|
| Status |
Public on Feb 06, 2012 |
| Title |
no_protein/D.mel_PB-seq_mock_seqB |
| Sample type |
SRA |
| |
|
| Source name |
mock IP (no HSF)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
library strategy: PB-Seq
library selection: immunoprecipitation
library source: naked, sheared genomic Drosophila S2 DNA
antibody: Anti-FLAG M2
|
| Growth protocol |
Prior to DNA extraction S2 were cultured as previously described (PMID: 20844575).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We performed an in vitro binding experiment with purified HSF and naked, sheared genomic Drosophila S2 DNA (PB-seq), to derive an accurate set of potential HSF binding sites in the Drosophila genome. HSF-bound DNA was specifically eluted and detected by high throughput sequencing. Drosophila HSF was N-terminally tagged with glutathione s-transferase and a tobacco etch virus (TEV) protease cleavage site. The C-terminus of the recombinant HSF was fused to the 3xFLAG epitope. Recombinant HSF was purified from E. coli with glutathione resin as previously described (PMID: 20078429) , with the following modifications: HSF-3xFLAG elution was achieved by addition of 6xHistidine tagged TEV protease and TEV protease was cleared from the HSF preparation using a Nickel-NTA column. We incubated 600pM HSF and 2500ng genomic DNA (sonicated to 100-600bp fragment size as previously described in PMID: 20844575) in 1500μl final volume of 1xHSF binding buffer and let it come to equilibrium for an hour at room temperature. We added 20μl ANTI-FLAG M2 affinity gel for 10 minutes, washed 8 times with 1xHSF binding buffer to remove unbound DNA; 3xFLAG peptide was added to a final concentration of 200ng/μl to specifically elute HSF and HSF-bound DNA. The mock IP was done in the absence of recombinant HSF.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
The PB-seq reads were aligned to the Drosophila Genome (BDGP R5/DM3) using BWA (v 0.5.8c)
|
| |
|
| Submission date |
Oct 03, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Joseph Guertin |
| E-mail(s) |
guertin@uchc.edu
|
| Organization name |
University of Connecticut
|
| Department |
Center for Cell Analysis and Modeling
|
| Lab |
Molecular Genomics
|
| Street address |
400 Farmington Ave
|
| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030 |
| Country |
USA |
| |
|
| Platform ID |
GPL13304 |
| Series (1) |
| GSE32570 |
Protein/DNA in vitro Binding seq (PB-seq) for Drosophila HSF and genomic Drosophila DNA |
|
| Relations |
| SRA |
SRX099637 |
| BioSample |
SAMN00736145 |
