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Sample GSM2111189

Query DataSets for GSM2111189

Status

Public on Dec 06, 2018

Title

MS2-GcvB

Sample type

SRA

Source name

Bacterium

Organism

Escherichia coli K-12

Characteristics

strain: MG1655 rne131 delta_gcvB
growth phase: exponential and stationary phases

Treatment protocol

0.1% arabinose was added to induce the expression of MS2-GcvB/GcvB during 10 min. Cells were chilled for 10 min on ice. Cells were then centrifuged, resuspended in 1 mL of buffer A (20 mMTris-HCl at pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT), and centrifuged again.

Growth protocol

Cells were harvested in exponential (OD600nm=0.5; 100 mL LB medium) and in stationary phase (OD600nm=1; 100 mL LB medium).

Extracted molecule

total RNA

Extraction protocol

Cells were resuspended in 3 mL of buffer A and lysed using a French Press (430 psi, four times). Lysate was then cleared by centrifugation. The soluble fraction was subjected to affinity chromatography. The column was prepared by adding 100 µL of amylose resin (New England Biolabs) to Bio-Spin disposable chromatography columns (Bio-Rad). The column was washed with 3 mL of buffer A. Next, 200 pmol of MS2-MPB protein was immobilized on the amylose resin, and the column was washed with 2 mL of buffer A. The cleared lysate was then loaded onto the column, which was washed with 8 mL of buffer A. RNA was eluted from the column with 1 mL of buffer A containing 15 mM maltose. Eluted RNA was extracted with phenol-chloroform, followed by ethanol (3 vol) precipitation of the aqueous phase in the presence of 20 mg of glycogen. RNA samples were treated with TURBO DNA-free (DNase treatment) and precipitated again.
cDNA libraries were prepared with ScriptSeq™ RNA-Seq Library Preparation Kit (Illumina)

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina MiSeq

Data processing

Basecalls performed using MCS Version 2.4.1.3
Reads were aligned to the Escherichia coli K12 genome assembly using Bowtie for Illumina (Galaxy Project)
Read counts were normalized by coverage according to Oshlack et al., 2010
Genome_build: Escherichia coli K12
Supplementary_files_format_and_content: tab-delimited text file include normalized reads for each sample and MS2-GcvB/GcvB ratio

Submission date

Apr 07, 2016

Last update date

May 15, 2019

Contact name

Eric Massé

E-mail(s)

eric.masse@usherbrooke.ca

Organization name

University of Sherbrooke

Department

Biochemistry