GSM9513983: SCA8_Met4; Mus musculus; RNA-Seq
Mouse tissue from cerebellum and brainstem was lysed and homogenized with 800 μL TRIzol reagent (Thermo Fisher Scientific, 15596018) in 2-mL tubes pre-filled with 1.5-mm high-impact zirconium beads (Benchmark Scientific, D1032-15). Tubes were agitated at 10,000 × g for 1 min using a Bead Ruptor 12 (OMNI International, 19-050A). Then, they were placed on ice for 1 min, for a total of four repetitions for complete tissue lysis. RNA was then extracted following the Direct-zol RNA Miniprep kit protocol, with DNase I treatment (Zymo Research, R2070). RNA concentration was measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific, ND-ONE-W). RNA integrity was assessed by measuring the RNA integrity number using capillary electrophoresis (Agilent, M5310AA) paired with the RNA kit (Agilent, DNF-471-0500). Samples with an RQN > 8 were further processed. RNA-seq libraries were constructed using the NEBNext Ultra II Directional RNA library prep kit for Illumina, using ribosomal RNA depletion followed by strand-specific RNA-seq preparation. Samples were amplified with PCR for 9–11 cycles and sequenced using the Illumina NextSeq 2000. A total of 300 ng RNA was used as input for library preparation. rRNA was depleted from samples (NEB, E7405), and libraries were prepared for paired-end Illumina sequencing (NEB, E7760L) with the following adjustments to the manufacturer's protocol: 1:40 adaptor dilution, universal and indexing primers at 1 μM final concentration, and 10 cycles for PCR enrichment of adaptor-ligated DNA. Library quality was assessed via capillary gel electrophoresis (Agilent, M5310AA) paired with the High Sensitivity Next Generation Sequencing Fragment Kit (Agilent, DNF-474-0500). Library concentrations were determined using a Library Quant Kit for Illumina (NEB, E7630L) following the manufacturer's protocol. Sequencing was performed on the Illumina NextSeq 2000 platform (Illumina, 20038897) using the onboard denature and dilute protocol, paired with P3 reagents for 200 cycles (Illumina, 20040560). Sequencing output was demultiplexed and converted to FASTQ format via Basespace BCL Convert (version 2.3.0).