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Sample GSM2893436

Query DataSets for GSM2893436

Status

Public on Jan 21, 2019

Title

MS2-SraL

Sample type

SRA

Source name

Bacterium

Organism

Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

Characteristics

strain: SL1344 sraL-
growth conditions: anaerobic shock and late stationary phase of growth

Treatment protocol

The expression of MS2-sraL (or sraL control) was induced by addition of 1mM IPTG for 10 min. Then, harvested cells were chilled for 20 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).

Growth protocol

Salmonella Typhimurium SL1344 strains were grown in LB medium at 37°C (100 mL). Cells were harvested in (A) exponential phase after anaerobic shock (with agitation until an OD600nm=0.3, and then transferred in a filled 50 ml tube, incubated without agitation at 37ºC for 30 min) and in (B) late stationary phase (with agitation until an OD600nm=2 + 6h).

Extracted molecule

total RNA

Extraction protocol

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). Cells were then transferred to screw cap tubes containing 200 μL of glass beads (0.1 mm bead diameter; BioSpec). Cells were disrupted using Precellys®24 Homogenizer (three cycles: 6500 rpm for 20 s, followed by 1 min in ice; Bertin). The lysate was centrifuged at 17,000g for 30 min at 4 °C and the supernatant was applied to the column for affinity purification. MS2-affinity purification was performed as previously described (Lalaouna et al., 2017). Both lysates were applied on the same column (200 pmol of the MS2-MBP protein immobilized on an amylose resin). Afterward, RNA samples (output) were treated with TURBO™DNase (Ambion).
cDNA libraries were prepared with ScriptSeq™ v3 RNA-Seq Library Preparation Kit (Illumina) ScriptSeq™

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

Basecalls performed using MCS Version 2.4.1.3
Reads were aligned to Salmonella Typhimurium SL1344 genome (chromosome and plasmids) using Bowtie for Illumina (Galaxy Project)
Read counts were normalized by coverage according to Oshlack et al., 2010
Genome_build: Salmonella enterica subsp. enterica serovar Typhimurium SL1344
Supplementary_files_format_and_content: tab-delimited text file including normalized reads for each sample and MS2-SraL/SraL ratio

Submission date

Dec 18, 2017

Last update date

Jan 21, 2019

Contact name

Eric Massé

E-mail(s)

eric.masse@usherbrooke.ca

Organization name

University of Sherbrooke

Department

Biochemistry