GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1513306

Query DataSets for GSM1513306

Status

Public on Aug 21, 2017

Title

Zbtb18-ES rep3

Sample type

SRA

Source name

J1 ES cells

Organism

Mus musculus

Characteristics

cell line: J1
cell type: embryonic stem cell
genotype: Zbtb18 stable expressed

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from cell or animal tissue by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Bioanalyzer 2200 (Aligent) and kept at -80℃.The RNA with RIN >8.0 is right for cDNA library construction.
RNA libraries were prepared for sequencing using IonProton,The cDNA libraries were then processed for the Proton Sequencing process according to the commercially available protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Data processing

Clean reads were obtained from the raw reads by removing the adaptor sequences, reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20 percent of bases with qualities of <13. The clean reads were then aligned to mouse genome (version: mm10) using the MapSplice program (v2.1.6). In alignment, preliminary experiments were performed to optimize the alignment parameters (-s 22 -p 15 --ins 6 --del 6 --non-canonical).
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.We applied DEseq algorithm to filter the differentially expressed genes, after the significant analysis and FDR analysis under the following criteria: i) Fold Change>2 or <0.5; ii) FDR<0.05.
Pathway analysis was used to find out the significant pathway of the differential genes according to KEGG database. We turn to the Fisher’s exact test to select the significant pathway, and the threshold of significance was defined by P-value and FDR.
Gene ontology (GO) analysis was performed to facilitate elucidating the biological implications of unique genes in the significant or representative profiles of the differentially expressed gene in the experiment. We downloaded the GO annotations from NCBI (http://www.ncbi.nlm.nih.gov/), UniProt (http://www.uniprot.org/) and the Gene Ontology (http://www.geneontology.org/). Fisher’s exact test was applied to identify the significant GO categories and FDR was used to correct the p-values.
We present Gene co-expression Networks to find the relations among genes. Gene co-expression Networks were built according to the normalized expression values of genes selected from genes in significant GO-terms. For each pair of genes, we calculate the Pearson Correlation and choose the significant correlation pairs (FDR<0.05) to constructed the network.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample …

Submission date

Sep 25, 2014

Last update date

May 15, 2019

Contact name

Haibo Wu

E-mail(s)

[email protected]

Organization name

Northwest A&F University

Street address

3rd, Taicheng Road