RNeasy Mini Kit Total RNA proceeds to library construction. The Poly-A containing mRNA molecules are purified using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA. Adapter ligation prepares the ds cDNA for hybridization onto a flow. The products are enriched with PCR and purified to create the final cDNA library. Final library was quantified and library quality was evaluated by TapeStation D1000 ScreenTape. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina NovaSeq S4 with a read length configuration of 150 PE for 20M PE reads per sample (20M in each direction) TruSeq Stranded mRNA Library