title: Total RNA extracted from minivette-collected capillary blood, middle finger
design: Total RNA was extracted from minivette-collected capillary whole blood samples using the Quick-RNA Whole Blood kit (Zymo Research, R1201), including on-column DNAse digestion.  200 µL of whole capillary blood in 600 µL 1x DNA/RNA shield was used for each extraction. DNA/RNA Shield-preserved samples were removed from -80⁰C storage and thawed on ice. Following thawing, samples were extracted according to the manufacturer's RNA Purification (Whole Blood) protocol with the following modifications: 16 µL proteinase K was added to the thawed preserved blood solution, 800 µL isopropanol was added to the resulting mixture and spun through the purification column in 800 µL increments.  The remainder of the protocol proceeded per manufacturer's instructions, but 21 µL of Nuclease-free water was used for final elution. 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.