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Sample GSM3122500

Query DataSets for GSM3122500

Status

Public on Jan 16, 2019

Title

S10978-6H-02

Sample type

SRA

Source name

Salmonella strain isolated from chicken

Organism

Salmonella enterica subsp. enterica serovar Enteritidis

Characteristics

strain: SJTUF 10978
genotype: wild type

Treatment protocol

Mid-log phase bacteria cultures that grown in M9FeS medium was added 2/5 of the culture volume ) ice-cold RNA-stabilizing solution (5% (v/v) water-saturated phenol pH 4.3 in 95% (v/v) ethanol), and immediately after harvest. The mixture was incubated on ice for 40 min, transferred to 2 mL RNase free ice-cold centrifuge tubes, and centrifuged at 3000 × g for 10 min at 4°C to pellet the bacteria. The cell pellets were then immediatelyfrozen in liquid nitrogen and stored at -70°C. This sample was defined as the 0 h sample for the RNAseq experiments. Four culture bottles of egg whites incubation samples (50 mL/bottle) were harvested for RNA extraction by centrifuging at 12857× g for 2 min at 4°C in 50 mL Falcon tubes to pellet the bacteria, the supernatant was discarded and tubes were inverted the on paper for 15 s to remove the residual liquid as completely as possible. . The tubes were placed on ice and immediately added 1 mL diluted ice-cold RNA-stabilizing solution (1.5% (v/v) water-saturated phenol pH 4.3, 19% (v/v) ethanol in water) to scatter the pellet, and the pellets were transferred to 2 mL RNase free centrifuge tubes and kept on ice for 40 min to inhibits the degradation of mRNA, then centrifuged,. The centrifugation step was repeated and the samples were frozen in liquid nitrogen and stored at -70°C. At each time point, RNA from 4 parallel samples were pooled and taken as an independent biological replicate. Three independent biologically repeated experiments were done at different times with different batches of SPF eggs.

Growth protocol

The mid-log phase bacteria cultures that grown in M9FeS medium at 37°C with shaking at 200 rpm, was harvest. And then 10 mL samples were centrifuged at 5000 × g for 3 min, washed twice and suspended in PBS. The cell concentration was adjusted to 5 × 108 CFU/mL and 0.5 mL cell suspension solution was added to 9.5 mL PBS, mixed and transferred to 40 mL egg white in a 250 mL cone bottle that had been preheated to 37°C. This achieved a final inoculum concentration of 5×106 CFU/mL and a final egg white concentration of 80% (V/V).These solutions were mixed and incubated at 37°C under a 65% humidified atmosphere.

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 2000

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 serovar Enteritidis SJTUF 10978 genome, the accession number is CP015524.1
Supplementary_files_format_and_content: Excel files include GeneID, gene Length, Normalized FPKM value of S10978-0H-Expression and S10978-6H-Expression, log2FoldChange(S10978-6H/S10978-0H), Up-Down-Regulation(S10978-6H/S10978-0H), Padj Pvalue type , KEGG Orthology, GO Component, GO Function, GO Process, Blast nr

Submission date

Apr 30, 2018

Last update date

Jan 16, 2019

Contact name

Xiaozhen Huang

E-mail(s)

wdhxz@aliyun.com

Organization name

Shanghai Jiao Tong University

Department

School of Agriculture & Biology