Computational protocol: DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells

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Protocol publication

[…] RNA from PEFs (at passage 5) and pig iPSCs (at passages 5 or 10) was extracted using a RNeasy Mini Kit (Qiagen, Cat#74104). Libraries for sequencing were generated using NEBNext® Ultra ™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, MA) following manufacturer’s instruction, and index codes added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, Champaign, IL) according to the manufacturer’s instruction. After cluster generation, the library was sequenced on an Illumina Hiseq 2,000 platform and 100 bp paired-end reads were generated. Quality control of raw data (FASTQ format) was firstly processed through in-house Perl scripts. Clean data (clean reads) were obtained by removing reads with adapter and poly-N, as well as low abundant reads from raw data. Subsequent analyses were based on the clean data with high quality. Index of the reference genome was built using Bowtie v2.0.6 which is an ultrafast, memory-efficient short read aligner. Paired-end clean reads were aligned to the reference genome using TopHat v2.0.9 which is a fast splice junction mapper for RNA-Seq reads.Multi-array log2 transformation, normalization, and t-test were performed to identify the differentially expressed genes between any two selected samples using genomic analysis software suite (http://www.geworkbench.org). Fragments per kilobase of exon per million fragments mapped (FPKM) were calculated for each gene. Hierarchical clustering was built using Pearson’s Correlation and total linkage algorithms. The FASTQ data described in this study have been uploaded to NCBI’s Gene Expression Omnibus (accession number: GSE87361). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis of differentially expressed genes were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID); a hypergeometric test with the Benjamini and Hochberg false discovery rate (FDR) was performed using the default parameters to adjust the P value. […]

Pipeline specifications

Software tools Bowtie, TopHat, geWorkbench
Application RNA-seq analysis
Diseases Teratoma