GSM9474891: cardiac tissue of aged mice treated with MLN4924, Rep3; Mus musculus; RNA-Seq
Total RNA was isolated from cardiac tissue of young and aged mice treated with vehicle, and aged mice treated with MLN4924 (n = 3 per group), and RNA quality was assessed using an Agilent 2100 Bioanalyzer. Poly(A)+ mRNA was purified using oligo(dT)-attached magnetic beads and fragmented into short pieces using a fragmentation buffer. First-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand cDNA synthesis. The resulting double-stranded cDNA underwent end repair, 3′ adenylation, and adaptor ligation. Libraries were amplified by PCR, purified with XP beads, eluted in EB buffer, and quality-checked on the Agilent 2100 Bioanalyzer. Double-stranded PCR products were heat-denatured and circularized using a splint oligonucleotide to generate single-stranded circular DNA (ssCir DNA), which served as the final library template. DNA nanoballs (DNBs), each containing >300 copies of a single ssCir DNA molecule, were produced by phi29 polymerase–mediated rolling circle amplification.