|
| Status |
Public on Sep 13, 2018 |
| Title |
Rif treated GapR-3xFLAG ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
Caulobacter crescentus NA1000
|
| Organism |
Caulobacter vibrioides NA1000 |
| Characteristics |
growth stage: exponential phase
genotype: gapR::gapR-3xFLAG
media: PYE
growth treatment: 25 µg/mL of rifampicin was added for 20 min prior to fixation
|
| Growth protocol |
Caulobacter crescentus was grown to OD 0.2-0.4 before treatment or harvest
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Libraries were constructed by end repairing DNA with T4 DNA polymerase (NEB), T4 PNK (NEB), and Klenow large fragment (NEB). 3’ overhangs were added with Klenow (3'→5' exo-) (NEB). Y-shaped Illumina adapters were ligated onto the DNA, and libraries were amplified with either Phusion (NEB) or Kapa HiFi Polymerase (Kapa Biosystems). Libraries were size selected to be 250-600 bp.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
gDNA
Cells were fixed with 1% formaldehyde and quenched with glycine. Cells were lysed with lysozyme and sonication, and cell debris cleared by centrifugation. Supernatants were diluted to use 500 μg of protein was used for ChIP and pre-cleared with 50 μL of Protein-A Dynabeads (Life Technologies) pre-blocked in ChIP buffer + 0.01% SDS and 100 μg ultrapure BSA (Ambion). Beads were pelleted and 90 μL of the supernatant was removed as input DNA. Remaining pre-cleared supernatant was incubated with 1 µL of FLAG antibody (Sigma), and immune complexes were captured with pre-blocked Protein-A Dynabeads. Beads were then washed and complexes eluted. Crosslinking was reversed at 65 °C in the presence of RNase A, and treated with Proteinase K (NEB). gDNA was recovered by phenol:chloroform:isoamyl alcohol extraction and precipitation.
|
| Data processing |
Reads were aligned to Caulobacter NC_011916.1 (NA1000) with bowtie (single-end reads) or bowtie2 (paired end reads) allowing for 1 mismatch
Bowtie alignments were converted to wiggle files by mapping the center of each read.
Genome_build: NC_011916.1
Supplementary_files_format_and_content: wig files were generated using custom python scripts, tab delimited files, first column represents genome position, second column represents value of position
|
| |
|
| Submission date |
Jun 29, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Monica S Guo |
| E-mail(s) |
msguo@uw.edu
|
| Organization name |
University of Washington
|
| Department |
Microbiology
|
| Lab |
Guo
|
| Street address |
750 Republican St
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL20784 |
| Series (1) |
| GSE100657 |
A bacterial chromosome structuring protein binds overtwisted DNA to stimulate type II topoisomerases and enable DNA replication |
|
| Relations |
| BioSample |
SAMN07299076 |
| SRA |
SRX2969616 |
