GSM8009671: Brain WT 3; Rattus norvegicus; RNA-Seq
Total RNA was isolated using Trizol from indicated tissues and C6 cells. Only samples with a 260/280 nm ratio within 1.8 to 2.1 and the intensity of a 28S/18S ratio between 1.5 to 2 were further processed. Briefly, rRNA depletion and RNA fragmentation were performed on total RNA samples (2 μg). Next, the RNA was reverse-transcribed into double-stranded cDNA with random primers. cDNA samples were end-repaired and polyadenylated before ligation of Y-shaped adapters containing the index for multiplexing, followed by dUTP strand degradation. Fragments containing Y-shaped adapters on both ends were enriched by PCR. The quantity of the cDNA library was validated using Qubit dsDNA HS Assay Kit (Invitrogen). The purity of the libraries was analyzed by a fragment analyzer automated CE system (Advanced Analytical) and the sequencing was further performed only when the libraries had a size of 300 to 400 bp and no adaptor dimer contamination. PE150 sequencing was performed on the Illumina NovaSeq 6000.