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| Status |
Public on Oct 30, 2015 |
| Title |
abo1_mono_nucleosomes |
| Sample type |
SRA |
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| Source name |
Cross-linked in vivo chomatin
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: HM463
genotype: h-, abo1::KanMX
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| Treatment protocol |
Cells were crosslinked for 20 min at 30°C using 1% formaldehyde and quenched by the addition of glycine to 125 mM. Cells were washed once in CES buffer (50 mM citric acid/50 mM Na2HPO4 [pH 5.6], 40 mM EDTA [pH 8.0], 1.2 M sorbitol and 10 mM β-mercaptoethanol) and resuspended in 500 μl of CES buffer with 0.5mg Zymolase 100-T. Cells were spheroplasted at 30°C for up to 1 h and then washed twice with ice cold 1.2M sorbitol. Spheroplasts were then resuspended in 800 μl NP-S buffer (1.2 M sorbitol, 10 mM CaCl2, 100 mM NaCl, 1 mM EDTA pH 8.0, 14 mM β-mercaptoethanol, 50 mM Tris pH 8.0, 0.075% NP-40, 5 mM spermidine, 0.1 mM PMSF, 1% Sigma Protease inhibitors cocktail [Sigma P8215]). Spheroplasts were then divided into four 200 μl aliquots and each aliquot was mixed with 300 μl of NP-S buffer. Three aliquots were digested with between 75-187.5 units of MNase (USB) for 10 min at 37°C. The fourth was retained as an undigested control. MNase digestion was terminated by adding EDTA [pH 8.0] to a final concentration of 50 mM and SDS to 0.2%.
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| Growth protocol |
Cells (100 ml) were grown to OD595= 0.75-8.0 in YE5S at 30°C
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Reactions were incubated at 65°C overnight with 0.2 mg/ml proteinase K and 10 μg RNAse. DNA was purified by extracting twice with phenol:chloroform followed by ethanol precipitation (0.1 volumes of 3 M sodium acetate followed by 2 volumes of ethanol). Pellets were washed in 70% ethanol and resuspended in water containing 10 μg/ml RNase and incubated at 37°C for 30 min. Triplicate digests were pooled and treated with with 100 U unmodified T4 polynucleotide kinase (NEB) for 30 min at 37°C to remove 3′-phosphate groups left by MNase. DNA was extracted once more with phenol:chloroform, re-precipitated with sodium acetate and propan-2-ol, washed with 80% ethanol, dried and re-suspended in TE (pH 7.5).
DNA fragments were end repaired, 3′-adenylated and ligated to indexed adapters without size selection using Nextflex reagents (Newmarket Scientific, UK). Libraries were amplified with 8 cycles PCR using Kapa HiFi PCR master mix (Anachem), primers removed with GeneRead size selection protocol (QIAgen) before quantification by Bioanalyser DNA 7500 assay. Libraries were pooled, denatured, diluted to 6 nM before clustering in a single lane of a high output Illumina flowcell. Sequencing (100nt) was undertaken on a HiSeq 2500 using TruSeq SBS v3 reagents (Illumina).
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
MNase-protected DNA
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| Data processing |
Paired reads were aligned to the ASM294v1.17 reference genome using Bowtie 0.12.7 with command line flags: -n 0 --trim3 64 --maxins 5000 --fr -k 1 --sam. Reads in the .bam files are therefore trimmed to 36bp.
Aligned read pairs for each fission yeast strain were combined (bash cat) from the two HiSeq lane data sets and then sorted according to chromosome using grep, and into a range of size classes based on the SAM format ISIZE value (difference between 5’ end of the mate read and the 5’ end of the first mapped read) plus or minus 20% using scripts described in Kent et al., (2011,Nucleic Acids Res. 39:e26) . E.g. Mono-nucleosome-sized reads are, therefore, represented as 150bp ± 30bp.
To define the genomic position of MNase-resistant chromatin entities we mapped the mid-point position of the read pairs in a particular size class. Frequency distributions of the mid-point positions, were then calculated using 10bp bins, and the distributions lightly smoothed to a 3 bin moving average using scripts decribed in Kent et al., (2011,Nucleic Acids Res. 39:e26).
Note: WT_7 is a re-analysis of wild-type S. pombe MNase-seq data previously published in Gal C., Moore K.M., Paszkiewicz K., Kent N.A., Whitehall S.K. (2015) The impact of the HIRA histone chaperone upon global nucleosome architecture. Cell Cycle 14:123-134. Raw paired-end sequence (rather than .bam alignment) available under SRA: SRS712792
Genome_build: ASM294v1.17
Supplementary_files_format_and_content: After unzipping, the files describe nucleosome and di-nucleosome mid point position frequency distributions as zero-referenced, chromosome base, three-column tab-delimited text files. The three columns are: chromosome number; bin position; 3MA mid-point frequency value. By adding the file extension .sgr these files can be rendered directly using the Integrated Genome Browser.
Supplementary_files_format_and_content: WT_7_Part150chr_10.txt: Genomic mono-nucleosome map: frequency distribution of 150bp CPSA size class paired-read mid-point positions, in 10bp bins, 3-bin moving average.
Supplementary_files_format_and_content: WT_7_Part300chr_10.txt: Genomic di-nucleosome map: frequency distribution of 300bp CPSA size class paired-read mid-point positions, in 10bp bins, 3-bin moving average.
Supplementary_files_format_and_content: abo1_7_Part150chr_10.txt: Genomic mono-nucleosome map: frequency distribution of 150bp CPSA size class paired-read mid-point positions, in 10bp bins, 3-bin moving average.
Supplementary_files_format_and_content: abo1_7_Part300chr_10.txt: Genomic di-nucleosome map: frequency distribution of 300bp CPSA size class paired-read mid-point positions, in 10bp bins, 3-bin moving average.
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| Submission date |
Mar 30, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicholas Kent |
| E-mail(s) |
kentn@cardiff.ac.uk
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| Organization name |
Cardiff University
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| Department |
School of Biosciences
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| Street address |
Museum Avenue
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| City |
Cardiff |
| ZIP/Postal code |
CF10 3AX |
| Country |
United Kingdom |
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| Platform ID |
GPL13988 |
| Series (1) |
| GSE67410 |
Abo1, a conserved bromodomain AAA-ATPase maintains global nucleosome occupancy and organization |
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| Relations |
| BioSample |
SAMN03450964 |
| SRA |
SRX972050 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1646416_abo1_7_Part150chr_10.txt.gz |
3.8 Mb |
(ftp)(http) |
TXT |
| GSM1646416_abo1_7_Part300chr_10.txt.gz |
3.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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