GSM9567163: Cerebellum, Sedentary Wt, 116; Mus musculus; RNA-Seq
Total RNA was extracted from the cerebellum using TRIzol reagent (Invitrogen). The tissues were first homogenized with RNase-free beads and then processed using the RNeasy kit (Qiagen), followed by DNase treatment. The NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) with NEBNext rRNA Depletion Kit (New England Biolabs) was used to prepare RNA-seq libraries, with a total of 250 ng input RNA from each sample. The manufacturer's protocols were followed, with the following exceptions: 40× adaptor dilutions were used, and 13 cycles of library amplification were performed. The resulting libraries were pooled in equimolar amounts, quantified using the NEBNext Library Quant Kit for Illumina (New England Biolabs), quality checked via capillary electrophoresis on the 5300 fragment analyzer (Agilent Bioanalzyer) using the DNF-474 HS NGS kit (Agilent). Libraries with sufficient quantity were loaded into P2 flow cell 1000/2000 by Illumina and were sequenced using paired-end, 151 base pair sequencing on the Illumina NextSeq 2000 sequencer at the University at Albany RNA Institute. All datasets had total read depths greater than 90 million paired-end reads.