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Sample GSM997760

Query DataSets for GSM997760

Status

Public on May 02, 2013

Title

SHX treated interactions S1B

Sample type

SRA

Source name

Escherichia coli MG1655

Organism

Escherichia coli

Characteristics

biological replicate: 1
strain: MG1655

Treatment protocol

For the serine hydroximate (SHX) treated samples the cultures were treated with SHX (500 µg/ml, 30 min) before harvesting.

Growth protocol

For Genome Conformation Capture (GCC)(1) analyses, E. coli strains were recovered from -80°C on LB agar (2%) plates for 24 hours. LB medium (3ml) starter cultures were inoculated and grown (37°C, 220rpm, 16h). The Optical Density (OD600) of cultures was measured and used to inoculate LB test cultures to an OD600 of ~0.02. The test cultures were grown (37°C, 220rpm) until the OD600 reached ~0.25 and then harvested.

Extracted molecule

genomic DNA

Extraction protocol

E. coli chromatin was prepared according to Rodley et al. (2009)(1) with the following modifications. A total of 5*109 cells were cross-linked with formaldehyde (1% final v/v, 20min, RT) and then quenched with glycine (125mM final, 10min). Cells were collected by centrifugation (4000rpm, 15min, 4°C), washed twice (1% PBS, 1% TritonX-100, 5ml/50ml culture) and pelleted (4000rpm, 15min, 4°C). Cell pellets were suspended in 800μl of B1 lysis buffer (10mM Tris pH 8.0, 50mM NaCl, 10mM EDTA, 20% (w/v) sucrose, 1mg/ml lysozyme) and incubated (37°C, 30min). 800μl of B2 lysis buffer (200mM Tris pH 8.0, 600mM NaCl, 4% TritonX-100, 1 protease inhibitor tablet (Roche) per 10ml of buffer added just before use) was gently added, mixed by inversion 3-4 times and incubated (37°C, 10min). The cell lysate was centrifuged (21,500g, 20min, 4°C) and the supernatant decanted. The chromatin was washed once with 1ml of chromatin digestion buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 0.1% TritonX-100) by inverting the tube 3-4 times and centrifuged (21,500g, 20min, 4°C). The supernatant was decanted and the chromatin pellet was suspended in 500μl chromatin digestion buffer. Chromatin samples were aliquoted into 10 sets of 5*108 cells. Samples were digested with HhaI (100U, New England Biolabs). Following digestion a ligation control was added (see below and Table 2) and the samples were ligated with T4 DNA ligase (20U, Invitrogen). Following ligation, cross-links were removed in the presence of proteinase K (0.45U, Fermentas). RNA was removed and pUC19 plasmid (27.4pg/2ml) was added as a sequencing control prior to three extractions with 1:1 Phenol:Chloroform. DNA was column purified (Zymo, DNA clean and concentratorTM-5 kit) according to the manufacturer’s instructions and eluted in milliQ H2O before combining for sequencing. 3μg of purified DNA was sent for paired-end sequencing at the ATC sequencing facility (Rockville, MD, USA) on an Illumina Hi-Seq.
Sequences were fragmented to 200 bp fragments. End filled and primers ligated on, following standard Illumina protocols.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

sample S1 was split and tagged with three mids. These were then run across two lanes on the hiseq to determine the effect of tag and sequence lane on the results
proximity ligation (Genome conformation capture) library
S1 sample tag B lane 6

Data processing

Sequences were intercalated
Network assembly was performed using the Topography suite v1.19. GCC networks were constructed from 100bp (E. coli) paired end Illumina Genome Analyser sequence reads. Topography uses the SOAP algorithm to position PE tags and single ends which contain a HhaI (E. coli) restriction site onto the E. coli (NC_000913). pUC19 (SYNPUC19CV), pRS426 plasmid, and Lambda phage ligation control sequences were also included in the genome reference file. No mismatches or unassigned bases (N) were allowed during positioning.
Except where indicated, bioinformatics and statistical analyses were performed on interactions in which the sequence reads were able to be mapped uniquely onto the reference genome and were above the FDR cut-off value (5).
Genome_build: ASM584v1
Supplementary_files_format_and_content: combined_L1_L2.txt includes LB grown (exponential) interactions. combined_S1_S2.txt includes serine hydroxamate treated (SHX) interactions.
Supplementary_files_format_and_content: The data in the columns is ordered in the following way: First interacting fragment (FIF) number/Second interacting fragment (SIF) number/Number of interactions between FIF and SIF/FIF Chromosome/ FIF Chromosome start/ FIF Chromosome stop/SIF Chromosome/SIF Chromosome Start/ SIF Chromosome Stop/FIF annotation/SIF annotation

Submission date

Sep 04, 2012

Last update date

May 15, 2019

Contact name

Justin M. O'Sullivan

E-mail(s)

justin.osullivan@auckland.ac.nz

Organization name

University of Auckland

Department

Liggins Institute