title: Natural Conditions Study: RNA-Seq of Papio anubis: Female Spleen (Week 47)
design: RNA-sequencing libraries were made using total RNA from PBMCs, spleen and lymph node cells.  1  g total RNA was used for the cDNA library construction using TruSeq  Stranded mRNA LT kit (Illumina, San Diego, USA) and epMotion robot (Eppendorf, Hamburg, Germany). Briefly, total RNA was incubated with magnetic oligo (dT) beads and heat denatured.  Fragmented mRNA was primed with random hexamers and transcribed into first strand cDNA using SuperScript  III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and random primers, followed by second strand cDNA synthesis using DNA polymerase I. The resulting blunt-ended dscDNAs were then 3  adenylated.  Libraries were then single-indexed using unique indexes for each sample. Indexed libraries were then enriched via PCR to amplify DNA fragments that contained adapter molecules on both ends. The libraries were then purified using AMPure XP purification beads (Beckman Coulter, Brea, CA, USA). Library construction produced single-indexed libraries with a median insert size of ~300 bp which was validated by gel electrophoresis on an Agilent 2200 TapeStation instrument using D1000 Screen Tapes (Santa Clara, CA, USA). All libraries were quantified in triplicate using SynergyH1 fluorescent plate reader (BioTek, Vermont, USA). Prior to cluster generation for sequencing, samples were normalized to an equimolar concentration. Stock libraries were diluted in 10 mM Tris-HCl to 10 nM concentrations, pooled and pools were quantified. The resulting libraries were then further diluted to 4 nM before denaturation with NaOH. The pooled denatured cDNA libraries were loaded on a cBot for cluster generation followed by 2X125 bp read length paired-end sequencing using HiSeq SBS Kits with V4 chemistry on a HiSeq 2500 sequencer (Illumina, San Diego, USA).