|
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 700L 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
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|
|
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
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|
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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 |