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| Status |
Public on Aug 22, 2017 |
| Title |
Hi-C in sap1-1mutant R2 |
| Sample type |
SRA |
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| Source name |
log. growing cells
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: sap1-1
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| Treatment protocol |
Cells (OD600 ~0.5) were fixed in 3% formaldehyde (Sigma) for 20 min at 26°C, and quenched with glycine for 5 min at 26°C.
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| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA). Cultures were first grown at 26˚C and then shifted to 33˚C for 6 hours.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinked cells were lysed by grinding in a cold mortar and pestle. Cell lysate was treated with 0.1% SDS for 10 min at 65˚C, and then quenched with 1% TritonX-100. Cell lysate was digested overnight with 100 U HindIII at 37˚C. The 5' overhang from the HindIII digestion was filled in using Klenow fragment in presence of biotin-14-dCTP and dATP, dGTP, and dTTP at 37˚C for 45 min. The reaction was terminated with 1.5% SDS. The DNA fragments were ligated by T4 DNA ligase in diluted conditions that favor the ligation between cross-linked DNA fragments in 16˚C for 8 hours (Hi-C DNA). The Hi-C DNA was reverse cross-linked at 65˚C overnight in the presence of proteinase K and purified by phenol/chrolorom extraction. Purified Hi-C DNA was treated with 1 mg/ml Rnase A for 30 min at 37˚C. Un-ligated biotin lablelled DNA was selectively removed by T4 DNA plymerase and reaction were purified by phenol/chloroform extraction.
Hi-C DNA was sheared by Covaris in the size range of <500 bp. The sheared Hi-C DNA was subjected to end-repair and 3' end adenylation. Hi-C DNA between 150 - 300 bp was selected with AMPure XP (Beckman-Coulter). The biotin-labled Hi-C DNA was selectively captured by Dynabeads Myone Streptavidin C1 (Invitrogen) and used for Illumina PE adapter ligation. Streptavidin beads containing bounds Hi-C DNA were used for the template for library amplification by PE-PCR primers (Illumina).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Hi-C data were mapped, and reads were filtered. Corrected contact probability matrices at 10kb resolution were obtained using iterative correction. Both steps were performed using the hiclib library for python, publicly available at https://bitbucket.org/mirnylab/hiclib.
Paired-end sequencing reads were mapped independently using Bowtie 2.1.0 to the S. pombe reference genome (ASM294v2). Mapping with iteratively increasing truncation length was used to maximize yield of valid Hi-C interactions.Only read pairs where both reads uniquely aligned to the genome were considered for subsequent steps. Read pairs corresponding to repeat instances of the same DNA molecule were removed. based upon their HindIII restriction fragment assignments and orientations, read pairs were classified as valid Hi-C products, non-ligation, or self-ligation products. The following fragment-level filters were then applied which remove read pairs: with one end adjacent to the restriction site, from restriction fragments with very high or low counts, from very large or small restriction fragments, and separated by very few restriction fragments.
These data is then binned at 10 kB resolution. The bins with lowest 3% coverage are discarded (in addition to bins with zero counts). Remove the diagonal and neighboring diagonal (i.e. interactions < 20kB). Remove the standalone bins. Removed potential biases in raw Hi-C contact maps by normalizing coverage using an iterative procedure. Interpolate the singleton bins using neighboring bins. Normalize the sum = 1 along each row and column.
Genome_build: ASM294v2
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| Submission date |
May 01, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Shiv Grewal |
| Phone |
2407607553
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| Organization name |
NCI
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| Department |
LBMB
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| Lab |
Shiv Grewal
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| Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL13988 |
| Series (2) |
| GSE96883 |
Shelterin Mediates Genome Reorganization in Response to Replication Stress |
| GSE98381 |
Genome-wide chromatin conformation capture (Hi-C) analysis in fission yeast. |
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| Relations |
| BioSample |
SAMN06854470 |
| SRA |
SRX2771301 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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