GSM8816767: MKL-1, DMSO, biol rep 1, PacBio Sequel IIe; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) Strand-specific libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit, following the manufacturer's instructions. ≥400 ng of total RNA (measured by Qubit) was used. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, first-strand cDNA was synthesized using random hexamer primers, followed by second-strand cDNA synthesis with dUTP instead of dTTP to maintain strand specificity. The strand-specific libraries then underwent end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Size selection was performed using AMPure XP beads to isolate fragments of approximately 300 bp. Library quality was assessed using Qubit and real-time PCR for quantification and a Bioanalyzer for size distribution analysis. Equimolar amounts of each quantified library were pooled for sequencing on the Illumina NovaSeq X Plus Series platform, generating paired-end 150 bp reads with 18 GB of raw data per sample (60 million reads).