title: BGISEQ-500 paired end sequencing; Acute effects on the human peripheral blood transcriptome of decompression sickness secondary to scuba diving
library_construction_protocol: Recruitment of DCS cases (n = 7) and controls (n = 6) was carried out by a hyperbaric medicine physician according to the following criteria. DCS cases were included in this study if they presented with the typical cutis marmorata rash within 8 hours of surfacing from an underwater dive. Control subjects were divers who surfaced from a deep dive (≥25 msw) without dive table violation and without any symptoms suggestive of DCS as ascertained by a hyperbaric medicine physician. The Francis and Smith classification for dysbaric illness was recorded for each subject (Francis and Smith, 1991). All study subjects were unrelated and of Caucasian ethnicity, and none suffered from diabetes mellitus, hypertension, asthma, ischemic heart disease or congestive heart failure at the time of sampling. For both DCS cases and controls, whole blood was sampled at two time points, T1 - within 8 hours of surfacing from diving and T2 - at 40-44 hours after surfacing. Prior to sampling at T2, subjects were fasted for 10 hours. Specific exclusion criteria included a) age < 18 years b) self-reported consumption of alcohol and/or strenuous physical activity before T2 c) symptoms suggestive of delayed DCS presentation in controls at T2 d) acute life-threatening clinical complications or death within 72 hours of surfacing. All cases received emergency HBO as per United States Navy Treatment Table Six between T1 and T2.  For RNA isolation, 2.5mL of whole blood was collected in a PAXgene® Blood RNA Tube (PreAnalytiX, Qiagen/BD) from DCS cases and controls at both T1 and T2. The samples were frozen within 24 hours and stored at -80ºC till further use. RNA was extracted using the PAXgene® blood kit (PreAnalytiX, Qiagen/BD) and quantified by 260/280nm absorbance using UV-spectrophotometry (Nanodrop®, ThermoFisher Scientific). The quality of RNA was evaluated by RNA Integrity Number (RIN) determination using the RNA6000 Nano protocol on an Agilent 2100 Bioanalyzer system (Agilent, USA). The RIN values for samples undergoing transcriptome analysis ranged from 7.8 to 9.3. Depletion of alpha and beta globin mRNA was carried out using the GLOBINclear™ kit (ThermoFisher Scientific). RNA samples were submitted for library generation and sequencing by the Beijing Genomics Institute (BGI-Shenzhen). Briefly, poly(A) mRNA was enriched using poly(T) oligo-attached magnetic beads, followed by fragmentation. First strand cDNA synthesis was carried out using random hexamer N6 primers and reverse transcriptase. Following adaptor ligation to cDNA fragments, PCR amplification and purification, single stranded DNA circles were generated in a final library. DNA nanoballs (DNBs) were subsequently generated by rolling circle replication, which underwent paired end sequencing (100bp) on the BGI DNBseq platform.