|
| Status |
Public on Jun 25, 2014 |
| Title |
WT_SOLID_313 |
| Sample type |
SRA |
| |
|
| Source name |
meiotic culture
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
epitope tag: Rec12-flag
genotype: pat1-114
ploidy: diploid
|
| Growth protocol |
Synchronized meiotic cultures were prepared at 30°C and harvested at 4.5 hr.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Based on Milman et al. 2009 Mol Cell Biol 29: 5998-6005 and Pan et al. 2011 Cell 144:719-731. Whole cell extracts were prepared by grinding cells with glass beads and extracting in 10%TCA. Pellets were then extracted with SDS and soluble protein was subjected to immunoprecipitation of Rec12-flag with anti-flag antibodies. Protein was digested with proteinase K and Rec12-associated oligonucleotides were recovered by ethanol precipitation.
Based on Pan et al. 2011 Cell 144:719-731. Oligonucleotides were tailed with ribo-G using rGTP and terminal transferase, then ligated to duplex DNA adaptors with a dC overhang using T4 RNA ligase 2. Complementary strands were synthesized with Klenow, then purified by denaturing polyacrylamide electrophoresis. Purified complementary strands were subjected to 3' tailing with ribo-G using rGTP and terminal transferase, then another duplex adaptor was ligated using T4 RNA ligase 2. Ligated material was amplified with 16 cycles of PCR to add 454 or SOLiD adapters.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
AB SOLiD 4 System |
| |
|
| Description |
Rec12-associated DNA oligonucleotides
|
| Data processing |
Base calls were made using 454 gsRunBrowser for samples run on 454, and SOLiD Analysis Tools (SAT) for those run on SOLID.
Sequenced reads were trimmed for adaptor sequence, then mapped to S. pombe whole genome using SHRiMP 2.2.2 gsmapper-cs with options -E --strata --global -o 10000 -V -n 1 -g -100 -e -100. Mapping was done in three steps: Step (1) Reads longer than 15bp; Default Seeds for gsmapper-cs; (2) Reads between 11 and 15bp: -s 1111101111 (ie seed values were reset); (3) Reads shorter than 11bp: -s 1111111
Maps of 5' ends of uniquely mapping sequence reads were compiled and analyzed in R
For reads that mapped to multiple locations, imputed maps ("multimap") were calculated by fractionally assigning each multi-mapper to each of its map positions in proportion to the density of uniquely mapped reads within +/- 250 bp
Genome_build: Sanger Center S. pombe genome, Aug 7, 2010 version
Supplementary_files_format_and_content: plain tab-delimited text files include raw Rec12 oligo counts for each sample at each non-zero base position in the genome
|
| |
|
| Submission date |
Aug 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Bic MSKCC |
| Organization name |
Memorial SLoan-Kettering Cancer Center
|
| Street address |
1275 York Ave.
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL17165 |
| Series (1) |
| GSE49977 |
Mapping of Rec12 oligonucleotides in Schizosaccharomyces pombe |
|
| Relations |
| BioSample |
SAMN02318952 |
| SRA |
SRX336221 |
