Paired-end RNA-Seq of mouse RPE (C57BL/6J), sample Cont-1-RPE (KAS24120986) (KAE24095448)
0.5 ug of total RNA was used to generate sequencing libraries with the Illumina TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, Inc., 20020599, San Diego, CA, USA). Ribosomal RNA was depleted, and the remaining RNA was fragmented by incubation with divalent cations at high temperature. The resulting RNA fragments were reverse-transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen, 18064014) and random hexamer primers. Second-strand synthesis was then carried out with DNA Polymerase I, RNase H, and dUTP incorporation. The double-stranded cDNA was subsequently processed for end repair, adenylation of 3 ends, and adapter ligation. Following PCR amplification, the libraries were purified, quantified using the KAPA Library Quantification Kit (KAPA BIOSYSTEMS, KK4854, Potters Bar, UK), and evaluated for quality with the Agilent TapeStation D1000 ScreenTape (Agilent Technologies, 5067-5582).