NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5605713 Query DataSets for GSM5605713
Status Public on Nov 11, 2021
Title S. Typhimurium WT_SPI-1 Inducing Conditions 1
Sample type SRA
 
Source name S. Typhimurium WT
Organism Salmonella enterica subsp. enterica serovar Typhimurium
Characteristics growth conditions: SPI-1 Inducing Conditions
genotype: S. Typhimurium WT
Treatment protocol N/A
Growth protocol For SPI-1 inducing conditions, bacteria were grown overnight at 37C in LB broth and subcultured 1:33 into LB broth before growth for 2 hours and 40 minutes. For SPI-2 inducing conditons, bacteria were grown overnight at 37C in LB broth and subcultured 1:50 in LPM media (see Coombes et al. 2004) for 4 hours.
Extracted molecule total RNA
Extraction protocol RNAprotect Bacteria Reagent (Qiagen) in order to stabilize transcripts. After 5 minutes, bacteria were re-pelleted, and resuspended in 200mL of TE Buffer containing lysozyme (15mg/mL) and 20mL of Proteinase K. After addition of 700uL of buffer RLT+ bME, RNA was extracted using an RNeasy extraction kit (Qiagen). DNase was degraded with two Turbo DNase (Thermo-Fisher) per manufacturer instructions. To remove DNase after treatment, the solution was mixed with 350 L of -mercaptoethanol-containing RLT buffer, and then 700L of 96% ethanol was added. The mixture was then added to a RNeasy MinElute column (Qiagen) and RNA was reisolated according to manufacturer instructions.
Illumina TruSeq Stranded total RNA-Seq Kit combined with Ribo-Zero rRNA removal kit (bacteria) was used to prepare total RNA-seq libraries.
The rRNA depleted RNA was then reverse transcribed.  During the 2nd strand synthesis, the cDNA:RNA hybrid is converted into to double-stranded cDNA (dscDNA) and dUTP incorporated into the 2nd cDNA strand, effectively marking the second strand.  Illumina sequencing adapters were then ligated to the dscDNA fragments and amplified to produce the final RNA-seq library. The strand marked with dUTP is not amplified, allowing strand-specificity sequencing. 
Libraries were indexed using a dual indexing approach allowing for multiple libraries to be pooled and sequenced on the same sequencing flow cell
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina MiSeq
 
Description Ribosomal RNA depleted RNA
Data processing RNA-seq data was processed using the TrimGalore toolkit (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore) which employs Cutadapt to trim low quality bases and Illumina sequencing adapters from the 3’ end of the reads. Only reads that were 20nt or longer after trimming were kept for further analysis. Reads were mapped to the ASM2216v1 version of the Salmonella enterica strain 14028S genome and transcriptome using the STAR RNA-seq alignment tool . Reads were kept for subsequent analysis if they mapped to a single genomic location. Gene counts were compiled using the HTSeq tool (http://www-huber.embl.de/users/anders/HTSeq/). Only genes that had at least 10 reads in any given library were used in subsequent analysis. Normalization and differential expression was carried out using the DESeq2 Bioconductor package with the R statistical programming environment (https://www.R-project.org/).The false discovery rate was calculated to control for multiple hypothesis testing.
Genome_build: ASM2216v1
Supplementary_files_format_and_content: Processed_yhdJ_RNAseq.xlsx
 
Submission date Sep 30, 2021
Last update date Nov 11, 2021
Contact name Jeffrey Bourgeois
E-mail(s) Jeffrey.Bourgeois@tufts.edu
Organization name Tufts University
Street address 136 Harrison Ave
City Boston
ZIP/Postal code 02111
Country USA
 
Platform ID GPL23683
Series (2)
GSE185073 RNA-seq of wild-type and ∆yhdJ Salmonella enterica serovar Typhimurium (14028s)
GSE185077 Whole-genome integration of DNA methylomics and transcriptomics in Salmonella enterica sero. Typhimurium (14028s)
Relations
BioSample SAMN21914002
SRA SRX12412514

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap