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| Status |
Public on Oct 11, 2019 |
| Title |
MNase-seq_G2_IAA |
| Sample type |
SRA |
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| Source name |
G2_IAA
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: AS499 (S288C)
treatment: G2_IAA
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| Growth protocol |
Exponentially growing cultures (OD600=0.5) were arrested in G1 in minimal media by adding α-factor to a final concentration of 3 x 10-8 M for 3 hours at 25 Celsius degrees. Cells were released from G1 by pelleting them (4,000 r.p.m, 1 min) and washing in YPD 3 times. The pellet was then resuspended in YPD containing 0.1 mg/ml pronase and to arrest cells in G2/M, Nocodazole (1.5 mg/ml stock in DMSO 100%) was added to a final concentration of 0.015 mg/ml for 2 hours. Then IAA (Stock at 800mM in DMSO) was added for 2 hours at a final concentration of 8mM to deplete the cells in Scc1-AID.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For MNase digestions 160 ml of cells (OD600=1) where fixed with 1 % formaldehyde for 30 minutes at 25 °C with 100 r.p.m. agitation and quenched with 125 mM glycine for 10 minutes at 25 °C with 100 r.p.m. agitation. Cells were spun and washed twice with PBS. Pellet was resuspended in 20 ml sorbitol/ tris buffer (1M sorbitol, 50 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol) containing 10 mg of Zymolyase 20T and incubated at 30 °C for 5 minutes or until spheroplast were formed. Spheroplasts were spun at 3,000 r.p.m. for 5 minutes and washed with sorbitol/tris buffer without b-mercaptoethanol. Spheroplasts were resuspended in NP buffer (1 M sorbitol, 50 mM NaCl, 10mM Tris pH 7.5, 5 mM MgCl2, 1mM CaCl2, 1mM b-mercaptoethanol, 500 μM spermidine, 0.075 % NP40) and digested with 600, 1200 or 1800 U/ ml of MNase (NEB) for 20 minutes at 37 °C. MNase digestion was stopped adding 200 μl of STOP solution ( 5% SDS, 50 mM EDTA). DNA was decrosslinked by adding 1 mg/ml proteinase K and incubating at 65 °C overnight. DNA was purified by adding 0.6M potassium acetate pH 5.5 and incubating on ice 5 minutes and centrifuging 10 minutes at 3,500 r.p.m followed by a phenol chloroform extraction. Finally DNA was precipitated by adding 25 μg/ml glycogen, 1/25 columes NaCl, 0.7 volumes Isopropanol, incubating 30 minutes at -20 °C and centrifuging 30 minutes at 13,200 r.p.m. The pellet was washed in 70 % ethanol and resuspended in TE 1X. Samples were run in parallel in a 1.5 % agarose gel and the mononucleosomes of a digestion with a ratio of 80:20 mononucleosomes to dinucleosomes were purified and used in Next Generation Sequencing
Libraries of mononucleosomal and ChIP DNA were constructed following the Illumina compatible NEXTflexTM ChIP-Seq kit protocol and sequenced in an Illumina NextSeq500 platform using the paired-end protocol. Reads were aligned to the S.cerevisiae (SacCer3) genome using Bowtie. Nucleosome occupancy maps were generated using the NUCwave algorithm. Nucleosome fuzziness was analyzed using the dpos utility of DANPOS2 application. To make it compatible with NUCwave maps, we used the following parameters: a span of 1 bp and a read extension of 50 bp. ChIP-seq data representation was performed using the Model-based Analysis of ChIP-Seq (MACS) with a read extension of 147 bp.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paired-end Sequencing reads were aligned to Yeast genome sacCer3 using bowtie version 2.2.9 with default parameters. DANPOS2 was applied for two separate pair-wise comparisons at each nucleosome between G1 arrest and G2/M arrest, and G2/M arrest and G2/M +IAA. DANPOS output for pair-wise comparison contains a set of reference positions (with fixed length of 200bp) for nucleosomes defined by integrating all the nucleosomal positions called in each of the two conditions under study. The DANPOS-generated reference positions in two pair-wise comparisons were very similar but there was a small fraction of positions that did not overlap well in two comparisons. DANPOS also calculated Fuzziness scores for each condition and FDR and log10 p-value for each pair-wise comparison. We reported the nucleosomes with significant fuzziness change as reference nucleosomes in each comparison with FDR < 0.01 and p-value<-2, recommended in DANPOS manual. The fuzziness of these nucleosomes may be increased or decreased from one condition to the other. We also used DNAPOS-generated wig files for visualization of Fuzziness on IGV genome browser.
Supplementary_files_format_and_content: .wig
Genome_build: SacCer3
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| Submission date |
Aug 14, 2018 |
| Last update date |
May 16, 2022 |
| Contact name |
Jonay Garcia-Luis |
| E-mail(s) |
jonaygl@gmail.com
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| Phone |
660220907
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| Organization name |
UKRI
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| Department |
London Institute of Medical Sciences
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| Street address |
Du Cane Road
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| City |
London |
| State/province |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE118534 |
FACT mediates cohesin function on chromatin |
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| Relations |
| BioSample |
SAMN09831989 |
| SRA |
SRX4551751 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3332094_MN-NZ-IAA-300-Exp1_depth_wl_trimmed_PE.wig.gz |
28.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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