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| Status |
Public on Dec 07, 2017 |
| Title |
3C-seq of E. coli wt cells harbouring plasmid pGBM2 in exponential phase 30°C -Replicate 1 |
| Sample type |
SRA |
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| Source name |
E coli cells
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
growth condition: MM + 0.12% casaminoacids +0.4% glucose at 30°C
genotype: F- lambda- ilvG- rfb-50 rph-1
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| Growth protocol |
E. coli cells were grown at 22°C, 30°C and 37°C in either Lennox Broth (LB) or liquid minimal medium A supplemented with 0.12% casamino acids and 0.4% glucose. The cultures were grown to OD600 = 0.2 (early exponential) or 2 (stationary phase).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For 3C-seq: ≈ 1-2 x 109 crosslinked cells (7% formaldehyde, unless otherwise stated ) are suspended in 600 μl Tris 10 mM EDTA 0.5 mM (TE) (pH 8) with 4 μl of lysozyme (35 U/μl; Tebu Bio), and incubated at RT for 20 minutes. SDS is added to the mix (final concentration 0.5%) and the cells are incubated for 10 minutes at RT. 50μl of lysed cells are then transferred in 8 tubes containing 450μL of digestion mix (1X NEB 1 buffer, 1% triton X-100, and 100U HpaII enzyme). DNA is digested for 3 hours at 37°C, split in 4 aliquots, and diluted in 8 ml ligation buffer (1X ligation buffer NEB without ATP, 1 mM ATP, 0.1 mg/ml BSA, 125 Units of T4 DNA ligase 5 U/μl). Ligation is then performed at 16°C for 4 hours, followed by incubation overnight (ON) at 65°C in presence of 250 μg/ml proteinase K and 5 mM EDTA. Precipitation of DNA is performed using an equal volume of 3 M Na-Acetate (pH 5.2) and two volumes of iso-propanol. After one hour at -80°C, DNA is pelleted, suspended in 500μl 1X TE buffer, and incubated for 30 minutes at 37°C in presence of RNAse A (0.03 mg/ml). DNA is then transferred into 2 ml centrifuge tubes, extracted twice with 500 μl phenol-chloroform pH 8.0, precipitated, washed with 1 ml cold ethanol 70% and diluted in 30 μl 1X TE buffer. All tubes are pooled and the resulting 3C library is quantified on gel using Quantity One software (BioRad).
3C-seq: 5 µg of a 3C library was suspended in water (final volume 130 µL) and sheared using a Covaris S220 instrument (Duty cycle 5, Intensity 5, cycles/burst 200, time 60 sec for 4 cycles). The DNA was purified and processed according to manufacturer instructions (Paired-End DNA sample Prep Kit – Illumina – PE-930-1001), except that DNA was ligated to custom-made adapters (see Marbouty M. et al, 2015, Mol Cell) for 4 hours at room temperature. Tubes were then incubated at 65°C for 20 minutes. DNA fragments ranging in size from 400 to 900 pb were purified using a PippinPrep apparatus (SAGE Science). For each library, four test PCR reactions were performed to determine the optimal number of PCR cycles (1 or 2 µL of the collected DNA, Illumina primers PE1.0 and PE2.0 using Taq Phusion [Finnzymes]). A large-scale PCR (8 reactions) was then set-up with the number of PCR cycles determined previously. The PCR product was finally purified on Qiagen MinElute columns and subject to paired-end sequenced on an Illumina sequencer.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: 3C-seq
3C-seq libraries were proccessed using the 3C-seq pipeline available at (https://github.com/koszullab/).
Removal of PCR duplicates based on the 20 first bp of the read containing a 6 nt random tag (home made script).
Iterative alignment, Min-leght=20, step=5, bowtie2 --very-sensitive
Filtering, Mapping quality=30, no ambiguous reads
Assignment to restriction fragment
Removal of un-informative events (uncuts, loops etc) as described in Cournac et al. BMC 2012.
Binning at 5kb.
Genome_build: E. coli MG1655 GenBank: U00096.2,total length : 4639675 bp
Supplementary_files_format_and_content: rna-seq: genomic position (middle of the 5kb-bin) to each count. 3C seq: contact maps (2D array of 928 5kb-bins x 928 5kb-bins).
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| Submission date |
Nov 29, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Axel Cournac |
| E-mail(s) |
acournac@pasteur.fr
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| Organization name |
Institut Pasteur
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| Street address |
25 rue Dr. Roux
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| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
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| Platform ID |
GPL21117 |
| Series (1) |
| GSE107301 |
Multiscale structuring of the E. coli chromosome by nucleoid-associated and condensin proteins |
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| Relations |
| BioSample |
SAMN08138985 |
| SRA |
SRX3451233 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2870431_mat_BC162_GACT_wt_pGBM2_MM_30C_rep1.txt.gz |
505.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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