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Sample GSM2049269

Query DataSets for GSM2049269

Status

Public on Feb 06, 2016

Title

IAI1 transcriptome from chemostat growth_2

Sample type

SRA

Source name

IAI1 chemostat

Organism

Escherichia coli

Characteristics

strain: IAI1
growth protocol: Bacteria liquid culture
treatment: chemostat growth

Growth protocol

A minimal growth medium as described in the study by Ihssen and Egli (2004) was used for all experiments. Bacterial stock cultures were streaked onto agar plates and incubated overnight. One colony was then transferred into 20 ml minimal medium, grown at 37 °C (250 r.p.m.) over night culture (ONC) and served as the inoculum for experiments. For continuous culture experiments we designed and constructed 500 ml bioreactors according to the study by Huwiler et al., (2012) that were half-filled with medium (0.5 g glucose per l) and incubated at 37 °C in a temperature controlled water bath. Before continuous cultivation (dilution rate=0.25), 1–2 ml of the ONC was transferred and grown in batch-mode until reactors became visibly turbid. Subsequently, cells were grown to steady-state (defined as constant optical density over time) and harvested for experimentation. For starvation experiments the medium flow was stopped during steady-state and bacteria were collected after 4 h. To avoid gene-expression signatures of stationary cells from the ONC, batch cultures (1000 ml Erlenmeyer flasks containing 100 ml of pre-warmed medium (1 g glucose per l); 37 °C; 250 r.p.m.)) were inoculated with 5 ml of an exponentially growing pre-culture that derived from the ONC.

Extracted molecule

total RNA

Extraction protocol

Harvested cells (4 × 6 ml) were immediately combined with 6 ml RNAlater (Life Technologies, Grand Island, NY, USA), centrifuged for 15 min at 12 000 r.p.m., washed with 1 ml RNAlater (3 min at 15 000 r.p.m.), re-suspended in 0.5 ml RNAlater and stored at −20 °C. For RNA extraction the RiboPure-Bacteria Kit (Life Technologies) was used according to the manufacturer’s instructions. To achieve high RNA yields several reactions for each sample were done in parallel and pooled at the end of the procedure. An additional DNase treatment step with TURBO DNase (Life Technologies) was included to assure no genomic DNA contamination. Messenger RNA was enriched using the RiboMinus Bacteria Kit (Life Technologies) according to the manufacturer’s instructions.
RNA quality was confirmed with the Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) and sequencing (50 cycles, pooling eight bar-coded samples per lane) was performed on the Illumina HiSeq platform (Illumina, San Diego, CA, USA) at the Research Technology Support Facility (RTSF) at Michigan State University.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Processed data: raw_count.txt, normalized_count.txt, panGeneMap.txt, Gene_annotations.txt
IAI1CC2

Data processing

Quality trimming of 3' end at Read Segment Quality Control Indicator.
Map RNA-Seq reads to reference genomes using Bowtie (Langmead et al., 2009) with alignment parameters (-n 2-e 70-l 28 –best)
Summarize the read count to each annotated gene
Remove non-protein coding counts
Differential expression analysis with DESeq (Anders and Huber 2010) with the default setting
Genome_build: NC_011741 (E.coli strain IAI1), NC_000913.2 (E.coli strain MG1655), TW09308 (E.coli strain TW09308), and TW11588 (E.coli strain TW11588)
Supplementary_files_format_and_content: All in tab-delimited ASCII format. raw_count.txt (pre-normalized read count), normalized_count.txt, panGeneMap.txt (geneID map between the strains and pangene ID), Gene_annotations.txt (annotation of genes)

Submission date

Jan 28, 2016

Last update date

May 15, 2019

Contact name

Benli Chai

E-mail(s)

chaibenl@msu.edu

Phone

517-353-3842

Organization name

Michigan State University