|
| Status |
Public on Dec 01, 2021 |
| Title |
Input (IN) |
| Sample type |
SRA |
| |
|
| Source name |
KRX E. coli
|
| Organism |
Escherichia coli KRX |
| Characteristics |
chip antibody: None
plasmid for expression: pPMS1259 (encodes Tus-GFP)
|
| Treatment protocol |
Culture aliquots were subject to formaldehyde solution (final concentration of 1%) and the bacterial suspensions were reacted for 20 minutes at room temperature.
|
| Growth protocol |
Competent E. coli KRX bacteria were transformed with pPMS1259 (His6-Tus-GFP) and grown overnight at 37°C. Colonies were resuspended and diluted to an OD600 of 0.1 in LB broth supplemented with ampicillin. All cultures were grown for 45 minutes at 37°C before inducing moderate levels of Tus-GFP with Rhamnose 0.02 % Rhamnose (w/v final culture concentration). Bacteria were incubated for 2 hours at 37°C, followed by 2 hours at 16°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The lysates were passed three times in a French press at 12,000 psi and each lysate was heated for 10 minutes at 50°C to denature free Tus-GFP. Supernatants were immunoprecipitated (excluding input) with goat anti-GFP IgG (Abcam; Ab6673) and Immunocaptured DNA was released by adding elution and de-crosslinking buffer containing proteinase k. Samples were incubated at 95°C to denature proteinase K and residual crosslinked proteins.
Each library was prepared using the NEBNext Ultra DNA library preparation kit for Illumina.Briefly, 55.5 µL of DNA suspensions were end repaired. Due to the low DNA concentrations in the suspensions, the NEB adaptors were diluted 10 fold in water to 1.5 µM for ligation as recommended. The adaptors were cleaved using uracil excision. Size selection was not recommended for sample < 50 ng. DNA was then cleaned up using Sera-Mag beads (ratio of 1.4) and eluted in 28 µL of 0.1xTE. Index primers were added by PCR using 18 cycles. The samples were pooled in a single library, denatured and loaded for sequencing with an Illumina MiSeq desktop sequencer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Sample type contains three biological replicates e.g IN-1 represents biological replicate 1. The processed data is of each replicate combined
|
| Data processing |
Removal of Illumina adapters and leading and tailing nucleotides with a Phred score ≤ 10 was performed with Trimmomatic (v.0.36)
The replicate data for Input and ChIP-Seq Tus-GFP (pos) were pooled into three separate fastqc files and aligned to the KRX E. coli genome using the default settings of Bowtie2
Samtools (version 1.9) was then used to organise each alignment file
Genome_build: krx.fsa (pending GenBank submission)
Supplementary_files_format_and_content: The single base read counts was calculated for ChIP and Input using genomeCoverageBed (Version: v2.26.0). Columns are as follows Chromosome I.e contig_2_pilon; chromosome position; depth (number) of reads overlapping this chromosome position
Ter coverage text files are averaged read counts over the 23 base Ter sequences. Columns are as follows Chromosome; chromosome position; depth (number) of reads overlapping this chromosome position
|
| |
|
| Submission date |
Dec 21, 2020 |
| Last update date |
Dec 01, 2021 |
| Contact name |
Patrick Schaeffer |
| E-mail(s) |
patrick.schaeffer@jcu.edu.au
|
| Organization name |
James Cook University
|
| Department |
CPHMVS
|
| Lab |
Schaeffer
|
| Street address |
1 James Cook Drive
|
| City |
Douglas |
| State/province |
QLD |
| ZIP/Postal code |
4814 |
| Country |
Australia |
| |
|
| Platform ID |
GPL29521 |
| Series (1) |
| GSE163680 |
Genome-wide distribution of Tus-GFP in KRX E. coli |
|
| Relations |
| BioSample |
SAMN17132169 |
| SRA |
SRX9713514 |
