|
| Status |
Public on Feb 12, 2015 |
| Title |
RpoH_chip_sample1 |
| Sample type |
SRA |
| |
|
| Source name |
RpoH_chip
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
strain: UCBP-PA14
genotype/background: harboring a plasmid encoding for the polyhistidine-tagged sigma factor gene
chip antibody: Anti-6X His tag antibody - ChIP Grade
chip antibody vendor: abcam
chip antibody cat. #: ab9108
chip antibody lot #: GR67062-1 and GR76800-1
|
| Treatment protocol |
DNA-protein crosslinking was performed by addition of formaldehyde to a final concentration of 0.5% for 5 minutes at ambient temperature. Reaction was quenched by addition of glycine to a final concentration of 137 mM. Cells were lysed in lysis buffer containing EDTA and lysozyme. Cells were disrupted and DNA was sheared by sonication.
|
| Growth protocol |
PA14 wild type harboring a plasmid encoding for the polyhistidine-tagged sigma factor gene was cultivated under optimal conditions regarding sigma factor activity. In addition, sigma factor expression was strongly induced by adding L-arabinose (0.5%) to the culture for at least 45 minutes prior to formaldehyde-mediated cross-linking.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Polyhistidine-tagged sigma-factors were isolated by using anti6Xhis-antibodies (abcam) and magnetic beads coupled with protein-G (Invitrogen) on a rotator.
Immunoprecipitated DNA was linear amplified similar to the LinDA-ChIP-seq protocol published by Shankaranarayanan et al., Nature potocols, 2012. Library was constructed using the TruSeq DNA sample preparation kit according to the low-throughput protocol (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
ChIP-seq treated rep1
|
| Data processing |
Sequenced ChIP-seq reads were trimmed for adaptor sequence allowing for a minimal quality of 10 at their ends, then mapped to the PA14 genome using bowtie v0.12.7 with default parameters
ChIP-seq peaks were called using the SAM files and the MACS algorithm with the following options: -g 5200000 –nomodel --shiftsize=30 -p 0.05
Comparisons were performed in the following way: sample1 vs control1; sample2 vs control2
Genome_build: gi|116048575|ref|NC_008463.1|
Supplementary_files_format_and_content: The MACS output files contain the coordinates of the enriched genomic regions as well as their P-values, false discovery rate (FDR) and fold enrichment
|
| |
|
| Submission date |
Feb 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Denitsa Eckweiler |
| E-mail(s) |
[email protected]
|
| Organization name |
Technische Universität Braunschweig
|
| Department |
Institute of Microbiology
|
| Lab |
Prof. Dieter Jahn
|
| Street address |
Spielmannstr. 7
|
| City |
Braunschweig |
| State/province |
Lower Saxony |
| ZIP/Postal code |
D-38106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18286 |
| Series (2) |
| GSE54997 |
ChIP-seq study of sigma factors in Pseudomonas aeruginosa |
| GSE54999 |
Elucidating the Sigmulome of P.aeruginosa PA14 by ChIP-Seq and RNA-Seq approaches |
|
| Relations |
| BioSample |
SAMN02641766 |
| SRA |
SRX470299 |
