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Sample GSM3739238

Query DataSets for GSM3739238

Status

Public on Apr 24, 2022

Title

IR5

Sample type

SRA

Source name

Ileum

Organism

Sus scrofa

Characteristics

breed: Y x L
developmental stage: piglet
age: 7-day old
tissue: ileum
infection: Clostridium perfringens type C
phenotype: resistant group (IR)

Extracted molecule

total RNA

Extraction protocol

Ileum were removed, flash frozen and total RNA was extracted from each sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 4000

Data processing

Firstly, clean reads were obtained by removing reads containing ploy-N, with 5’ adapter contaminants, without 3’ adapter or the insert tag, containing ploy A /T / G / C and low-quality reads from raw data. Simultaneously, Q20, Q30, and GC-content of the raw data were calculated. Then, we chose a certain range of length from clean reads to do all the downstream analyses. Lastly, the miRNA tags were mapped to pig reference genome (Sus scrofa 10.2) by Bowtie [18] without mismatch to analyze their expression and distribution on the reference genome.
Mapped small RNA tags were used to looking for known miRNA. miRBase 20.0 was used as reference, modified software mirdeep2 and srna-tools-cli were used to obtain the potential miRNA and draw the secondary structures. The characteristics of hairpin structure of miRNA precursor can be used to predict novel miRNA. The available software miREvo and mirdeep2 were integrated to predict novel miRNA through exploring the secondary structure, the Dicer cleavage site and the minimum free energy of the small RNA tags unannotated in the former steps.
miRNA expression levels were estimated by TPM (transcript per million) through the following criteria : Normalization formula: Normalized expression = mapped readcount/total reads*1000000. Differential expression analysis was performed using the DESeq R package. The P-values was adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.05 was set as the threshold for significantly differential expression by default.
Predicting the target gene of miRNA was performed by miRanda , RNAhybrid and PITA.
GOseq software was used to analyze the GO function of differentially expressed miRNA target genes, and KOBAS software was used to analyze the KEGG signal pathway of miRNA target genes to understand the function of differentially expressed miRNA.
Genome_build: Sus scrofa 10.2
Supplementary_files_format_and_content: TPMs and readcounts

Submission date

Apr 30, 2019

Last update date

Apr 24, 2022

Contact name

Pengfei Wang

E-mail(s)

wangpf815@163.com

Phone

18153683410

Organization name

Gansu Agricultural University