Ambion globinclear kit

RNA was extracted and globin-cleared using the Ambion GLOBINclear kit (Thermo Fisher, Waltham, MA). RNA-Seq libraries were prepared with 0.1–1 μg total RNA using Illumina TruSeq RNA-Seq v2 kit (Illumina, San Diego, CA) according to manufacturer protocol. Ambion External RNA Controls Consortium (ERCC) RNA Spike-In Control Mix 1 (Thermo Fisher, Waltham, MA) was added to the samples. Quality control of libraries involved picogreen and size analysis on an Agilent Bioanalyzer or Tapestation 2200 (Agilent, Santa Clara, CA) and qPCR quantitation against a standard curve. Samples with an initial low RIN (i.e. < 6) were re-extracted; any samples with a persistent RIN < 6 were run in duplicate. Samples were randomized to one of 4 pools of 5–6 subjects, and each pool was sequenced in two different lanes. Sequencing of 75 base pair, paired-end reads was performed with an Illumina HiSeq 2500 instrument at Partners Personalized Medicine (Boston, MA) under rapid mode using PhiX spike-in and processed using Real-Time Analysis v1.18.64, Control Software v2.2.58 (Illumina, San Diego, CA).

Total RNA was extracted from human whole blood collected in Tempus Blood RNA tubes (ThermoFisher) using the MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit (ThermoFisher). Total RNA was subjected to globin mRNA depletion using the Ambion GLOBINclear kit (ThermoFisher). Agilent Bioanalyzer was used to evaluate total RNA integrity and the RNA Integrity Number (RIN) was calculated. All samples had RIN in the range of 4.7–8.8; however, the median value was 7. For genome-wide gene expression profiling, fluorescent-labeled PCR products were prepared according to Illumina Human Whole-Genome Gene Expression DASL Assay Guide. The labeled products were hybridized onto the Illumina HumanHT-12-v4 Expression Bead ChIP for 16 h at 58 °C. These arrays were washed, coated based on the Assay Guide, and scanned using Bead Array Scanner 500GX at BSF Microarray Facility.