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Sample GSM1510152

Query DataSets for GSM1510152

Status

Public on Sep 01, 2015

Title

Sample_WT_stat_w-o_no-2

Sample type

SRA

Source name

E. coli MG1655 stationary phase, non-crosslink

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

growth phase: stationary phase

Growth protocol

Cells were grown in M9 minimal medium with glycerol as carbon source and harvested during exponential or stationary growth phase.

Extracted molecule

genomic DNA

Extraction protocol

Hi-C was performed similar to a previous study by van Berkum et al. (2010) with the following changes. 5x10^9 cells grown in M9 minimal medium with glycerol as carbon source were fixed in 1% formaldehyde in PBS buffer for 20 min at room temperature. In the case of non-crosslink controls, cells were incubated in pure PBS buffer. After quenching of fixation in 0,125 M glycine, cells were lysed with Ready-Lyse lysozyme (Epicentre Biotechnologies) in TE buffer for 20 min at room temperature. Lysis was stopped by incubation in 1% SDS for 30 min at room temperature. DNA was digested with 500 U MluI (NEB). DNA was extracted with PCIAA pH7.9 (roth) twice and chloroform once. DNA was precipitated in TE with 50 µg/ml glycogen and 0.3 M sodium acetate and 1 volume isopropanol. DNA was sheared to a size of 300-500 bp in a Nebulizer according to the Illumina Kit protocol and purified using the QIAquick PCR Purification Kit. Raw data files with denomination „no-3“ are from samples in which biotin-labeling and subsequent enrichment of ligation products was omitted.
Libraries were prepared for sequencing according to the Illumina TruSeq DNA Sample Prep v2 Low Throughput protocol.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

without crosslink

Data processing

Library strategy: Hi-C
Paired end reads were seperately aligned to the MG1655 reference genome NC_000913.3 using RazerS3.
Uniquely aligning reads were assigned to their corresponding MluI restriction fragment by means of their position on the genome.
Paired reads which have been assigned to their restriction fragments are then rejoined by means of their identical read-id.
Only pairs where both reads were assigned to different restriction fragments were used for further analysis.
Interactions between restriction fragments (paired reads that were assigned to different restriction fragments count as one interaction of those restriction fragments) were then converted into interactions between equally sized bins with a size of 20 kb, each, resulting in an interaction matrix of interactions between bins.
The interaction matrix was normalized using a method similar to Imakaev et al., 2012.
Normalized Hi-C contact maps from samples obtained under the same conditions (crosslink or non-crosslink; exponential or stationary phase) were compared by Pearson correlation and yielded values above 0.92.
Binning and normalization was thus performed on pooled data sets (all crosslink exponential phase samples, all non-crosslink exponential phase samples, all crosslink stationary phase samples and all non-crosslink stationary phase samples).
In order to detect crosslink-independent interactions, we divided the Hi-C contact matrices of the crosslinked samples by the Hi-C contact matrices of the corresponding non-crosslinked samples and logarithmized the resulting values. The resulting Hi-C contact matrices show the genomic crosslink-independent interactions.
Genome_build: NC_000913.3

Submission date

Sep 23, 2014

Last update date

May 15, 2019

Contact name

Jörg Hartig

Organization name

Universität Konstanz

Department

Chemistry

Street address

Universitätsstraße 10