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| Status |
Public on May 16, 2024 |
| Title |
ZM532, RM, Fur, rep2 |
| Sample type |
SRA |
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| Source name |
ZM532
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| Organism |
Zymomonas mobilis |
| Characteristics |
strain: ZM532
chip antibody: anti-Fur
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| Growth protocol |
Z. mobilis were grown to an OD600 of 0.6 in RM.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Formaldehyde was added to cultures to a final concentration of 1% and cells were crosslinked for 25 min at 30 °C with 100 rpm rotation. Quench formaldehyde was carried out by adding 0.25 M glycine. After crosslink, cells were collected by centrifugation at 4,000 rpm for 10 min and washed twice with 1× ice-cold Phosphate Buffered Saline. The crosslinked pellets were stored at -80 °C until use, or resuspended in 1 ml FA lysis buffer (50 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) per 50 ml cultures containing 2 mg ml-1 lysozyme and 1× Protease Inhibitor Cocktail II (Abcam), and then incubated at 37 °C for 30 min to lyse cells. Sonication was used to shred genomic DNA into 200-500 bp fragments, which were then centrifuged at 14,000 rpm for 15 min. The supernatant was retained and either used for immunoprecipitation or stored at -80 °C. Before starting the IP assays, ~ 2% of the supernatant was kept as the input sample, and the remaining extract was pre-cleared with 30 μl agarose protein G beads (CST, 9007s) at 4 °C for 1 h to reduce nonspecific background. The beads can be pelleted by centrifuging at 6,000 rpm for 1 min as per manufacturer’s instructions. 4 μg ml-1 anti-Zur or 10 μg ml-1 anti-Fur monoclonal antibody was added to the supernatant above and incubated at 4 °C overnight. After that, 30 μl agarose protein G beads were added and rocked at 4 °C for 1 h. After centrifugation, the beads were washed twice with 1 ml FA lysis buffer, once with high salt buffer (lysis buffer + 500 mM NaCl), once with LiCl buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 250 mM LiCl, 0.5 % Na deoxycholate), and twice with TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA). Finally, the beads were incubated with 100 μl elution buffer (50 mM Tris, pH 7.5, 10 mM EDTA, 1% SDS) for 20 min at 65 °C and washed once with 150 μl of TE + 0.67% SDS. The supernatant of eluted DNA complexes above was collected and then blended. In addition, 150 μl of TE + 0.67% SDS was added to the input sample. To reverse the crosslinking, all IP and input samples were incubated at 65 °C overnight. After treated with 1 μl of RNase A (10 mg ml-1) and 5 μl of Proteinase K (10 mg ml-1) at 37 °C for 1 h respectively, the immunoprecipitated chromatin was purified using phenol-chloroform-isoamyl alcohol method and resuspend in 50 μl TE buffer pH 7.5.
The DNA-sequencing library was generated by NEBNext Ultra II DNA Library Prep Kit (NEB) as per manufacturer’s instructions and subsequently sequenced on the Illumina HiSeq X Ten platform in 150PE mode.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Data processing |
The raw sequencing reads of fastq format were first processed by in-house Perl scripts.
Reads were subsequently checked by FastQC (v1.2) before proceeding with downstream processing.
Clean reads were then mapped to Z. mobilis ZM4 reference genome using Bowtie2 and further subjected to peak calling using MACS2.
The mapping results were visualized using the Integrative Genomics Viewer
Assembly: NC_006526.2
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)
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| Submission date |
Dec 21, 2022 |
| Last update date |
May 16, 2024 |
| Contact name |
Mao Chen |
| E-mail(s) |
chenmao92@hotmail.com
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| Organization name |
Biogas Institute of Ministry of Agriculture and Rural Affairs
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| Street address |
Section 4-13, Renmin Rd. South
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| City |
Chengdu |
| ZIP/Postal code |
610041 |
| Country |
China |
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| Platform ID |
GPL32966 |
| Series (2) |
| GSE221496 |
Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation [ChIP-Seq] |
| GSE221499 |
Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation |
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| Relations |
| BioSample |
SAMN32347297 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6876589_ZM532_RM_Fur_R2_peaks.narrowPeak.gz |
9.6 Kb |
(ftp)(http) |
NARROWPEAK |
| GSM6876589_ZM532_RM_Fur_R2_treat_pileup.bw |
6.6 Mb |
(ftp)(http) |
BW |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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