|
| Status |
Public on Apr 19, 2011 |
| Title |
Granulocyte nucleosome cores_1 |
| Sample type |
SRA |
| |
|
| Source name |
primary granulocytes (mostly neutrophils)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Granulocytes
|
| Growth protocol |
Cells were obtained from donor buffy coat and purified using sucrose gradient and Ig-beads
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The ends of isolated mononucleosome core DNAs (Granulocyte neutrophils) were processed by treating 0.3 ug - 0.5 ug of the DNA samples with T4 polynucleotide kinase (New England Biolabs) at 37oC for 2.5 hours followed by ethanol precipitation and subsequent treatment with T4 DNA polymerase (New England Biolabs) in the presence of dNTPs for 15 min at 12oC. After purification using either the QIAquick Gel Extraction Kit as described above or the QIAquick PCR Purification Kit (Qiagen), linkering of previously annealed duplexes AF-SJ-47 (5' OH-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT 3’) / AF-SJ-48 (5' Pi-ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGGTT 3’) and AF-SJ-49 (5' OH-CTGCCCCGGGTTCCTCATTCTCT 3’) / AF-SJ-50 (5' Pi-AGAGAATGAGGAACCCGGGGCAGTT 3’) to the samples was accomplished by T4 DNA ligase during a 6.5-hour room-temperature incubation. The ligation reactions were separated on a 2% agarose gel, and the relevant band isolated as described above. Amplification of the linkered libraries was accomplished with 8 (granulocyte mononucleosome library), 10 (CD4+ lymphocytes, CD8+ lymphocytes and genomic control libraries) or 12 (in vitro library) cycles of polymerase chair reaction (PCR) using primers AF-SJ-47 (SOLiD P1 primer) and AF-SJ-49 (SOLiD P2 primer) with subsequent separation and purification using a 2% agarose gel and the QIAquick Gel Extraction Kit as described above. The number of cycles used in the PCR amplification were monitored and selected as described in (Gu and Fire Chromosoma 2009).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 2.0 |
| |
|
| Description |
Gran_TC_cores_1
Nucleosome-bound DNA isolated by micrococcal-treatment of chromatin, followed by gel purification
|
| Data processing |
All sequence reads were aligned using SOLiD pipeline to human hg18 reference genome using the first 25 bps and allowing for up to two mismatches. RNA-Seq data was processed using custom scripts to obtain RPKM values for each gene isoform based on hg19 RefSeq annotation.
|
| |
|
| Submission date |
Feb 20, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Anton Valouev |
| Organization name |
Stanford University
|
| Department |
Pathology
|
| Lab |
Sidow Lab
|
| Street address |
300 Pasteur Dr.
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94035 |
| Country |
USA |
| |
|
| Platform ID |
GPL9138 |
| Series (1) |
| GSE25133 |
Determinants of nucleosome organization in primary human cells |
|
| Relations |
| SRA |
SRX046670 |
| BioSample |
SAMN00217090 |
