GSM8426699
ESCs infected with LCMV at a MOI of 0.1 or transfected with a plasmid expressing a MosIR dsRNA hairpin were used for isolation of RISC complexes by Trans-kingdom, rapid, affordable Purification of RISCs (TrapR, Lexogen, Austria) according to manufacturer's instructions 24 hpi and 48 hpi, respectively. RISC-associated small RNAs were extracted by a mixture of acidic phenol, chloroform and isoamylalcohol and precipitated by cold isopropanol. RISC-associated small RNAs were immediately used for small RNA library preparation. After isolation, RNA quality was verified by electrophoresis on 1% agarose gel and RNA concentration was determined by Qubit Broad Range Assay (Invitrogen). Small RNA libraries were prepared using NEXTFLEX® Small RNA-Seq Kit v3 for Illumina (PerkinElmer) according to the manufacturer's protocol; 3′ adapter ligation was performed overnight at 20 °C, 15 cycles were used for PCR amplification and gel purification was performed for size selection. For gel purification, libraries were separated on a 2.5% agarose gel using 1× lithium borate buffer and visualized with ethidium bromide. The 150–160 bp fraction was cut off the gel and DNA was isolated using the MinElute Gel Extraction Kit (Qiagen). Final libraries were sequenced by 75-nucleotide single-end reading using the Illumina NextSeq500/550 platform.