GSM5826962: Exd2+/+ rep 2 [small RNA-seq]; Mus musculus; ncRNA-Seq
RNA was extracted from testes with TRIzol™ Reagent (Invitrogen). RNA pellets were resuspended in 100 µl RNase free water. RNA concentration was quantified with NanoDrop (Thermo Fisher Scientific). For isolation of small RNAs, 10 µg of total RNA was run into a 15% denaturating Urea-PAGE gel, the region of gel containing small RNAs was cut out, and small RNAs were extracted rom the Urea PAGE gel slices with 400µl of NaCl 0.4M. After incubation, the solution was filtered by spinning full speed for 2 min, and RNA was precipitated by adding 40 µl of 7.5 M ammonium acetate, pH 5.5, 1 µl of Glycogen RNA grade (20 ng/ml) and 1 ml of 100% EtOH, incubating 5 h at -20°C. After centrifugation, RNA pellets were washed with 80% EtOH and suspended in RNase free H2O for Illumina library preparation. Small RNA-seq libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina®, and the quality of the libraries were validated in 2% agarose gel. For size selection, libraries were run into a 3.6% Agarose (MetaPhorTM) gel, region of desired size was cut out and DNA was extracted from gel slices with QIAGEN MinElute Gel extraction Kit. For the preparation of RNA-seq libraries, ribosomal RNA was depleted from 1 µg of total RNA using NEBNext® rRNA Depletion Kit v2 and libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®. Both small RNA-seq and RNA-seq libraries were sequenced at the GENOMICS Platform of the University of Geneva using Illumina HiSeq 4000 platform with single-end run with a read length of 50 nt.