RNA-seq of Pediatric Patients: 22-0107 D1
Bulk messenger RNA sequencing data were available from a subset of the cohort recruited between 2017 and 2019 with RNA processed from whole blood collected in PAXgene tubes on day 1 of septic shock. Prior to processing, samples in PAXgene Blood RNA tubes were removed from -80C to thaw at room temperature for two hours. The samples were then inverted several times to achieve homogeneity, after which 3 mL aliquots were removed for processing. RNA was extracted from these samples using a modified version of the RNeasy Mini Kit (QIAgen) protocol executed on the a QIAcube automated workstation. PAXgene samples comprise of whole blood in PAXgene stabilizing solution. The sample is diluted with PBS, then centrifuged at 3,000 x g to pellet precipitated nucleic acids. Pellets were washed with molecular biology grade water and again pelleted via centrifugation at 3,000 x g. Pelleted material is resuspended in Buffer RLT (QIAgen). Using the automated QIAcube, samples are then subjected to treatment by Proteinase K and gDNA elimination via columns (QIAgen). Flow-through was mixed with isopropanol and passed over a MinElute (QIAgen) spin column. The column was washed with 80% ethanol and purified nucleic acid was eluted in RNase-free water. Purified RNA was heat denatured at 55 C for 5 minutes, then snap-cooled on ice. RNA was quantitated using a Qubit fluorimeter with the Quant-iT RNA Assay kit (Thermo-Fisher). Samples with an RNA integrity number (RIN) below 7 (BioAnalyzer, Agilent) did not proceed to sequencing.  Total RNA samples were depleted of globin RNA using the GLOBINclear kit (Invitrogen) following the procedure described by the manufacturer. Globin-depleted RNA was quantified using the Qubit RNA High Sensitivity kit (Life Technologies) and 10ng of globin-depleted RNA was then used for rRNA depletion and RNAseq library preparation using the SMARTer Stranded Total RNAseq kit v2 Pico Input Mammalian (Takara Bio) following the manufacturers protocol. RNAseq libraries were then quantified using the Qubit dsDNA High Sensitivity kit (Life Technologies) and their quality and size evaluated by a Fragment Analyzer High Sensitivity Small Fragment kit (Agilent Technologies).