|
| Status |
Public on Sep 28, 2017 |
| Title |
JJC1392_LB_BR2 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial Cell Lysates
|
| Organism |
Escherichia coli |
| Characteristics |
strain: K-12 MG1655
growth medium: LB
chip antibody: anti-RecA antibody (Abcam, ab63797)
|
| Treatment protocol |
Protein DNA interactions were crosslinked for 10 min at 22.5C with 1% formaldehyde and quenched using glycine to a final concentration of 0.5M
|
| Growth protocol |
Cells were grown in either M9 minimal medium supplemented with 0.4% glucose, 5 μM, CaCl2 and 1 mM MgSO4 or LB medium supplemented with 0.5% glucose at 37 °C to an OD600nm of 0.2-0.25
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were collected by centrifugation and washed three times in ice-cold 1X PBS. The pellet was then re-suspended in 250 μl ChIP buffer (200 mM Tris-HCl (pH 8.0), 600 mM NaCl 4% Triton X, Complete protease inhibitor cocktail EDTA-free (Roche)). Sonication of crosslinked samples was performed using the Diagenode Bioruptor® at 30s intervals for 10 min at high amplitude. After sonication, 350 μl of ChIP buffer was added to each sample, the samples were mixed by gentle pipetting and 100 μl of each lysate was removed and stored as ‘input’. Immunoprecipitation was performed overnight at 4°C using 1/100 anti-RecA antibody (Abcam, ab63797). IP samples were then incubated with Protein G Dynabeads® (Life Technologies) for 2 h at room temperature. All samples were washed 3 times with 1 X PBS + 0.02% Tween-20 before re-suspending the Protein G dynabeads in 200 μl of TE buffer (10 mM Tris (pH 7.4), 1 mM EDTA) + 1% SDS. 100 μl of TE buffer was added to the input samples and all samples were then incubated at 65°C for 10 h to reverse formaldehyde crosslinks. DNA was isolated using the MinElute PCR purification kit (Qiagen). DNA was eluted in 50 μl of TE buffer using a 2-step elution. Samples were stored at -20°C.
All samples were processed following NEB’s protocol from the NEBNext® ChIP-Seq library preparation kit.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
files in fastq format were converted to fasta format - Example: fastq_to_fasta -i [FILE_NAME].sanfastq -o [FILE_NAME].fasta
reads were mapped to the E. coli K12 MG1655 (NC000913.3) genome using Novoalign version 2.07 (www.novocraft.com) - Example: (novoalign -f [FILE_NAME].fasta -d NC000913.3.nix -r Random > [FILE_NAME].novo)
pyCRAC software (http://sandergranneman.bio.ed.ac.uk/Granneman_Lab/pyCRAC_software.html) was used to convert the novo file into a GTF file and hit tables - Example: pyReadCounters.py -f [FILE_NAME].novo --gtf=Ecoli_K12.gtf --ignorestrand
bedtools version 2.26.0 (http://bedtools.readthedocs.io/en/latest/) were used to generate bedgraph files that were viewed in IGB version 9.0.0 (bioviz.org/igb) - Example: bedtools genomecov -bg -i [FILE_NAME]_count_output_reads.gtf -g chromosome.txt > [FILE_NAME].bedgraph
Genome_build: Escherichia coli K12 MG1655 NC000913.3
Supplementary_files_format_and_content: bedgraph files including count data for the whole genome
|
| |
|
| Submission date |
Jul 05, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
David Leach |
| E-mail(s) |
D.Leach@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Street address |
KIng's Building
|
| City |
Edinburgh |
| ZIP/Postal code |
EH9 3FF |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18133 |
| Series (1) |
| GSE100817 |
Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme |
|
| Relations |
| BioSample |
SAMN07322327 |
| SRA |
SRX2984785 |
