GSM2753170: 2328908_TimePoint1_BoneMarrow; Macaca mulatta; Plasmodium coatneyi; RNA-Seq
Bone marrow (1ml) was collected into 1.5 ml tubes with EDTA, and the mononuclear cells were purified by density gradient centrifugation on Lymphoprep (Stem Cell Technologies) solution and preserved in RLT buffer (Qiagen) to stabilize mRNA.  Whole blood (3 ml) was collected in Tempus tubes (Applied Biosystems) that also preserve mRNA; these samples include erythrocytes, platelets and granulocytes in addition to mononuclear lymphocytes.  RNA was extracted from the BM samples using Qiagen RNEasy Mini-Plus kits following the manufacturer-recommended procedures, and from PB samples using Tempus-Spin RNA isolation kits. The quality of all RNA samples was confirmed using a Bioanalyzer, with an RNA Integrity Number (RIN) greater than 8 recorded for all samples. Approximately 1 μg of total RNA per sample was converted to double-stranded cDNA using poly-A beads to enrich for mRNA, and Illumina TruSeq Stranded mRNA Sample Prep kits to generate strand-specific libraries.   As a quality control, 92 spike-in RNAs of known concentration and GC proportions (ERCC Spike-In Control, Life Technologies) were added to constitute approximately 1% of the total RNA for each library.  Adapters were ligated to facilitate multiplexed sequencing on an Illumina HiSeq1000 at the Yerkes Genomics Core, aiming for 50 million paired-end 100 base pair (bp) reads per library.