Kapa pure magnetic beads

Libraries were prepared using a SciClone G3 NGSx workstation (Perkin Elmer) using the Kapa mRNA HyperPrep kit (Roche Applied Science). Polyadenylated mRNAs were captured using oligo-dT-conjugated magnetic beads (Kapa mRNA HyperPrep kit, Roche Sequencing) from 500 ng of total RNA on a Perkin Elmer SciClone G3 NGSx automated workstation. Poly-adenylated mRNA samples were immediately fragmented to 200-300bp using heat and magnesium. First strand synthesis was completed using random priming followed by second-strand synthesis and A-tailing. A dUTP was incorporated into the second strand to allow strand-specific sequencing of the library. Libraries were enriched and indexed using 9 cycles of amplification (Kapa mRNA HyperPrep kit, Roche Sequencing) with PCR primers, which included dual 8bp index sequences to allow for multiplexing (IDT for Illumina unique dual 8bp indexes). Excess PCR reagents were removed through magnetic bead-based cleanup using Kapa Pure magnetic beads on a SciClone G3 NGSx workstation (Perkin Elmer). The resulting libraries were assessed using a 4200 TapeStation (Agilent Technologies) and quantified by QPCR (Roche Sequencing). Libraries were pooled and sequenced on one Illumina NovaSeq SP flow cell using paired-end, 50 bp reads.

Libraries were prepared using a SciClone G3 NGSx workstation (Perkin Elmer) using the Kapa mRNA HyperPrep kit (Roche Sequencing). Polyadenylated mRNAs were captured using oligo‐dT‐conjugated magnetic beads (Kapa mRNA HyperPrep kit, Roche Sequencing) from 300 ng of total RNA on a Perkin Elmer SciClone G3 NGSx automated workstation. Polyadenylated mRNA samples were immediately fragmented to 200–300 bp using heat and magnesium. First‐strand synthesis was completed using random priming followed by second‐strand synthesis and A tailing. dUTP was incorporated into the second strand to allow strand‐specific sequencing of the library. Libraries were enriched and indexed using 12 cycles of amplification (Kapa mRNA HyperPrep kit, Roche Sequencing) with PCR primers, which included dual 8bp index sequences to allow for multiplexing (IDT for Illumina unique dual 8bp indexes). Excess PCR reagents were removed through magnetic bead‐based cleanup using Kapa Pure magnetic beads on a SciClone G3 NGSx workstation (Perkin Elmer). Resulting libraries were assessed using a 4200 TapeStation (Agilent Technologies) and quantified by QPCR (Roche Sequencing). Libraries were pooled and sequenced on one Illumina NovaSeq SP flow cell using paired‐end, 75 bp reads.