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| Status |
Public on Sep 17, 2018 |
| Title |
Rec27-GFP_rec8D_interacting |
| Sample type |
SRA |
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| Source name |
Meiotic culture
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
epitope tag: Rec27-GFP
relevant genotype: pat1-114 rec8D
material ligated: Crosslinked DNA complexes
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| Growth protocol |
S. pombe cells were grown in minimal media without nitrogen for ~18 hours to synchronize the cells. Nitrogen was added and the temperature shifted to 34°C, marking the start of meiosis, and cells harvested at 3.5hr.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Formaldehyde was added to a final concentration of 1% in 500 mL of culture. Chromatin was prepared and sonicated as in Fowler et al. 2013 Mol. Cell 49:983-996. DNA was sheared to ~1000 bp and the epitope tagged protein of interest was immunoprecipitated. For conformation capture samples ("interacting"), the immunoprecipitate was ligated while immobilized on magnetic beads and subsequently eluted. Crosslinks were removed with high temperature incubation and protein digested using proteinase K. For "de-crosslinked" samples, immunoprecipitated chromatin was eluted, crosslinks removed, and protein digested prior to DNA ligation. In both cases, the ligated DNA was finally ethanol precipitated and amplified using one of two different kits (Sequenase Version 2.0 DNA Sequencing Kit, Affymetrix; or REPLI-g Single Cell Kit, Qiagen).
DNA from all samples was amplified using either a Sequenase Version 2.0 DNA Sequencing Kit (for anti-GFP) or a Qiagen REPLI-g Single Cell Kit (for anti-FLAG). DNA was quantified using a Bioanalyzer (Invitrogen). Sequencing libraries were each prepared from 1 µg of DNA. DNA was initially fragmented with a Covaris LE220 Focused-ultrasonicator (Covaris, Woburn, MA) using factory settings for an average size of 300 bp. Library DNA was prepared using the KAPA DNA Library Preparation and HiFi PCR Kits (Kapa Biosystems, Wilmington, MA) on a PerkinElmer Sciclone NGSx Workstation (PerkinElmer, Waltham, MA) for Rec25-FLAG and Rec27-FLAG IPs, or the Epicentre Nextera DNA Sample Prep kit (Illumina, Inc., San Diego, CA) manually for Rec27-GFP IPs.
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| Library strategy |
ChIA-PET |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Base calling: Illumina Real Time Analysis software, v1.12.4 or v1.17.21.3
De-multiplexing: Illumina CASAVA software, v1.8.0 or 1.8.2
Read alignment: Burrows-Wheeler Aligner, v 0.7.9a
Terminal base (bp 50) removed from each read; only first 49 used for alignment. One mismatch was permitted in the 49 bp. Each paired-end read was mapped separately and only analyzed further if both ends successfully mapped to single chromosomal position.
Uniquely mapping paired-reads were compiled and analyzed in R
Genome_build: Sanger Center S. pombe genome, August 2010 version
Supplementary_files_format_and_content: Unique ligation list
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| Submission date |
Sep 11, 2018 |
| Last update date |
Sep 17, 2018 |
| Contact name |
Kyle Fowler |
| Organization name |
University of California San Francisco
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| Street address |
600 16th Street
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| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL13988 |
| Series (2) |
| GSE119745 |
Physical basis for long-distance communication along meiotic chromosomes (ChIP-seq) |
| GSE119921 |
Physical basis for long-distance communication along meiotic chromosomes |
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| Relations |
| BioSample |
SAMN10034078 |
| SRA |
SRX4670920 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data not provided for this record |
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