Computational protocol: Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment

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Protocol publication

[…] Raw reads from each sequencing library were firstly cleaned using FASTX-Toolkit suite (http://hannonlab.cshl.edu/fastx_toolkit/) to remove adaptor sequences, reads with unknown sequences ‘N’ and low-quality sequences (the percentage of low-quality bases with a Phred quality score less than 20 was greater than 50% in a read). The clean reads were aligned to Ensembl 70 gene annotation of the NCBI38/mm10 genome using Bowtie with default parameters. The number of annotated clean reads for each gene was calculated and normalized to reads per kilobase per million (RPKM) []. Expression differences between the samples were quantified with DESeq []. The ‘false discovery rate (FDR) ≤ 0.05 and the value of shBcl6/shNC >1.5 or <0.7 and Bcl6/control >1.5 or <0.7′ were set as thresholds to judge the significance of gene expression difference. Gene ontology (GO) analysis was performed to further understand the biological functions of the genes within coordinate expression using the online bioinformatics database DAVID []. Significant GO categories with p < 0.05 were selected. […]

Pipeline specifications

Software tools FASTX-Toolkit, Bowtie, DESeq, DAVID
Application RNA-seq analysis
Chemicals Zinc