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Sample GSM6598761

Query DataSets for GSM6598761

Status

Public on Sep 28, 2022

Title

WT_1

Sample type

SRA

Source name

serotype 1

Organism

Actinobacillus pleuropneumoniae

Characteristics

tissue: serotype 1
genotype: WT

Extracted molecule

polyA RNA

Extraction protocol

The WT (S4074) and ∆cpxAR mutant strains were grown in TSB overnight, then transferred (at a 1:1000 dilution) into fresh medium and grown to an OD600 of 0.6 at 37°C or 42°C. Total RNA was extracted from the bacterial solution using a Bacteria Total RNA Isolation Kit (Sangon Biotech, China) following the manufacturer’s protocol.
Total RNA was used as input material for the RNA sample preparations. For prokaryotic samples, mRNA was purified from total RNA using probes to remove rRNA. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. And in the DNA polymerase I system, use dUTP to replace the dNTP of dTTP as the raw material to synthesize the second strand of cDNA. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. Then USER Enzyme was used to degrade the second strand of cDNA containing U, In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing N base and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Both building index of reference genome and aligning clean reads to reference genome were used Bowtie2-2.2.3.
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then 2 FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels .
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed.
Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes.
Assembly: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/003/290/385/GCF_003290385.1_ASM329038v1/
Supplementary files format and content: tab-delimited text files include FPKM values for all sample
Supplementary files format and content: tab-delimited text files include FPKM values for all sample

Submission date

Sep 23, 2022

Last update date

Sep 28, 2022

Contact name

Feng Liu

E-mail(s)

521026@yangtzeu.edu.cn

Phone

13407106056

Organization name

Huazhong Agricultural University