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| Status |
Public on Dec 25, 2017 |
| Title |
6F_GCC |
| Sample type |
SRA |
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| Source name |
Dividing t=2 hours
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| Organism |
Escherichia coli |
| Characteristics |
time post release: E. coli cells 2 hour following release from Stationary phase (t=2)
biological replicate: Replicate 2
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| Treatment protocol |
Released cultures were grown (37°C, 200 rpm) and harvested (time = 0, 1 hr, or 2 hr) for GCC and RNA isolation.
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| Growth protocol |
Synchronization was achieved by passaging cells through one round of stationary phase according to Cutler and Evans 1966. Briefly: Individual colonies (LB plate, 37°C, 24 h) were inoculated into M9 media and grown O/N (37°C, 200 rpm). M9 media was inoculated to a final OD600 = 0.25 (early-logarithmic phase). Cultures were grown (37°C, 200 rpm) and maintained in stationary phase (OD600 1.8) for approximately 2 hours. An appropriate amount of each culture was used to inoculate pre-warmed (37°C) M9 media to a final OD600 = 0.25 (approximate 7-fold dilution).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
E. coli chromatin was prepared according to Cagliero et al. 2013 with the following modifications. For each successive time point (T0, T60, T120), 100 mL of culture was cross-linked with formaldehyde (1% final v/v, 20 min, RT) and then quenched with glycine (125 mM final concentration, 10 min); 30 mL of culture was used for RNA extraction (see below); 2 x 1 mL samples were harvested for FACS and fluorescence microscopy. Following cross-linking and quenching, cells were collected in a 50 mL falcon tube by two successive rounds of centrifugation (4000 rpm, 15 min, 4°C). Pelleted cells were washed twice with 5 mL of 1% PBS, 1% Triton X-100, and then sedimented (4000 rpm, 15 min, 4°C). To break cells open, the cell pellets for each time point were suspended in 800 µL B1 lysis buffer (10 mM Tris pH 8.0, 50mM NaCl, 10mM EDTA, 20% (w/v) sucrose, 1mg/ml lysozyme) and transferred to 2 ml tubes. Each tube was incubated for 30 min at 37°C. 800 µL of B2 lysis buffer (200 mM Tris pH 8.0, 600 mM NaCL, 4 % TritonX-100, with 1 protease inhibitor tablet [Roche] per 10 ml of buffer added just prior to use) was gently added to each 2 mL tube, mixed by inversion 3-4 times, and incubated (30 min, 37°C). Cell lysates were centrifuged (21,500 g, 20 min, 4°C) and the supernatant removed. Chromatin pellets were washed once with 1 ml FA-lysis buffer (5 mM HEPES-potassium hydroxide pH 7.5, 14 mM NaCl, 100 µm EDTA pH 8.0, 9% of 11% Triton-x-100 (v/v), 1% of 10% deoxychlolic acid (v/v), with 1 protease inhibitor tablet [Roche] per 10 mL of buffer added just prior to use) by inversion (3-4 times) before centrifugation (21,500 g, 20 min, 4°C). The supernatant was decanted and the chromatin pellet suspended in 500 µL chromatin digestion buffer (10 mM Tris-HCL pH 8.0, 5 mM MgCL2, 0.1% TritonX-100). At this point samples were stored at -80°C until further use. 5 µL of 10% SDS was added to each tube and incubated for 10 min at 37°C. 45 µL of 11% TritonX-100 was added to make a final concentration of 1%. Each chromatin sample was aliquoted into 5 sets of 2 ml tubes (~5*108 cells per 55 µL aliquot) in order to carry out restriction enzyme digestion. A ligation control was added to the un-digested chromatin (either pRS426 or Lambda phage – see below). Samples were digested (37°C) with 5 µL HhaI (100U, New England Biolabs) for 2 hr with inversion every 20 min. 10% SDS (10 µL) was added to each tube and incubated for 20 min (65°C) to inactivate the restriction enzyme. Following restriction enzyme digestion, samples were diluted ~20 fold and ligated (2 hr at 16°C) with 20 µL T4 DNA ligase (20 U, Invitrogen). Ligation reactions were inactivated by adding: 20 µL 0.5 M EDTA (pH 8.0), 12 µL 5 M NaCl, 1.2 µL 1 M Tris-HCL (pH 7.5). Cross-links were removed by incubation O/N at 65°C in the presence of proteinase K (0.45 U, Fermentas). RNA was removed by the addition of 2 µL of RNAseA (10 mg/ml) to each tube and incubation for 15 min at 37°C. 2 µL of 13.7 ng/mL pUC19 plasmid was added to each 2 mL tube (a concentration that was equivalent to one plasmid per E. coli genome) to act as a sequencing control. Three extractions were carried out with equal volumes of phenol:chloroform (1:1) in large Oakridge tubes. DNA was column purified (Zymo, DNA clean and concentratorTM-5 kit) according to the manufacturer’s instructions. Each of the 12 columns was eluted using 11 µL of 65°C mQ H2O into 4 tubes, giving a final volume of 33 µL in each tube. Approximately 10 µg of purified DNA for each sample was sent for paired-end sequencing (100 bp) at the BGI Hong Kong sequencing facility.
