RNA-Seq of Human iPSC derived RPE cells
mRNA-seq libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module following the manufacturers standard workflow. Total RNA was enriched for polyadenylated mRNA using oligo d(T)25 magnetic beads with incubation at 65 C, followed by cooling on ice, magnetic separation, and multiple washes at room temperature. Purified mRNA was eluted at 80 C and subjected to fragmentation and priming at 94 C, then cooled to 4 C. First strand cDNA synthesis was performed with incubations at 25 C, 42 C, and 70 C. Strand-specific second strand cDNA synthesis incorporating dUTP was carried out at 16 C, followed by purification using magnetic beads. End repair and dA-tailing were performed at 20 C and 65 C respectively. Adapter ligation was carried out at 20 C, followed by USER enzyme treatment at 37 C. Libraries were PCR-enriched using indexed primers, purified with magnetic beads, assessed for fragment size distribution using a TapeStation 4150, quantified by Invitrogen Qubit 4, and sequenced on Illumina NovaSeq 6000 platform.