|
| Status |
Public on Oct 22, 2014 |
| Title |
clr4D_HIC |
| Sample type |
SRA |
| |
|
| Source name |
Clr4D cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: SPK567
|
| Treatment protocol |
The temperature was shifted in the temperature sensitive mutants.
|
| Growth protocol |
The Cells were grown on YEA at optimal temperature (30 C). Wherever temperature sensitive mutatnts were used, the cells were incubated at elevated temperatures (33 C) for 4 - 6 hours before library preparation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A total of 500 ml of cells (OD at 600nm ~ 0.5) was fixed in 3% formaldehyde (Sigma) for 20 min at 26 C, and quenched with glycine for 5 min at 26 C. Cells were poured into liquid nitrogen using 1x NEB2, disrupted by nitrogen grinding. 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 HiC-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 then sheared by ultrasonication (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).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
The pipeline for processing of each data-set was assembled around the library (see https://bitbucket.org/mirnylab). Briefly, each read of the sample was treated separately for alignment purposes and the information was then put together to make the HiC contact maps. The bowtie2 aligner was used to align each reads from each end. Only uniquely mapping reads were considered for further analysis.
The mapping is interative and starts with first 25 bases of each read, this length is extended by 5 bases till 75 bases to look for a unique mapping.
The assembled fragments are then filtered at the restriction fragment level and all the reads that span more than 4 restrction sites are discarded. All the double sided reads are discarded. All the reads right next to the restriction site (possible dangling ends) are discarded. All the duplicates are discarded. All the fragments with very high or very low counts are discarded (these are usually possible PCR artefacts). Very large and very small fragments are discarded. All the interactions separated by very few restriction fragments are also discarded. 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. Do the iteractive correction. Interpolate the singleton bins (singleton bins are the filtered out bins that are flanked by non-filtered out bins). Normalize the sum = 1 along each row and column.
For replicate samples, an average matrix was used.
Genome_build: S. pombe Sept 2007 version from PomBase.
Supplementary_files_format_and_content: Each file contains a square matrix. Each row and column has three parts, the
Supplementary_files_format_and_content: bin number, the number of a 10 kB bin. Each bin number begins with number of
Supplementary_files_format_and_content: the chromosome on which that bin belongs. Then there is name of the organism,
Supplementary_files_format_and_content: and finally the genomic coordinates of the regions represented by that bin.
Supplementary_files_format_and_content: Each cell in the matrix denotes score of interaction between the bins denotes by
Supplementary_files_format_and_content: the defining row and column. The NA and NANs are the values that were discarded
Supplementary_files_format_and_content: during the process of generation of the interaction scores.
|
| |
|
| Submission date |
May 05, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Shiv Grewal |
| Phone |
2407607553
|
| Organization name |
NCI
|
| Department |
LBMB
|
| Lab |
Shiv Grewal
|
| Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL13988 |
| Series (2) |
| GSE56849 |
High resolution Hi-C analysis in S. pombe reveals fundamental elements of genome architecture |
| GSE57316 |
High resolution Hi-C analysis in S. pombe reveals fundamental elements of genome architecture (NGS) |
|
| Relations |
| BioSample |
SAMN02744538 |
| SRA |
SRX533439 |
