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Sample GSM2757255

Query DataSets for GSM2757255

Status

Public on Sep 29, 2017

Title

0x58 replicate 2 state 1 (IPTG-/aTc-/Ara-)

Sample type

SRA

Source name

Culture grown in 14 ml tube

Organism

Escherichia coli

Characteristics

strain: NEB 10-beta

Growth protocol

Individual colonies were inoculated into MOPS EZ Rich Defined Medium (Teknova, CA, M2105) with 0.2% glycerol carbon source and 50 μg/mL kanamycin (Gold Biotechnology, MO, K-120-5) and grown overnight for 16 hours at 37 °C and 1000 RPM in V-bottom 96-well plates (Nunc, Roskilde, Denmark, 249952) in an ELMI Digital Thermos Microplates shaker incubator (Elmi Ltd, Riga, Latvia). The following day, cultures were diluted 178-fold (two serial dilutions of 15 µL into 185 µL) into EZ Rich glycerol with kanamycin, and grown under the same ELMI shaker incubator conditions for three hours. Cells were diluted 658-fold (4.56 µL into 3 mL) into culture tubes (Falcon 14 mL round-bottom polypropylene tubes; Corning, MA, 352059) containing EZ Rich glycerol with kanamycin and inducers. For Erlenmeyer flask assays (Pyrex 250 mL; Cole-Palmer, IL, 4980-250), cells were diluted 658-fold (76 µL into 50 mL) into EZ Rich glycerol with kanamycin and inducers. Eight inducer combinations were used that cover the presence or absence of 0.5 mM IPTG, 10 ng/mL aTc, and 5 mM L-arabinose. Culture tubes were then grown in an Innova 44 shaker (Eppendorf, CT) at 37 °C and 250 rpm for five hours.

Extracted molecule

total RNA

Extraction protocol

Cultures were spun down at 4 °C, 15,000 × g for 3 minutes. Supernatants were discarded after centrifugation and cell pellets were flash frozen in liquid nitrogen for storage at -80 °C. Cells were lysed with 1 mg of lysozme (Sigma Aldrich L6871) in 10 mM Tris-HCl (pH 8.0) (USB 75825) supplemented with 0.1 mM EDTA (USB 15694). RNA was extracted with PureLink RNA Mini Kit (Life Technologies) and further purified and concentrated with RNA Clean & Concentrator-5 (Zymo Research) to assure sample quality. The purified RNA samples were analyzed using a Bioanalyzer (Agilent) and Ribo-Zero rRNA Removal Kit for bacteria (Illumina) was used to deplete rRNA from the samples.
Strand specific RNAtag-seq libraries were created by the Broad Technology Labs specialized service facility (SSF) using the standard protocol described in Shishkin et al. Nature Methods 2016.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Reads mapped to reference using BWA versiopn 0.7.4 with default settings
Read counts for each host and circuit gene was carried out using the htseq-count command of the HTSeq toolkit with user-defined GFF annotations of the reference sequences and the options '-s reverse -a 10 -m union'.
Normalized FPKM values were generated from the raw gene counts by custom scripts that calculated and applied a trimmed mean of M-values (TMM) factor using edgeR version 3.8.6.
Genome_build: NC_010473.1
Supplementary_files_format_and_content: CSV files of raw read counts and normalized gene expression in FPKM units.

Submission date

Aug 28, 2017

Last update date

May 15, 2019

Contact name

Thomas Edward Gorochowski

E-mail(s)

tom@chofski.co.uk

Organization name

University of Bristol

Department

Biological Sciences