Total RNA extracted from microvette-collected capillary blood, middle finger
Total RNA was extracted from EDTA microvette-collected capillary blood using the Total RNA Purification Plus Micro kit (Norgen Biotek Corp., 48500), with the following modifications: 700 µL RL buffer with β-mercaptoethanol was mixed with each 100 µL EDTA-preserved blood sample in the microvette tube and mixed by vortexing 30s. 600 µL of the resulting solution was spun through a gDNA removal column, and the flow-through was spun through a new gDNA removal column. 0.6 volumes ethanol were added to each sample, mixed, and 600 µL of those samples was spun through the purification column in successive spins until the entire sample passed through the column. Samples were eluted in 25 µL of RNAse-free water. All RNA samples were assessed for purity and yield using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific, ND-2000c) and a Qubit 3.0 fluorometer (ThermoFisher Scientific, Q33216) using the Broad Range Assay kit (ThermoFisher Scientific, Q10210).  RNA integrity numbers were calculated using a 4200 TapeStation system (Agilent, G2991BA) and RNA ScreenTape tapes and reagents (Agilent, 5067-5576, 5067-5578, 5067-5577). Samples were diluted to 11 ng/uL (for a total of 99 ng) in RNase-free water (ThermoFisher, AM9938) and then shipped on dry ice to the Baylor College of Medicine Human Genome Sequencing Center for library preparation and sequencing.  Samples were randomly re-arrayed to minimize potential batch effect and spiked with ERCC synthetic RNA (ThermoFisher, 4456740).  Libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Globin kit (Illumina Inc., 20020612) following the manufacturer's protocol (Illumina Inc., RS-122-9007DOC, Part # 15031048 Rev. E, October 2013). Ribosomal RNA/Globin depleted RNA samples were purified using Agencourt RNAClean XP beads (Beckman Coulter, A63987) and then used for first and second strand cDNA synthesis. The cDNA was A-tailed and ligated with the TruSeq UD Indexes V2 (Illumina Inc., 20042113), PCR amplified using the Illumina Primer Cocktail Mix included in the library kit, and then purified using AMPure XP beads (Beckman Coulter, A63882). Libraries were quantified using the Fragment Analyzer 5300, pooled in equimolar ratios, and then the pools were quantified using qPCR.  Libraries were sequenced on the Illumina NovaSeq 6000 platform using 2x150 bp paired-end reads to generate a minimum of 60 million reads per library.