Illumina NovaSeq 6000 paired-end QuantSeq 3&#39; mRNA Sequencing of MCAOR_2 Mouse Brain (KAS24118980) (KAE24094486)
Total RNA was extracted from mouse brain tissues using TRIzol reagent (Invitrogen, CA, USA) according to instructions of manufacturer. RNA quantity and quality were assessed using a NanoDrop One spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). mRNA-Seq libraries were prepared from 500 ng of total RNA using the QuantSeq mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, cat. no. 015.96) following the manufacturer's instructions. Libraries were quantified using a Qubit 4 Fluorometer (Invitrogen, CA, USA) and their size distribution was analyzed using an Agilent 2100 Bioanalyzer. sinlge-end sequencing was performed on an Illumina NovaSeq 6000 platform by Ebiogen (Seoul, Korea).Raw FASTQ reads were subjected to quality control using FastQC version 0.11.9 to assess read quality and adapter content. Adapter sequences and low-quality bases (Phred quality score below 20, minimum read length 50 bp) were removed using Trimmomatic version 0.39 in single-end mode. Cleaned reads were then mapped onto the Mus musculus genome (mm10 assembly, GRCm38.p6) using STAR version 2.7.9a. Gene expression quantification from uniquely aligned reads was performed using featureCounts version 2.0.1 (part of the Subread package) with annotations from Ensembl version 102. The 'union' mode was used for counting, and only non-ambiguously assigned reads were retained for further analyses. Differential expression analysis was conducted using the DESeq2 R package version 1.38.3. Assembly: mm10. Supplementary files format and content: TSV file containing raw counts for each sample.