Computational protocol: The histone demethylase dKDM5/LID interacts with the SIN3 histone deacetylase complex and shares functional similarities with SIN3

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Protocol publication

[…] The TruSeq RNA Sample Preparation Kit (Illumina) was used to prepare adapter ligated PCR fragments for sequencing. In brief, mRNA was purified from total RNA and fragmented. The cleaved mRNA was primed with random hexamers and reverse transcribed into first strand cDNA. The RNA template was then removed and a replacement, complementary strand was generated. The ends of the double-stranded cDNA was repaired and adenylated. Then sequencing adapters were ligated to the prepared cDNA. PCR was used to selectively enrich the fragments containing the adapters. The PCR fragments were validated using Agilent 2200 TapeStation. Single indexed samples were multiplexed and sequenced on an Illumina HiSeq 2500 sequencing system in paired-end mode with a read length of 2 × 50 bp. Samples were demultiplexed using Illumina bcl2fastq converter (v1.8.3). Read quality was assessed with FastQC. The RNAseq experiments were conducted in triplicate. Depth of coverage of ~25–30 million reads was obtained. [...] The Tuxedo pipeline was utilized for analysis []. The high-performance GRID computing system at Wayne State University was used for the Tuxedo pipeline analysis. In brief, reads obtained from RNA sequencing were aligned to the UCSC reference genome (dm3) using Bowtie/Tophat. Cufflinks was used to assemble the aligned reads into transcripts. The obtained reads were mapped to a total of 14,542 Refseq genes and FPKM (fragments per kilobase per million fragments mapped) values reflecting mRNA expression levels were generated through Cufflinks. Cuffdiff, an integrated package of Cufflinks, was used to identify statistically significant genes that were differentially expressed in treatment conditions compared to control. The default false discovery rate (FDR) of 5 % was used for the differential expression analysis. The R statistics environment was used to visualize the data. The correlation plots were generated using the Lattice package. The volcano plots were generated using the CummeRbund package. The heatmap was generated using gplots package (heatmap.2). Gene ontology analysis was performed using DAVID []. The functional annotation tool of DAVID was utilized where gene ontology term, GOTERM_BP_FAT was utilized to identify enriched biological processes and KEGG_PATHWAY was used to identify enriched pathways. The RNAseq data are available in the NCBI gene expression omnibus (GEO) database under accession number GSE68775 []. […]

Pipeline specifications