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Sample GSM2299960

Query DataSets for GSM2299960

Status

Public on Aug 31, 2019

Title

GH_243_4_TAGCTT_L005

Sample type

SRA

Source name

Salmonella enterica serovar strain SG111

Organism

Salmonella enterica subsp. enterica serovar Typhimurium

Characteristics

strain: opgGH mutant

Growth protocol

Salmonella enterica serovar strain SL1344, its opgGH mutant and plasmid borne (opgGH) complemented strains (pBK16) [Microbiology 155, 229-237 (2009); Arch Microbiol. 192, 167-174 (2010)] were streaked on LB agar plates from freezer stocks, and a single colony was inoculated in LB broth and grown at 37 oC in a shaker incubator for 18-20 h. The medium was supplemented as needed with antibiotics at the following concentrations: ampicillin (100 µg ml-1), kanamycin (50 µg ml-1), nalidixic acid (10 µg ml-1). Osmolarity of growth media was measured with Wescor vapor pressure osmometer (model 5500, Wescor, Inc., Logan UT).

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from Salmonella cells from swarm edges, 7 h from the time cells were spotted on swarm agar plates. Cells were suspended in RNA stabilization reagent [J. Microbiol. Methods 55, 399-409 (2003)] and stored at -70 0 C till proceeding for RNA isolation. Cells were suspended in hot TRIzol RNA isolation reagent (LifeTechnologies, Carlsbad, CA) and processed for RNA isolation as described [Microbiol Discov. 2013; 1:10. http://dx.doi.org/10.7243/2052-6180-1-10]. RNA was treated with DNase three times (twice on column and once in solution) and DNA contamination was checked by PCR using primers against rpoD and 16S RNA genes.
The messenger RNA population from all RNA samples was enriched by depleting rRNA sequences and strand-specific libraries were constructed as described earlier [Adv Microbiol 4, 25-32 (2014); Arch Microbiol 198, 353-362 (2016)]. Briefly, starting with 8 - 50 ng rRNA-depleted RNA, random-primed cDNA synthesis was done using ScriptSeq v2 RNA-Seq library preparation kit (Epicenter, WI). cDNA was purified using Agencourt AMPure XP system (BeckmanCoulter, NJ). Libraries were amplified using FailSafe PCR enzyme kit (Epicenter, WI). Typically 12 (50-100 ng starting RNA) or 15 (8 ng starting RNA) PCR cycles were used and reverse primer from the kit was replaced with one of the ScriptSeq Index primers. After PCR amplification, libraries were purified and size selected (~280 bp) using the Agencourt AMPure XP system (BeckmanCoulter, NJ). Profiles of library insert sizes were verified on the Experion microfluidic platform using 1K DNA chip (Bio-Rad Laboratories, CA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

GH_243_4_TAGCTT_L005

Data processing

Illumina RealTime Analysis Software, version 2 was used for basecalling.
Raw data were inspected with the quality control tool, FastQC [http://www.bioinformatics.babraham.ac.uk/projects/fastqc/].
The reads were aligned to the reference using BWA 0.6.2 aln [Bioinformatics, 25, 1754-1760, (2009)] with a parameter for read trimming of 30 and default parameters otherwise.
Gene expression was caculated using the Partek Genomics Suite 6.6 RNA-Seq Workflow (Partek Inc., St. Louis, MO, USA; http://www.partek.com) and normalized using reads per kilobase of exon model per million mapped reads, RPKM.
Genes with RPKM values less than 5.0 across all samples were filtered, and then the RPKM values were then log base 2 transformed.
Genome_build: NC_016810, S. enterica subsp. enterica serovar Typhimurium SL1344 complete genome; NC_017718, plasmid pCol1B9_SL1344 complete sequence; NC_017719, plasmid pRSF1010_SL1344 complete sequence; and NC_017720, plasmid pSLT_S1344 complete sequence
Supplementary_files_format_and_content: The file aabhagwatprocessed.txt has the format tab-delimited text and contains the filtered and transformed data for the samples in columns and genes in rows.

Submission date

Aug 31, 2016

Last update date

Aug 31, 2019

Contact name

Arvind A Bhagwat

E-mail(s)

arvind.bhagwat@ars.usda.gov

Phone

301-504-6443

Organization name

United States Department of Agriculture