GSM5739068: LN REP2 (lanes 1-4); Macaca mulatta; ssRNA-seq
GSM5739068
Rhesus monkey total RNA was purchased from a vendor (Zyagen). We prepared strand-specific RNA-Seq libraries using a dUTP-based method published previously (Levin JZ et al. Nat Methods 2010) with modifications. Starting from 100–1000 ng total RNA per sample, we performed ribosomal RNA depletion with antisense DNA oligos and RNase H (Matranga CB et al. Genome Biol 2014). During the cleanup of ribo-depleted RNA with RNAClean XP magnetic beads (Beckman Coulter), we eluted ribo-depleted RNA with 2X SuperScript IV Buffer and random hexamers (both Thermo Fisher Scientific) and fragmented the RNA at 94 °C for 3 min. We then added dNTPs, DTT, Superase-In RNase Inhibitor, actinomycin D to 0.2 µg/µL, and SSIV reverse transcriptase to 10 U/µL, incubated the reaction at room temperature for 10 min followed by slow ramping to 55 °C in an air incubator for 15 min, then 60 °C for 15 min, 65 °C for 15 min, and heat inactivation at 80 °C for 15 min. We performed second-strand synthesis with the Gubler-Hoffman method (aka nick translation; RNase H, DNA polymerase, and DNA ligase), with dUTP instead of dTTP to mark the second strand, at 16 °C for 2 h. Following cleanup on Ampure XP magnetic beads (Beckman Coulter), we treated cDNA with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB) and then performed adaptor ligation with high-efficiency conditions detailed in SPRITE (Quinodoz SA et al. Cell 2018) at 20 °C for 1 h. To mark unique cDNA fragments, we ligated IDT xGen Dual Index UMI Adapters (MacConaill LE et al. BMC Genomics 2018), which include a random 9-mer unique molecular identifier (UMI) after the 8-bp i7 (Index 1) sample index. We then degraded the dUTP-marked second strand with Thermolabile USER II enzyme (NEB), purified the remaining first-strand cDNA on Ampure XP magnetic beads, and amplified each library with primers annealing to Illumina P5 and Illumina P7 sequences (IDT) with the NEBNext Ultra II Q5 Master Mix for 6–8 PCR cycles.