GSM6738839: Cerebellum WT 10wk replicate 2; Mus musculus; RNA-Seq
Collected tissue was stored in RNAlater solution (Thermo Fisher Scientific). Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer's protocol. Quality control involved quantification using fluorimetry via RiboGreen assay kit (Thermo Fisher Scientific) and RNA integrity was assessed via capillary electrophoresis using an Agilent BioAnalyzer 2100 to generate an RNA integrity number (RIN). Library creation was completed using oligo-dT purification of polyadenylated RNA, which was reverse transcribed to create cDNA. cDNA was fragmented, blunt ended, and ligated to barcode adaptors. Libraries were size selected to 320 bp ± 5% to produce average inserts of approximately 200 bp, and size distribution was validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen, Thermo Fisher Scientific) and qPCR. Libraries were then normalized, pooled, and sequenced on an S4 flow cell by an Illumina NovaSeq 6000 using a 150-nucleotide, paired-end read strategy. Libraries were prepared and sequenced in two batches based on mouse age; 10-week mice were sequenced to a minimum of 70 million reads/sample, while 26-week mice were sequenced to  a minimum pf 40 million reads/sample.