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Sample GSM6112165

Query DataSets for GSM6112165

Status

Public on May 04, 2023

Title

young-3

Sample type

SRA

Source name

heart ventricle

Organism

Mus musculus

Characteristics

tissue: heart ventricle
strain: C57BL6
genotype: C57BL6
age: 6-8 week

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from 3 separate samples of adult and aged C57BL6 mouse heart ventricle using Trizol(Life science)and DNA-free kit(Ambion).
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and pair end 100 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Data processing

The sequencing data was filtered with SOAPnuke (v1.5.2)by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set then expression level of gene was calculated by RSEM (v1.2.12)
The heatmap was drawn by pheatmap (v1.0.8) (https://cran.r-project.org/web/packages/pheatmap/index.html)according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.4.5) (http://www.bioconductor.org/packages/release/bioc/html/ DESeq2.html)with Q value ≤ 0.05.
To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expressed gene was performed by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on Hypergeometric test . The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05)
Assembly: mm10
Supplementary files format and content: xls file include FPKM values for each Sample

Submission date

May 06, 2022

Last update date

May 04, 2023

Contact name

zhenglong guo

E-mail(s)

[email protected]

Organization name

Henan Provincial People's Hospital

Street address

NO.7, Weiwu road