Computational protocol: PADI4 acts as a coactivator ofTal1 by counteracting repressivehistone arginine methylation

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Protocol publication

[…] Gene expression array analysis of shTal1 and shPADI4 HEL cells was done with a whole-genome 4 × 44K gene array (GPL13497, Agilent Technologies, Santa Clara, CA, USA). Array analysis was performed in triplicate. Microarrays were done using the ‘Low RNA Input linear Amplification Kit Plus, One Color’ protocol (Agilent Technologies) and the Agilent RNA Spike-In Kit for One color (Agilent Technologies) following the manufacturer’s standard protocol. Total RNA (200 ng) were used as a starting material to prepare cDNA. cDNA synthesis and in vitro transcription were performed according to the manufacturer’s recommendation. Quantity and efficiency of the labelled amplified cDNA were determined using the NanoDrop ND-1000 UV–vis Spectrophotometer version 3.2.1. The hybridizations were performed for 17 h at 10 r.p.m. and 65 °C in the Hybridization Oven (Agilent Technologies). Washing and staining of the arrays were done according to the manufacturer’s recommendation. Cy3 intensities were detected by one-colour scanning using an Agilent DNA microarray scanner. Scanned image files were visually inspected for artifacts and then analysed.Intensity data were extracted using Agilent’s Feature Extraction software (version 11.5.1.1) including a quality control based on internal controls using Agilent’s protocol GE1_107_Sep09. All chips passed the quality control and were analszed using the Limma package of Bioconductor. Candidate genes were filtered to a minimum of twofold change and false discovery rate-corrected P-value <0.05. For functional characterization of the differentially expressed transcripts, GO enrichment analysis was conducted via goseq accounting for gene length bias and correcting for multiple testing. Further functional association of candidate genes was performed with the webtool DAVID. […]

Pipeline specifications

Software tools limma, GOseq, DAVID
Application Gene expression microarray analysis
Diseases Leukemia