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Sample GSM2723766

Query DataSets for GSM2723766

Status

Public on Aug 10, 2017

Title

Exp 2_IP sample

Sample type

SRA

Source name

Bacterial cells

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

growth stage: log-phase
medium: M9 medium supplemented with 0.2% glucose and 5% LB
antibody: Anti-Dps antibodies
ngs platform: Illumina MiSeq
sequencing mode: paired-end 2*150 nt protocol

Treatment protocol

ChIP-seq experiments were performed in duplicate using similar growth condition and protocols for chromatin immunoprecipitation. The first experiment was done in the Centre for Genomic Regulation (CRG, Barcelona, Spain), while the second one in the Immanuel Kant Baltic Federal University (Kaliningrad, Russia). In both cases, bacterial cells were grown aerobically at 37°C until OD600~0.6 were treated with formaldehyde (final concentration of 1%, 20 min incubation). Cross-linking was stopped with glycine (final concentration of 450 mM). After 5 minutes of incubation the cells were pelleted by centrifugation at 14,000 rpm for 15 minutes (+4°C), washed twice with 5 ml of PBS and resuspended in 1.3 ml of ice-cold immunoprecipitation buffer prepared from 50 ml of buffer containing 100 mM NaCl, 50 mM Tris-HCl (pH 8.1), 5 mM EDTA, 0.2% NaN3, 0.5% SDS, and 25 ml of buffer containing 100 mM Tris-HCl (pH 8.6); 100 mM NaCl; 5 mM EDTA; 0,2% NaN3, 5% Triton-X-100. Then phenylmethylsulfonyl fluoride (final concentration of 1 mM) or 20 μl of Protease Inhibitor Cocktail (PIC, Sigma) for the first and the second experiment, respectively, were added followed by incubation for 30 min at +4°C.

Growth protocol

Bacterial cells were grown aerobically in M9 medium supplemented with 0.2% glucose and 5% LB at 37°C under constant shaking (~120 rpm) in a water bath and harvested at OD ~0.6.

Extracted molecule

genomic DNA

Extraction protocol

For immunoprecipitation, 800 μl of collected chromatin (~1000 μg of protein) were incubated on a rotating wheel over night at 4°C with 10 μg of either rabbit anti-Dps antibodies (experimental sample) or rabbit pre-immune IgG (negative control). Next day 30 μl of Ultra Link Protein A/G beads (Thermo Scientific, USA) were added to the samples, and incubation was allowed for a further 2 hours at 4°C on a rotating wheel. The beads were washed 3 times with 1 ml of the low-salt buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1% Triton X-100, 1xPIC) and then with 1ml of the high-salt buffer (500 mM NaCl). Immunoprecipitated DNA-protein complexes were removed from the beads by 3 h shaking at 65°C and 1000 rpm in 110 μl of freshly prepared elution buffer, containing 100 mM NaHCO3 and 1% SDS. After centrifugation at 3000 rpm for 5 minutes, 100 μl of the supernatant was transferred to a new tube, and the DNA was purified with a PCR Purification Kit (Qiagen, Germany). The DNA concentration was measured on Qubit 2.0 using Qubit dsDNA HS Assay kit. A total of 8-10 identical samples were combined and concentrated to a volume of 30-50 μl containing 5-10 ng of the DNA.
ChIP-seq libraries were prepared from 5-10 ng of the DNA samples with the NebNext® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) following the manufacturer’s instructions. For the final amplification of the library 15 PCR cycles were used. Size distribution and concentration of the amplicons was checked on the Bioanalyzer 2100 (Agilent, USA). In the first experiment, the maximum was at about 300 bp, and ChIP libraries were sequenced using 50 nt single-end read protocol on the Illumina HiSeq system (Illumina, USA) of the Genomics Facility in the Centre for Genomic Regulation (Barcelona). In the second experiment, the maximum was at about 450 bp, and samples were sequenced using standard paired-end 2*150 nt protocol on the MiSeq system (Illumina, USA) in the Immanuel Kant Baltic Federal University (Kaliningrad).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina MiSeq

Description

Experimental sample
Experiment_2_CLC_GW_peaks.txt
Experiment_2_Matcher_peaks.txt

Data processing

Raw data were quality filtered on the Galaxy server (Filter by Quality tool, Q≥20) providing 99% probability of correct sequencing for all nucleotides in each read. As a result, the sets contain 31,656,551 and 45,396,252 sequences in the control and immunoprecipitated samples of the first experiment, and, respectively, 7,493,528 and 8,214,737 reads for the second experiment.
Two different approaches were used to align sequence reads to the genome of E. coli K-12 MG1655 (U00096.3). First, quality controlled reads were mapped using the CLC Genomics Workbench version 7.5.1 (CLC GW, Bio-Qiagen, Aarhus, Denmark) with either default settings for the length and similarity fractions (0.5 and 0.8, respectively, see supplemental Table 2 of the paper) or with the most stringent criteria (1.0 and 1.0, respectively, deposited). Reads with sequences aligned to multiple genomic regions were ignored.
To apply the second approach variable in length reads from Illumina MiSeq were first trimmed from both sides to obtain a set of standard 50 nt sequences taken from the middle of longer reads. Reads shorter than 50 nt were discarded. Then, all four sets from both experiments were aligned to the genome using the Matcher program (available at: http://www.mathcell.ru/DnaRnaTools/Matcher.zip). This software maps only 5’-ends if the reads correspond to the top strand of genomic DNA, or only 3’-ends, if they are aligned to the bottom strand. As such, the signals from fully complementary reads will match the same (left) position. Reporting the distribution of matching reads across the genome the program also evaluates reads with multiple occurrence and their positions.
Only ideal correspondence of sequence reads to the genome in both cases was permitted. The profiles obtained for experimental and control samples were normalized by the scaling method initially offered by Affymetrix for microarray data analysis and later implemented in several other approaches. This method assumes unaffected protein occupancy at most genomic positions and quantifies the scaling factor on the basis of corrected mean values obtained after removal of 2% signals with highest and lowest intensities from both control and experimental sets.
After normalization, the read counts were estimated in the running windows of 35 bp, and the ratios R between values obtained for experimental and control libraries were calculated. Peaks were localized requiring R≥1.5 for at least 50% positions of at least 60 bp genomic region in length.
Genome_build: E. coli K-12 MG1655 (U00096.3)
Supplementary_files_format_and_content: Processed data include read pileups obtained in two ChIP-seq experiments by two different approaches (files 1-4), the set of regions bound by Dps in both experiments (file 5) and the set of regions unbound by Dps in both experiments (file 6). All processed data are provided as tab-delimited .txt files.

Submission date

Jul 31, 2017

Last update date

May 15, 2019

Contact name

Olga N Ozoline

E-mail(s)

[email protected]

Organization name

Institute of Cell Biophysics of Russian Academy of Sciences

Department

Functional Genomics and Cellular Stress