title: Illumina HiSeq 4000 paired end sequencing; Transcriptomic profile of children with septic shock with versus without immunoparalysis
design: Transcriptomic profile of children with septic shock with versus without immunoparalysis
library_construction_protocol: Whole blood (0.6 to 1ml) was drawn into tubes containing 1 ml of TempusTM RNA stabilization solution (Applied Biosystems).  The tubes were shaken vigorously then immediately frozen and stored at -20°C for batch analysis.  RNA was isolated using the MagMax for Stabilized Blood Tubes RNA Isolation kit (Life Technologies) via a modification of the manufacture's protocol to permit small volume blood sampling as follows (15).  100 ul of Tempus 1x PBS was added to each blood sample and vortexed for 30 seconds.  Tubes were centrifuged at 4,000 x g for 20 minutes at 4°C to pellet the crude RNA.  Pellets were washed with 4ml Tempus Pre-Digestions Wash, centrifuged at 4,000 x g, resuspended in 120 ul of Tempus Resuspension Solution and Proteinase and vortexed for 10 seconds to resuspend the pellet. 10 ul of TURBOTM DNase was added and the tubes and vortexed for 10 minutes. 50 ul of Binding Solution Concentrate and 20 ul of RNA binding beads were then added and the tube was vortexed for 1 minute. 200 ul of 100% isopropanol was added for another 3-minute vortex. A magnetic stand was used to capture the RNA binding beads with the supernatant discarded. 150 ul of Wash Solution 1 was added to the tube and vortexed for 1 minute. The tube was then placed on the magnetic stand to capture the RNA Binding beads, discarding the supernatant. This wash was repeated once. 150 ul of Wash Solution 2 was added to the tube and vortexed for 1 minute then put back on the magnetic stand. The supernatant was carefully discarded, and the wash was repeated. The tubes were inverted on absorbent paper for 2 minutes to dry the beads then 60 ul of Elution Buffer was added. The tubes were vortexed for 4 minutes and put back on the magnetic stand. The RNA-containing supernatant was extracted from the tubes and stored at -20 °C. Strand-specific RNA-seq libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, following the manufacturer's recommendations.  In summary, total RNA quality was assessed using RNA 6000 Nano kit on Agilent 2100 Bioanalyzer (Agilent Biotechnologies) and concentration measured using Qubit RNA HS assay kit (Life Technologies). An aliquot of total RNA was rRNA depleted using NEB's Human/Mouse/Rat RNAse-H based Depletion kit (New England BioLabs).  Following rRNA removal, mRNA was fragmented and then used for first- and second-strand cDNA synthesis with random hexamer primers. ds cDNA fragments underwent end-repair and a-tailing and ligated to dual-unique adapters (Integrated DNA Technologies). Adaptor-ligated cDNA was amplified by limit-cycle PCR.  Library quality was analyzed on Tapestation High-Sensitivity D1000 ScreenTape (Agilent Biotechnologies) and quantified by KAPA qPCR (KAPA BioSystems).