|
| Status |
Public on May 15, 2022 |
| Title |
LaubLab_ChIPseq_antiFLAG_driD-3xFlag_pluszeo |
| Sample type |
SRA |
| |
|
| Source name |
Caulobacter crecentus CB15N
|
| Organism |
Caulobacter vibrioides |
| Characteristics |
growth phase: mixed population, mid-exponential phase
strain: CB15N
genotype: CB15N driD::driD-3xFlag
agent: 15ug/mL zeocin
antibody: antiFLAG
|
| Treatment protocol |
For samples 2 and 4, 15ug/mL of zeocin was added to induced double strand breaks. For sample 6, 0.5mM vanillate was added to induce expression of the restriction enzyme I-SceI to cause a single double strand break at the engineered I-SceI site on the genome.
|
| Growth protocol |
Cultures of Caulobacter crescentus were grown at 30oC in PYE to mid-exponential phase for all samples. Subsequently, formaldehyde was added to fix cells for ChIP-seq.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For samples 1-6, cultures were treated with formaldehyde to fix cells, and glycine was added to quench. Further washes and protein-DNA complex isolation was performed following protocols in the Methods.
Standard library construction for Illumina NextSeq500 sequencing platform.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
cells were grown to mid exponential phase (in PYE at 30 degC) treated with or without DNA damage (zeocin to cause DSBs or vanillate to induce I-SceI) before DriD-3xFLAG-DNA complexes were extracted and immunoprecipitated with anti-FLAG beads
|
| Data processing |
For samples 1-6, paired-end reads were mapped to Caulobacter crescentus NC011916.1 using Bowtie2 (Langmead and Salzberg) with the following command: bowtie2 -x NA1000 -1 *sequence_1.fastq -2 *sequence_2.fastq -S output.sam. Subsequently, sam files were converted to bams samtools: samtools view -Sbo *.bam *.sam. Bam files were ordered with samtools: samtools sort *.bam _sorted.bam. Genome coverage and conversion to wig files were performed with custom python scripts. Final visualization was performed in MochiView, with read count normalized by total number of reads, relative to Sample 1.
|
| |
|
| Submission date |
Mar 06, 2022 |
| Last update date |
May 17, 2022 |
| Contact name |
Kevin Gozzi |
| E-mail(s) |
kgozzi@mit.edu
|
| Phone |
2038153995
|
| Organization name |
Massachusetts Institute of Techonology
|
| Street address |
31 Ames St
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL24555 |
| Series (1) |
| GSE197978 |
ssDNA is an allosteric regulator of the C. crescentus SOS-independent DNA damage response transcription activator, DriD |
|
| Relations |
| BioSample |
SAMN26490140 |
| SRA |
SRX14388043 |
