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Sample GSM8652994

Query DataSets for GSM8652994

Status

Public on Dec 01, 2024

Title

T5.415, 50 mM PPLO medium repeat1

Sample type

SRA

Source name

bacterial cell

Organism

Mycoplasmopsis bovis

Characteristics

cell type: bacterial cell
genotype: T5.415
treatment: inoculate under 50 mM PPLO medium

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies). Quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 system (Agilent).
Zymo-Seq RiboFree Total RNA Library Kit was used to remove rRNA from total RNA. Random oligonucleotides and SuperScript III were used to synthesize the first strand cDNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). T

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Library name: T_5415_1
T5.415_allSamples

Data processing

The quality information of raw data in FASTQ format was calculated and then the raw data was filtered using fastp(0.22.0) software, clean data was obtained by removing reads containing adapter, reads containing ploy-N and low quality reads. All the subsequent analysis was based on high quality clean data
The reference genome and gene annotation files were downloaded from genome website. Reference genome index was built by Bowtie2(v2.5.1) and the filtered reads were mapping to the reference genome using Bowtie2
The gene read count value was counted using HTSeq(v0.9.1) as the original expression level of the gene. In order to make the gene expression levels of different genes and different samples comparable, FPKM(fragments per kilobaseof exon per million fragments mapped) is used to normalize the expression.
DESeq(v1.38.3) was conducted to analyze the differential expressed mRNA, transcripts with |log2FoldChange|>1 and P-value<0.05 were considered as differentially expressed mRNA.
we mapped all the genes to Terms in the Gene Ontology database and calculated the numbers of differentially enriched genes in each Term. Using topGO to perform GO enrichment analysis on the differential genes, calculate P-value by hypergeometric distribution method (the standard of significant enrichment is P-value <0.05), and find the GO term with significantly enriched differential genes to determine the main biological functions performed by differential genes. ClusterProfiler (v4.6.0) software was used to carry out the enrichment analysis of the KEGG pathway of differential genes, focusing on the significant enrichment pathway with P-value <0.05.
Assembly: GCF_000270525.1_ASM27052v1_genomic.fna
Supplementary files format and content: rawdata data is fastq Format file
Supplementary files format and content: This table contains the corresponding FPKM values for each gene identified on the genome for each sample

Submission date

Nov 26, 2024

Last update date

Dec 01, 2024

Contact name

Jiongxi Chen

E-mail(s)

[email protected]

Organization name

Huazhong Agriclutural University

Department

College of Animal Science & Technology