER4 cells were treated with 100 nM estradiol for 22h prior to harvest. Then, for mitotic arrest, both G1E and G1E-ER4 cells were treated with nocodazole (200 ng/ml) for 7h prior to harvest.
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| Growth protocol |
G1E and G1E-ER4 cells were grown as described in Weiss et al., 1997.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
At harvest, nocodazole-treated and asynchronous cells were cross-linked with 0.1% formaldehyde at room temperature for 10 minutes, then quenched with 1 M glycine. Fixed cells were washed with PBS and resuspended in 1X Cell Lysis Buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4, 300 mM sucrose, 0.1 mM EGTA, 0.5 mM PMSF, 0.1% NP40, and 2 μL/mL protease inhibitor cocktail (Sigma)). For mitotic samples, cells were then stained with anti-H3S10Phos antibody (Millipore 04-817) and Dy488 F(ab’)2 anti-rabbit IgG secondary antibody (Jackson 711-485-152), and sorted by FACS for H3S10Phos-positive cells as shown in Fig. 1. DNase I digestion was performed based on protocol from (Cockerill, 1999), with details outlined as follows. 2-10 million asynchronous cells (in Cell Lysis Buffer) or 2 million mitotic cells (collected from the FACS machine in PBS) were resuspended in 50 μL Nuclei Lysis Buffer (300 mM sodium acetate, 5mM EDTA pH 7.4, 0.5% SDS), added to 5 μL of 100 mM CaCl2, equilibrated at room temperature for 10minutes. A range of units of DNase I were added (see DNase-seq library preparation below for the range of units selected for sequencing) and the digestion reaction proceeded for 10 minutes at room temperature, then terminated by adding 350 μL of 0.1 mg/mL proteinase K in Nuclei Lysis Buffer. Samples were gently mixed by inversion, then incubated at 55◦Cfor 5 minutes, then overnight at 65◦Cfor reversal of formaldehyde crosslinks. Additional proteinase K was added to a final concentration of 0.1 mg/mL and incubated at 55◦Cfor 1h. DNA fragments were isolated by phenol-chloroform extraction and ethanol precipitation.
DNase-seq library construction was performed as described in (Song and Crawford, 2010), with the following modifications. Standard 0.8% agarose gels were run for 2h at 80V with 5 μL of each sample and stained with ethidium bromide to check extent of chromatin digestion. A range of 3 different DNase I concentrations were chosen for each condition that best matched digestion patterns between conditions. For mitotic samples, this was 2U, 4U, and 8U of DNase. For asynchronous samples, this was between 4U to 40U, adjusted proportionally to the number of cells in the sample. 70 μL of each sample (for each DNase I concentration) were subjected to blunt-end reaction containing 20 μL NEB Buffer 2, 7 μL 10 mM dNTP’s, 6 μL T4 DNA polymerase (NEB M0203), 2 μL BSA (100x), 4 μL 50mMMgCl2, and 95 μL dH2O, incubated at room temperature for 3.25h. 200 μL TE buffer was added and samples placed at 65◦Cfor 15 minutes to deactivate enzyme. Reactions were cleaned up by phenol-chloroform extraction and DNA resuspended in 30 μL 10 mM Tris-Cl. The samples corresponding to the three chosen DNase I concentrations chosen for each condition were measured by nano drop and pooled at equimolar concentrations into a single tube for overnight ligations to the first DNase-seq linker, as per (Song and Crawford, 2010). The one modification from (Song and Crawford, 2010) is that we added a 5’ phosphate to linker 1 to increase ligation efficiency.
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| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
processed data file: G1E-ER4_Mitotic_pooled_peaks.bed.gz; G1E-ER4_Mitotic_pooled.bigWig
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| Data processing |
Raw reads from DNase-seq libraries were first groomed using FASTQ Groomer on Galaxy Giardine (2005); Blankenberg et al. (2001);Goecks et al. (2010). This program verifies that each base call has a corresponding quality value, and that the quality value is in the Sanger, Phred+33 format. Groomed reads were then trimmed as only the first twenty bases of each read are genomic DNA. The trimmed reads were then mapped to mouse mm9 genome using Bowtie Langmead et al. (2009) using the parameters -m = 4 , -k = 1, and -best, thus allowing the 20 basepair reads to map to at most 4 locations, but reporting only the single, best alignment. This option was chosen to allow reads to map in duplicated regions, while still excluding reads to highly repetitive DNA. The mapped reads from Bowtie were then filtered to retain only those reads successfully mapped to the mm9 genome.
Using DNase2Hotspots as described in Baek et al., 2011, we initially called hotspots using mapped reads pooled from biological triplicates for each experimental condition. After testing a range of z-score thresholds, we decided on a final stringent threshold of 0% FDR that still captured many mildly DNase-sensitive areas. From within hotspots that meet the 0% FDR threshold, we called DNase peaks further enriched from the surrounding hotspot, using a thresholded at a z-score >20.
Reads pooled from biological triplicates for each experimental condition were converted to bed files that were used as input to F-seq (Boyle et al., 2008), which generates smoothed signals based on library-size normalized density of reads. From these pooled reads we used F-seq to identify a set of top 100,000 read-enriched regions. For visualization in browser tracks, genome-wide signal track produced by F-seq for individual replicates as well as the reads pooled from replicates are available as bigwig files at https://mery.genome-browser.bx.psu.edu/cgi-bin/ hgTrackUi?hgsid=187170_w5iejxXAHeIanNii3mA1Otjcfthv&c=chr17&g=chopDnase2. Note that F-seq signal is linearly proportional to RPKM.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files contain genome wide DNase signals calculated by F-seq as described above. Bed files contain the chromosone, start, and end positions of the peak signal regions called by F-seq.
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| Submission date |
Sep 30, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ross Hardison |
| E-mail(s) |
rch8@psu.edu
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| Organization name |
Pennsylvania State University
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| Street address |
303 Wartik Lab
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE61885 |
Genome accessibility is widely preserved and locally modulated during mitosis [Dnase-seq] |
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| Relations |
| BioSample |
SAMN03084400 |
| SRA |
SRX717118 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1516381_ER4_Mitotic_rep1.bigWig |
2.8 Gb |
(ftp)(http) |
BIGWIG |
| GSM1516381_ER4_Mitotic_rep1_peaks.bed.gz |
1.9 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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