Computational protocol: RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems

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Protocol publication

[…] Small-RNA sequencing from Aag2 cells and mosquitoes was carried out by Edinburgh Genomics (University of Edinburgh) using an Illumina HighSeq 2000 platform and a HighSeq 2500 platform, respectively, as previously described (). Cells were either infected with RVFV strain MP12 at a multiplicity of infection (MOI) of 5 or left uninfected. At 24 h p.i., RNA was isolated using 1 ml TRIzol reagent (Life Technologies, Inc.) per flask followed by purification.Similarly, S2 cells were infected at an MOI of 1, and the small-RNA fraction was isolated 96 h p.i. using a mirVana miRNA isolation kit (Ambion). The small-RNA fraction derived from purification was further size selected by fractionation on a 15% PAGE gel (7 M urea, 0.5× Tris-borate-EDTA [TBE]). RNAs (18 to 24 nucleotides in size) were excised using a RiboReady Colour Micro RNA ladder (Amresco) for size control and eluted from gel overnight in 0.3 M NaCl buffer at 4°C with horizontal shaking (200 rpm). Purified 18-nt to 24-nt small-RNA fractions were suspended in 10 μl H2O, and library preparation was performed using an NEB Next Multiplex small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer’s protocol. Sequencing was performed using a MiSeq reagent kit (v3) and an Illumina MiSeq platform according to the manufacturer’s protocol.Analysis of small-RNA reads was performed by the use of clipping adapters, and identical sequences were collapsed using the fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Reads were then aligned to RVFV (NCBI accession numbers: for the L segment, DQ375404; for the M segment, DQ380208; for the S segment, DQ380154) using BWA-backtrack (). Length distributions of the reads and positioning on the RVFV genome were analyzed using in-house perl (version 5.20.1) and R () (version 3.1.1) scripts. To examine the preferred overlap between reads mapping on opposite strands, for each position of the genomic strand, the numbers of reads on the antigenomic strand with overlaps of between 1 and 20 nucleotides were counted, normalized to 1, and summed up (weighted with the numbers of reads mapping at the respective positions of the genomic strand). […]

Pipeline specifications

Software tools FASTX-Toolkit, BWA
Application RNA-seq analysis
Organisms Rift Valley fever virus, Homo sapiens, Viruses, Human poliovirus 1 Mahoney, Drosophila melanogaster, Culex quinquefasciatus