RNA-Seq of Pristimantis sp. 5: liver
We dual indexed each library using unique combinations of i5 and i7 Illumina adapters with 8 bp indexes (Glennet al., 2019). Adapters were added via limited cycle PCR using 25ul of Kapa HiFi Master Mix (KappaRoche), 2.5ul of each 10uM i5 and i7 barcoded adapters, 10ul of double-distilled water, 10ul of DNA and Kapa HiFi Taq (Roche) per reaction. The PCR conditions were as follows: initial step for 45s at 98 C,followed by 20 cycles of denaturation for 10s at 98C, annealing for 30s at 60 C, and extension for 30s at 72C; with a final extension for 3m at 72C. Each library was quantified with KAPA Library Quantification Kits (Roache), equimolarly pooled, then the final pool was re-quantified.18 Libraries were sequenced on the Illumina HiSeq plataform using 2 x150 bp chemistry.