Total RNA extracted from post-mortem lung stabilized with RNAlater
Tissue samples were homogenized using a TissueLyser II bead mill (QIAGEN, 85300) using the Adapter Set 2 x 24 and 5 mm stainless steel beads (QIAGEN, 69989) in QIAzol Lysis Reagent (brain) or Buffer RLT (lung and muscle). RNA was isolated from the homogenized tissue samples using either the RNeasy Lipid Tissue Mini kit (QIAGEN, 74804) for brain samples or the RNeasy Fibrous Tissue Mini kit (QIAGEN, 74704) for lung and muscle samples. On-column DNase I treatment was performed for all tissue extractions using the RNase-Free DNase Set (QIAGEN, 79254). RNA was isolated from whole blood samples using the Quick-RNA Whole Blood kit and treated with on-column DNase I (Zymo Research, R1201). RNA was analyzed 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 with sufficient concentration were diluted in RNase-free water and then shipped on dry ice to the Baylor College of Medicine Human Genome Sequencing Center (HGSC) for library preparation and sequencing. At the HGSC, RNA quality was assessed using a Fragment Analyzer 5300 (Agilent, M5311AA) and RNA Kit (15NT) reagents (Agilent, DNA-471-0500). Samples were randomly re-arrayed to minimize potential batch effects and spiked with ERCC synthetic RNA (ThermoFisher Scientific, 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, 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 qPCR quantified. Libraries were sequenced on the Illumina NovaSeq 6000 platform using 2x150 bp paired-end reads to generate a minimum of 100 million reads per library.