title: GSM8426747: brain DicerDHEL1het PKRnull TBEV 6dpi; Mus musculus; ncRNA-Seq
library_construction_protocol: Organs collected from sacrificed animals were either directly used for analysis or stored at -80° C for later use. Mouse organs were washed with PBS, homogenized in Qiazol lysis reagent (Qiagen) and total RNA was isolated by Qiazol-chloroform extraction and ethanol precipitation method. 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.