Computational protocol: A fungal Argonaute interferes with RNA interference

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Protocol publication

[…] Fungal mycelia were ground with mortar and pestle in liquid nitrogen, and transferred into lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid and 0.1% Triton X-100]. After mixing by vortex, cell lysate was collected by centrifugation at 12 000 × g for 3min at 4°C, and incubated with Anti-FLAG M2 affinity agarose gel (Sigma-Aldrich) for 3hr on rotation at 4°C. After washing with TBS [50 mM Tris-Cl (pH 7.5), 150 mM NaCl] three times, FLAG-tagged protein was eluted from the agarose gel by incubating with 150 ng/μl FLAG peptide for 30 min at 4°C. The elute was collected by centrifugation at 8000 × g for 30 s at 4°C.For AGO-associated sRNA sequencing, sRNAs were recovered from the FLAG-immunoprecipitates by phenol–chloroform extraction and ethanol precipitation. For construction of the input library, sRNAs were purified from total RNA by High Pure miRNA isolation kit. Indexed cDNA libraries were prepared with the NEXTflex Small RNA-Seq v3 kit for Illumina (Bioo Scientific) according to the manufacturer’s instructions. The cDNA products were purified using Agencourt AMPure XP beads, and enriched with PCR to create the final double stranded cDNA library. The resulting libraries were sequenced on the MiSeq system (Illumina) using single end sequencing with a 75 cycle read length. Sequencing data were analyzed using Genomics workbench software v10.1 (CLCbio). For sRNA mapping, the genome of the Magnaporthe oryzae strain 70–15 (release 8.0, http://www.broadinstitute.org/) was used as a reference sequence. […]

Pipeline specifications

Software tools geWorkbench, CLC Assembly Cell
Application sRNA-seq analysis