Production of external ligation controls for GCC library preparation Two sets of primers were designed to obtain two PCR products (of approximately the same length between 150-200 bp) to act as ligation controls from the pRS426 and Lambda phage genome templates (Table S2). Both sets of primers were designed to include HhaI site at one end of the final product. Putative sequences of the ligation controls were aligned against the E. coli MG1655 genome using BLAST software to check for regions of homology. External ligation controls were produced by PCR amplification of short regions of the Lambda phase genome and the pRS426 plasmid using the two sets of primers. PCR amplification (95oC, 2min; [95oC, 30 s; 58oC, 20s;; 72oC, 20s] x35; 72oC, 2min) was performed on a GeneAmp® PCR System 9700. PCR products were analyzed following electrophoresis through a 2.0 % (w/v) agarose and subsequently viewed under ultraviolet illumination. PCR products were then gel purified using QIAquick Gel Extraction Kit (Quiagen), following the manufacturer’s instructions. Purified PCR products were digested with 4 U of HhaI and incubated for 2 hr at 37°C. Purified, digested PCR products were introduced into the GCC samples at a 1:1 ratio with the number of genomes prior to the restriction digestion step during GCC preparation. The Lambda ligation control was added to all time points of biological replicate one, and pRS426 ligation control was added to all time points of biological replicate two. Except where indicated, bioinformatics and statistical analyses were performed on interactions identified by sequence reads that were uniquely mapped onto the reference genome and were above the FDR cut-off value derived from the ligation control interactions (see below). All bioinformatics analysis was performed using in house Perl scripts, as previously described22.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Genome conformation capture
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| Data processing |
Library strategy: GCC
To identify interacting DNA fragments from the paired-end sequence reads, network assembly was performed using the Topography suite v1.19 (Rodley et al. 2012). GCC networks were constructed from 100 bp paired-end Illumina Genome Analyser sequence reads. Except where indicated, bioinformatics and statistical analyses were performed on interactions identified by sequence reads that were uniquely mapped onto the reference genome and were above the cut-off value derived from the ligation control interactions . All bioinformatics analysis was performed using in house Perl and Python scripts . Statistical analyses were performed in R.
Bioinformatics and statistical analyses were performed on interactions identified by sequence reads that were uniquely mapped onto the reference genome and were above the FDR cut-off value derived from the ligation control interactions. All bioinformatics analysis was performed using in house Perl scripts, as previously described (Cagliero, C., Grand, R. S., Jones, M. B., Jin, D. J. & O’Sullivan, J. M. Genome conformation capture reveals that the Escherichia coli chromosome is organized by replication and transcription. Nucleic Acids Res. 41, 6058–6071 (2013)).
During the preparation of the GCC samples, random ligation events can occur during: 1) the ligation of the cross-linked fragments, and 2) the sequencing preparation step (i.e. the addition of linkers during sequencing library preparation). Two methods were employed for the identification of significant interactions: 1) a statistical method that calculates a false detection rate (FDR) cut off; and 2) external controls were added during the GCC library preparation to obtain estimates of the rates of inter-molecular ligation events. Two inter-molecular ligations events were detected which was the same frequency as the calculated significance cut-off value. Therefore, the cut-off for filtering out random ligations was set to 3 interactions; only fragment pairs that occurred 3 or more times were considered for analysis.
Genome_build: Escherichia coli MG1655 reference genome (NC_00091313-Feb-2011
Supplementary_file: unique interaction file: *.dat.txt
Supplementary_file: all interactions file: *.datcomb.txt
Supplementary_file: multiple interactions file: *.datmult.txt
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| Submission date |
Sep 20, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Justin M. O'Sullivan |
| E-mail(s) |
justin.osullivan@auckland.ac.nz
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| Organization name |
University of Auckland
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| Department |
Liggins Institute
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| Street address |
85 Park Road, Grafton
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| City |
Auckland |
| ZIP/Postal code |
1023 |
| Country |
New Zealand |
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| Platform ID |
GPL14548 |
| Series (2) |
| GSE87117 |
The Escherichia coli nucleoid is shaped around replication and transcription [GCC] |
| GSE87118 |
The Escherichia coli nucleoid is shaped around replication and transcription |
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| Relations |
| BioSample |
SAMN05791203 |
| SRA |
SRX2178818 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2322593_6F_interlacedshortened_ra.out.dat.txt.gz |
18.5 Mb |
(ftp)(http) |
TXT |
| GSM2322593_6F_interlacedshortened_ra.out.datcomb.txt.gz |
25.7 Mb |
(ftp)(http) |
TXT |
| GSM2322593_6F_interlacedshortened_ra.out.datmult.txt.gz |
8.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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