GSM8907896: EAE_Brain3; Mus musculus; RNA-Seq
Total RNA was extracted from cortical tissues using TRIzol Reagent followed by purification with the RNeasy Mini Kit , including on-column DNase I digestion to remove genomic DNA. RNA purity was assessed by NanoDrop 2000 with A260/A280 = 1.9-2.1 and A260/A230 >1.8. RNA integrity (RIN ≥7) was confirmed using an Agilent 2100 Bioanalyzer RNA Nano Chip. Genomic DNA contamination was excluded by PCR amplification of GAPDH intronic regions. After total RNA extraction, eukaryotic mRNA was enriched using Oligo(dT) magnetic beads. The mRNA was fragmented into short segments using fragmentation buffer. First-strand cDNA was synthesized from the mRNA template with random hexamer primers, followed by second-strand cDNA synthesis using buffer, dNTPs, RNase H, and DNA polymerase I. The double-stranded cDNA was purified with the QiaQuick PCR Purification Kit and eluted in EB buffer. End repair, poly(A) tailing, and sequencing adapter ligation were performed. Size selection was conducted via agarose gel electrophoresis. The libraries were amplified by PCR and sequenced on an Illumina HiSeq platform.