GSM5743471: SABio_LN-mes_CtrlAb_NHP6; Macaca mulatta; RNA-Seq
We extracted total RNA (preferentially enriched for long RNA >200 nt) using a hybrid TRIzol/column-based extraction published previously (Barnes KG et al. Clin Inf Dis 2017). Briefly, we added chloroform to TRIzol homogenates, centrifuged to separate the aqueous and organic phases, and transferred the aqueous phase to a new microfuge tube. We added AVL Buffer (QIAamp Viral RNA Mini Kit, QIAgen) and ethanol to the aqueous phase, bound RNA on QIAamp Mini Spin columns by centrifugation, washed the column with Buffers AW1, AW2, and ethanol, and eluted in nuclease-free water. We further treated samples with TURBO DNase (Thermo Fisher Scientific) and purified RNA using RNAClean XP magnetic beads (Beckman Coulter). We prepared strand-specific RNA-Seq libraries using the TruSeq Stranded mRNA Kit (Illumina) 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 FPF (Illumina) and fragmented the RNA at 94 °C for 2 min. We then proceeded with the TruSeq Stranded mRNA Kit per manufacturer's protocol, starting from the reverse transcription step. The remainder of the manufacturer's protocol includes dUTP marking of the second strand, end repair and dA-tailing of the cDNA, ligation with Y-shape adaptors, USER digestion of the second strand, and 15 cycles of PCR.