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| Status |
Public on Jun 07, 2023 |
| Title |
∆ntrC xylX::pPTM057-3xFLAG-ntrC output DNA |
| Sample type |
SRA |
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| Source name |
cultures
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| Organism |
Caulobacter vibrioides NA1000 |
| Characteristics |
tissue: cultures
treatment: untreated
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| Growth protocol |
The ∆ntrC xylX::pPTM057-3xFLAG-ntrC strain was grown overnight in PYE at 30˚C. The overnight culture was diluted to OD660 0.1 in PYE and outgrown for 2 h at 30˚C. This culture was back-diluted to OD660 0.1 in PYE supplemented with 50 µM cumate and grown for 3.25 h at 37˚C to induce 3xFLAG-ntrC during log-phase growth.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To crosslink 3xFLAG-NtrC to DNA, formaldehyde was added to 125 ml of culture to a final concentration of 1% (w/v) and shaken at 37˚C for 10 min. The crosslinking was quenched using a final concentration of 125 mM glycine and shaken at 37˚C for 5 min. Cells were pelleted by centrifugation at 7,196 x g for 5 min at 4˚C. Supernatant was removed and the pellet was washed 4 times with ice-cold PBS pH 7.5. To lyse the cells, the washed pellet was resuspended in 1 ml lysis buffer [10 mM Tris pH 8, 1 mM EDTA, protease inhibitor tablet (Roche), 1 mg/ml lysozyme]. After a 30 min incubation at 37˚C for 0.1% (w/v) sodium dodecyl sulfate (SDS) was added. To shear the genomic DNA to 300-500 bp fragments, the lysate was sonicated on ice for 10 cycles (20% magnitude for 20 sec on/off pulses using a Branson Sonicator). Cell debris was cleared by centrifugation (15,000 x g for 10 min at 4°C). Supernatant was transferred to a clean tube and Triton X-100 was added to a final concentration of 1% (v/v). The sample was pre-cleared via incubation with 30 µl of SureBeads Protein A magnetic agarose beads (BioRad) for 30 min at RT. The supernatant was transferred to a clean tube and 5% of the total lysate was saved as the input DNA reference sample. Pulldown was performed as previously described (58). Briefly, 100 ul magnetic agarose anti-FLAG beads (Pierce / Thermo) were pre-equilibrated in binding buffer [10 mM Tris pH 8 at 4°C, 1 mM EDTA, 0.1% (w/v) SDS, 1% (v/v) Triton X-100] supplemented with 1% (w/v) bovine serum albumin (BSA) overnight at 4˚C, washed with binding buffer and incubated in the lysate for 3 hours at room temperature. Beads were cleared from the lysate with a magnet, and washed with a low-salt buffer [50 mM HEPES pH 7.5, 1% (v/v) Triton X-100, 150 mM NaCl], followed by a high-salt buffer [50 mM HEPES pH 7.5, 1% (v/v) Triton X-100, 500 mM NaCl], and then LiCl buffer [10 mM Tris pH 8 at 4°C, 1 mM EDTA, 1% (w/v) Triton X-100, 0.5% (v/v) IGEPAL CA-630, 150 mM LiCl]. Finally, beads were incubated with 100 ul elution buffer [10 mM Tris pH 8 at 4°C, 1 mM EDTA, 1% (w/v) SDS, 100 ng/μl3xFLAG peptide] for 30 minutes at room temperature. After pulldown, the input sample was brought to equal volume as the output/pulldown sample using elution buffer [10 mM Tris pH 8, 1 mM EDTA pH 8, 1% SDS, 100 ng/µl 3xFLAG peptide]. Input and output samples were supplemented with 300 mM NaCl and 100 µg/ml RNAse A and incubated at 37°C for 30 min. Proteinase K was added to samples at a final concentration of 200 µg/ml and samples were incubated overnight at 65°C to reverse crosslinks. Samples were purified using the Zymo ChIP DNA Clean & Concentrator kit. ChIP DNA was sequenced at SeqCenter (Pittsburgh, PA). Briefly, sequencing libraries were prepared using the Illumina DNA prep kit and sequenced (150 bp paired end reads) on an Illumina Nextseq 2000.
Library preparation was performed using the Illumina DNA prep kit and 10bp UDI indices
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Paired-end reads were mapped to the C. crescentus NA1000 reference genome (GenBank accession number CP001340) with CLC Genomics Workbench 20 (Qiagen). Peak calling was performed with the Genrich tool (https://github.com/jsh58/Genrich) on Galaxy; peaks are presented in Table S2. Briefly, PCR duplicates were removed from mapped reads, replicates were pooled, input reads were used as the control dataset, and peak were called using the default peak calling option [Maximum q-value: 0.05, Minimum area under the curve (AUC): 20, Minimum peak length: 0, Maximum distance between significant sites: 100]. To identify promoters that contained NtrC peaks, promoters were designated as 300 bp upstream and 100 bp downstream of the transcription start sites (TSS) annotated for each operon (65, 66). For genes/operons that did not have an annotated TSS, the +1 nucleotide of the first gene in the operon was designated as the TSS. Promoters were defined as containing an NtrC peak if there was any overlap between the NtrC ChIP-seq peak and the indicated promoter. To compare the relative location of NtrC binding sites with various cell cycle regulators, ChIPpeakAnno (67) was used to determine distance from the summit of the NtrC peaks to the nearest CtrA, SciP, MucR1, and GapR peak summit. To compare the relative location of NtrC binding sites with various cell cycle regulators, ChIPpeakAnno (67) was used to determine distance from the summit of the NtrC peaks to the nearest CtrA, SciP, MucR1, and GapR peak summit. ChIP-seq peaks (50 bp windows) for CtrA, SciP, and MucR1 were derived from (17) and the summits were considered the center of the 50 bp window. ChIP-seq summits for GapR were derived from (16). For motif discovery, sequences of the ChIP-seq peaks were submitted to MEME suite (68). Sequences were scanned for enriched motifs between 6 and 30 bp in length that had any number of occurrences per sequence.
Assembly: NA1000 Caulobacter crescentus
Supplementary files format and content: Processed data file contains log2 fold enrichment of reads (output DNA/input DNA) (SCD_346/SCD_345)
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| Submission date |
Jun 05, 2023 |
| Last update date |
Jun 07, 2023 |
| Contact name |
Sean Crosson |
| E-mail(s) |
crosson4@msu.edu
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| Phone |
5178845345
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| Organization name |
Michigan State University
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| Department |
Dept. Microbiology and Molecular Genetics
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| Street address |
567 Wilson Rd
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| City |
East Lansing |
| State/province |
Michigan |
| ZIP/Postal code |
48824 |
| Country |
USA |
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| Platform ID |
GPL33463 |
| Series (2) |
| GSE234096 |
The Caulobacter NtrB-NtrC two-component system bridges nitrogen assimilation and cell development [ChIP-seq] |
| GSE234097 |
The Caulobacter NtrB-NtrC two-component system bridges nitrogen assimilation and cell development |
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| Relations |
| BioSample |
SAMN35621727 |
| SRA |
SRX20592331 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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