Computational protocol: Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells

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Protocol publication

[…] A detailed description of RNA sequencing analysis, including RNA isolation; library preparation; quality assessment, filtering and alignment of RNA sequencing; gene expression filtering; and transcriptome analysis are provided in accompanying research paper by Ignatius Irudayam et al. . Sequencing was performed on an Illumina HiSeq 2500 and obtained an average read depth of 26 million reads per sample. Quality control of raw illumina reads was done by RNASEQC . TOPHAT program was used for aligning reads to the UCSC human reference genome (hg19) . Reference-guided transcript assembly using a gene transfer file (GTF) from UCSC genes was used for calculating gene expression. For all samples, normalized quantification of reads as RPKM (# of Reads Per Kilobase of ORF per Million reads aligned) was calculated with Cufflinks and subsequently was compiled into a gene counts table. For gene expression filtering, we utilized raw FPKM (Fragments Per kilobase of transcript per Million fragments mapped) values of over 1.1 to prevent overestimating gene expression differences between groups of samples and to permit downstream analyses. Raw FPKM values less than 1.1 were considered poorly measured. For calculating uniquely expressed genes, we used FPKM 5 as cut-off which represents 1 transcript copy per cell . Genes with minimum FPKM value of 5 in one of the time points were included for downstream analysis. Data can be accessed form Gene Expression Omnibus (GEO) repository using accession number GSE67848. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Databases GEO
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Neoplasms