Computational protocol: In Vivo Ligands of MDA5 and RIG-I in Measles Virus-Infected Cells

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Protocol publication

[…] Isolated RNA was prepared for Illumina sequencing using the mRNA-Seq library preparation kit (Epicentre) according to manufacturer's protocol. To remove ribosomal RNA species from the sequencing libraries a Ribo-Zero rRNA removal kit (Epicentre) was used. Quality of RNA-Seq libraries was validated on a DNA1500 chip for the Bioanalyzer 2100 (Agilent). Sequencing was performed on the Illumina Genome Analyzer in the Gene Center sequencing facility (LAFUGA). Obtained sequences were processed with the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) in order to remove adapter sequences and reads with PHRED scores below 30. Remaining sequences were mapped to human and viral genomes by utilization of the Bowtie algorithm , allowing maximal one mismatch per unique read. The Bowtie sequence alignments were converted with SAMtools to pileup format, which was subsequently used for further data analysis. Relative sequence abundances were analyzed between RLR pull-down samples and the GFP control. Specific read enrichments were calculated by determining the relative sequence abundance at each position on the genomic segment and calculating the average of the RLR/GFP ratios over a dynamic window of 200 reads. Relative sequence abundances with log2 ratios above +1 were defined as significantly enriched in the RLR library. [...] RNA secondary structure prediction from measles virus genome or in vitro transcripts was performed by utilization of RNAfold from the ViennaRNA package using standard parameter settings. For this purpose, the genome was divided into 201 nt fragments with a shifting window size of 5 nt. The sequences were folded in silico and the linear relationship between different data sets was quantified with the Pearson correlation coefficient. […]

Pipeline specifications

Software tools RNAfold, ViennaRNA
Application RNA structure analysis
Organisms Measles morbillivirus, Homo sapiens
Diseases Virus Diseases
Chemicals Adenosine Triphosphate