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Sample GSM5462576

Query DataSets for GSM5462576

Status

Public on Feb 09, 2022

Title

WT_TREAT_2

Sample type

SRA

Source name

wild-type

Organism

Salmonella enterica subsp. enterica serovar Typhimurium

Characteristics

strain: ATCC14028
genotype: wild-type
agent: H2O2

Treatment protocol

The culture of mid-log phase bacteria was treated with 3 mM H2O2 for 30 min.

Growth protocol

A single colony of WT or mutant was inoculated into fresh LB broth and grown at 37 °C with rotation at 200 rpm until an optical density at 600 nm (OD600) of 1.0 was achieved. Each culture was then inoculated at a dilution of 1:100 into 5 mL fresh LB and grown to mid-log phase.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using TRIzol Reagent (Invitrogen, CAS 15596026) according to the manual.RNA quality and integrity was verified by electrophoretic analysis and UV spectroscopy using NanoDrop and Agilent 2100 instruments. The RNA integrity number for all samples were between 8.2 and 9.7.
The Strand-specific transcriptome library construction and RNA sequencing was done as follow. Briefly, 1 µg total RNA was digested with DNase I (Thermo Scientific) and treated with Ribo-Zero Magnetic Gold Kit (Epicenter) to deplete rRNA. The protocol of TruSeq RNA Sample Prep Kit v2 (Illumina) was used to construct the libraries. The RNA was fragmented into small pieces and first-strand cDNA was generated using Super Script II (Invitrogen). The products were purified and used in a Second Strand Master Mix containing dATP, dGTP, dCTP and dUTP. The purified fragmented cDNA was end-repaired and purified and A-tailed and adapters were attached. The purified product was digested with uracil-N-glycosylase to remove the dUTP containing second strand cDNA, followed by several rounds of PCR amplification to enrich the cDNA fragments. The PCR products were purified and the libraries were assessed using the Agilent 2100 Bioanalyzer and qPCR.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence to get clean reads,the clean reads was compared to reference genomic sequences using HISAT, and the average ratio of each sample reached 97.51%.
Prediction of New transcript.The new transcription product was predicted by comparing the sequence alignment results with the reference gene sequences.We first selected potential regions with a length greater than 100 bp, an average coverage greater than 8 bp, and 60 bp away from upstream and downstream genes as candidate new transcripts. The genomic annotation information together with the new transcription products made up the integral reference genes that were used in the subsequent analyses.
The clean reads were mapped to the integral reference genes with Bowtie2 v2.2.5(Parameter: ：-q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant -p 1 -k 200) and the gene expression level was calculated by RSEM v1.2.12(Parameter：--forward-prob=0.0)
The fragments per kilobase of transcript per million mapped fragments (FPKM) incorporate normalization steps to ensure that expression levels for different genes and transcripts can be compared across runs. The DESeq2 method(parameter: abs log2(FoldChange) >= 1 && Adjusted Pvalue <= 0.05) was used to calculate the differentially expressed genes (DEG). Genes with fold change ≥ 2, Bonferroni-corrected P value (Padj) ≤0.05 and FPKM≥30 in at least one sample were determined to be DGEs.
Genome_build: Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S, complete genome GenBank: CP001363.1
Supplementary_files_format_and_content: Excel files include GeneID, Normalized FPKM value of each sample, Padj Pvalue type , KEGG Orthology, GO Component, GO Function, GO Process, Blast nr

Submission date

Jul 20, 2021

Last update date

Feb 09, 2022

Contact name

Rui Dong

E-mail(s)

ruidong518@126.com

Organization name

Shanghai Jiao Tong University

Street address

Dongchuan Road