GSM5831357
RNA was extracted from snap frozen, unfractionated PBMCs using the Qiagen RNeasy Plus Mini Kit (Qiagen 74134). Briefly, cells were first thawed and pelleted, then lysed following the kit protocol.  gDNA was removed using a gDNA Eliminator spin column, and remaining flow through was added to a RNeasy spin column to bind RNA.  The spin column/RNA was washed, RNA was eluted and stored at -80oC.  Later, samples were concentrated using Zymo Clean & Concentrate-5 Kit (R1013).  Briefly, RNA was thawed, bound, and added to the Zymo-Spin IC Column.  Then RNA bound to the column was washed, eluted, aliquoted and stored at -80oC. Samples were then submitted for RNA-Seq analysis using a high output, and single read 75 cycle run. Prior to Illumina total RNA library construction, DNase treated RNA was quantitated using the Qubit RNA BR assay kit (ThermoFisher Scientific, #Q10210). RNA quality (RIN) was determined on an Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano kit (Agilent, #5067-1511). Cytoplasmic and mitochondrial rRNA was removed from each sample following the Ribo-Zero Gold rRNA Removal Kit Reference Guide from Illumina. After RNA clean up, samples were resuspended in 11ul Elution buffer followed by prime and fragmentation. Illumina compatible total RNA cDNA libraries were generated following the TruSeq Stranded Total RNA Sample Preparation Guide (Illumina #20020596).